Large-bodied organisms have more cells that can potentially turn cancerous than small-bodied organisms, imposing an increased risk of developing cancer. This expectation predicts a positive correlation between body size and cancer risk; however, there is no correlation between body size and cancer risk across species (“Peto’s paradox”). Here, we show that elephants and their extinct relatives (proboscideans) may have resolved Peto’s paradox in part through refunctionalizing a leukemia inhibitory factor pseudogene (LIF6) with pro-apoptotic functions. LIF6 is transcriptionally upregulated by TP53 in response to DNA damage and translocates to the mitochondria where it induces apoptosis. Phylogenetic analyses of living and extinct proboscidean LIF6 genes indicates that its TP53 response element evolved coincident with the evolution of large body sizes in the proboscidean stem lineage. These results suggest that refunctionalizing of a pro-apoptotic LIF pseudogene may have been permissive (although not sufficient) for the evolution of large body sizes in proboscideans.

Here, we show that the genomes of paenungulates (elephant, hyrax, and manatee) contain numerous duplicate LIF pseudogenes, at least one (LIF6) of which is expressed in elephant cells and is upregulated by TP53 in response to DNA damage. LIF6 encodes a separation of function isoform structurally similar to LIF-T that induces apoptosis when overexpressed in multiple cell types and is required for the elephant-specific enhanced cell death in response to DNA damage. These results suggest that the origin of a zombie LIF gene (a reanimated pseudogene that kills cells when expressed) may have contributed to the evolution of enhanced cancer resistance in the elephant lineage and thus the evolution large body sizes and long life spans.

Among the most parsimonious mechanisms to resolve Peto’s paradox are a reduced number of oncogenes and/or an increased number of tumor suppressor genes (), but even these relatively simple scenarios are complicated by transcriptional complexity and context dependence. The multifunctional interleukin-6 class cytokine leukemia inhibitory factor (LIF), for example, can function as either a tumor suppressor or an oncogene depending on the context. Classically, LIF functions as an extracellular cytokine by binding the LIF receptor (LIFR) complex, which activates downstream phosphatidylinositol 3-kinase (PI3K)/AKT, Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3), and transforming growth factor β (TGFβ signaling pathways. The LIF gene encodes at least three transcripts, LIF-D, LIF-M, and LIF-T, which contain alternative first exons spliced to common second and third exons (). Remarkably, while the LIF-D and LIF-M isoforms are secreted proteins that interact with the LIFR (), the LIF-T isoform lacks the propeptide sequence and is an exclusively intracellular protein () that induces caspase-dependent apoptosis through an unknown mechanism ().

While the ultimate resolution to Peto’s paradox is that large-bodied and/or long-lived species evolved enhanced cancer protection mechanisms, identifying and characterizing those mechanisms is essential for elucidating how enhanced cancer resistance and thus large bodies and long life spans evolved. Numerous and diverse mechanisms have been proposed to resolve Peto’s paradox (), but discovering those mechanisms has been challenging because the ideal study system is one in which a large, long-lived species is deeply nested within a clade of smaller, short-lived species—all of which have sequenced genomes. Unfortunately, few lineages fit this pattern. Furthermore, while comparative genomics can identify genetic changes that are phylogenetically associated with the evolution of enhanced cancer protection, determining which of those genetic changes are causally related to cancer biology through traditional reverse- and forward-genetics approaches is not realistic for large species such as whales and elephants. Thus, we must use other methods to demonstrate causality.

Importance of metabolic rate to the relationship between the number of genes in a functional category and body size in Peto’s paradox for cancer.

The risk of developing cancer places severe constraints on the evolution of large body sizes and long life spans in animals. If all cells have a similar risk of malignant transformation and equivalent cancer suppression mechanisms, organisms with many cells should have a higher risk of developing cancer than organisms with fewer cells. Similarly, organisms with long life spans have more time to accumulate cancer-causing mutations than organisms with shorter life spans and therefore should also be at an increased risk of developing cancer, a risk that is compounded in large-bodied, long-lived organisms (). Consistent with these expectations, there is a strong positive correlation between body size and cancer incidence within species. Larger dog breeds, for example, have higher rates of cancer than smaller breeds (), and human cancer incidence increases with increasing adult height for numerous cancer types (). In stark contrast, there are no correlations between body size or life span and cancer risk between species (); this lack of correlation is often referred to as “Peto’s paradox” ().

Finally, we inferred a Bayesian time-calibrated phylogeny of atlantogenatan LIF genes, including LIF6 from African and Asian elephant, woolly and Columbian mammoth, straight-tusked elephant, and American mastodon, to place upper and lower bounds on when the proboscidean LIF6 gene may have refunctionalized ( Figure 7 A). We found that estimated divergence date of the proboscideans LIF6 lineage was ∼59 million years ago (mya) (95% highest posterior density [HPD], 61–57 mya), whereas the divergence of proboscideans was ∼26 mya (95% HPD, 23.28 mya). These data indicate that the proboscidean LIF6 gene refunctionalized during the evolutionary origin of large body sizes in this lineage, although precisely when within this time interval is unclear ( Figure 7 B). Thus, LIF6 was reanimated sometime before the demands of maintaining a larger body existed in the proboscidean lineage, suggesting LIF6 is permissive for the origin of large bodies but is not sufficient.

(A) Time-calibrated Bayesian phylogeny of atlantogenatan LIF genes. The proboscidean LIF6 clade is highlighted in red, canonical LIF genes in black, and LIF duplicates in gray. The 95% highest posterior density (HPD) of estimated divergence dates is shown as blue bars. Nodes used to calibrate divergence dates are shown with black circles.

While functional genes evolve under selective constraints that reduce their d N /d S (ω) ratio to below 1, pseudogenes are generally free of such constraints and experience a relaxation in the intensity of purifying selection and an elevation in their d N /d S ratio. Therefore, we used a random-effects branch-site model (RELAX) to test for relaxed selection on duplicate LIF genes compared to canonical LIF genes. The RELAX method fits a codon model with three ω rate classes to the phylogeny (null model), and then tests for relaxed/intensified selection along lineages by incorporating a selection intensity parameter (K) to the inferred ω values; relaxed selection (both positive and negative) intensity is inferred when K < 1 and increased selection intensity is inferred when K > 1. As expected for pseudogenes, LIF duplicates (other than proboscidean LIF6 genes) had significant evidence for a relaxation in the intensity of selection (K = 0.36, likelihood ratio test [LRT] = 42.19, p = 8.26 × 10 −11 ) as did the proboscidean LIF6 stem lineage (K = 0.00, LRT = 3.84, p = 0.05). In contrast, proboscidean LIF6 genes had significant evidence for selection intensification (K = 50, LRT = 4.46, p = 0.03). We also found that the branch-site unrestricted statistical test for episodic diversification (BUSTED), which can detect gene-wide (not site-specific) positive selection on at least one site and on at least one branch, inferred a class of strongly constrained sites in (ω = 0.00, 23.74%), a class of moderately constrained sites (ω = 0.64, 75.85%), and a few sites that may have experienced positive selection in proboscidean LIF6 genes (ω = 10000.00, 0.41%; LRT = 48.81, p ≤ 0.001). These data are consistent with the reacquisition of constraints after refunctionalization.

We reasoned that most duplicate LIF genes are (likely) pseudogenes because elephant LIF6 is deeply nested within the duplicate LIF clade, is the only expressed duplicate, and is the only duplicate with a TP53 response element, suggesting elephant LIF6 re-evolved into a functional gene from a pseudogene ancestor. To test this hypothesis and reconstruct the evolutionary history of the LIF6 gene in the proboscideans with greater phylogenetic resolution, we annotated the LIF6 locus in the genomes of elephantids including the African savannah elephant (Loxodonta africana), African forest elephant (Loxodonta cyclotis), Asian elephant (Elephas maximus), woolly mammoth (Mammuthus primigenius), Columbian mammoth (Mammuthus columbi), and straight-tusked elephant (Palaeoloxodon antiquus), as well as the American mastodon (Mammut americanum), an extinct mammutid. We found that the genomes of each extinct proboscidean contained a LIF6 gene with coding potential similar to the African and Asian elephant LIF6 genes as well as the TP53 binding site, indicating that LIF6 evolved to be a TP53 target gene in the stem lineage of proboscideans.

To test whether LIF6-induced apoptosis was specific to elephant cells and independent of LIFR-mediated signaling, we transiently transfected Chinese hamster (Cricetulus griseus) ovary (CHO) cells, which do not express LIFR (), with the LIF6 expression vector and assayed the induction of apoptosis with the ApoTox-Glo assay. Overexpression of LIF6 induced apoptosis 5.38-fold (Wilcox test, p = 3.33 × 10) 24 hr after transfection, consistent with a pro-apoptotic function independent of LIFR ( Figure 6 B). Induction of apoptosis by LIF6, however, was almost completely blocked by co-treatment with Z-VAD-FMK ( Figure 6 B) but not cyclosporine A (CsA) ( Figure 6 B). To test whether LIF6-induced apoptosis is dependent upon Bax and Bak, we overexpressed LIF6 in Bax/Bak knockout mouse embryonic fibroblasts (MEFs) but did not observe an induction of apoptosis (Wilcox test, p = 0.14; Figures 6 C and S5 ). In contrast LIF6 overexpression induced apoptosis in wild-type MEFs (Wilcox test, p = 0. 3.10 × 10 Figures 6 C and S5 ). During apoptosis, collapse of the mitochondrial membrane potential (MMP) coincides with the opening of the mitochondrial transition pores, leading to the release of pro-apoptotic factors into the cytosol. Consistent with this mechanism, we found that LIF6 overexpression, treatment with DOX, or with nutlin-3a induced loss of MMP in CHO cells 48 hr after transfection (Wilcox test, p = 7.40 × 10 Figure 6 D). Thus, LIF6 is sufficient to induce mitochondrial dysfunction and apoptosis mediated through Bax/Bak and independent of MPTP opening.

To infer the mechanism(s) by which LIF6 contributes to the induction of apoptosis, we first determined the sub-cellular localization of a LIF6-eGFP fusion protein in African elephant dermal fibroblasts. Unlike LIF-T, which has diffuse cytoplasmic and nuclear localization (), LIF6-eGFP was located in discrete foci that co-localized with MitoTracker Red CM-H2XRos-stained mitochondria ( Figure 6 A). Mitochondria are critical mediators of cell death, with distinct pathways and molecular effectors underlying death through either apoptosis () or necrosis (). During apoptosis, for example, the Bcl-2 family members Bax/Bak form large pores in the outer mitochondrial membrane that allow cytochrome c to be released into the cytosol, thereby activating the caspase cascade (). In contrast, during necrosis, Bax/Bak in the outer membrane interact with the cyclophilin D (CypD) and the inner membrane complex, leading to the opening of the mitochondrial permeability transition pore (MPTP), swelling, and eventual rupture ().

(B) Chinese hamster ovary (CHO) cells (which do not express LIFR) were transiently transfected with an expression vector encoding the African elephant LIF6 gene and assayed for the induction of apoptosis with an ApoTox-Glo assay 24 hr after transfection. Induction of apoptosis by LIF6 was inhibited by co-treatment with the irreversible broad-spectrum caspase inhibitor Z-VAD-FMK but not CsA. Treatment of CHO cells with Z-VAD-FMK or CsA alone reduced apoptosis. N = 16; Wilcox test.

To determine whether LIF6 expression was sufficient to induce apoptosis, we transiently transfected a LIF6 expression vector into African elephant dermal fibroblasts and assayed cell viability, cytotoxicity, and apoptosis using the ApoTox-Glo assay 24 hr after transfection. We again found that LIF6 overexpression induced apoptosis in the absence of either DNA damage by DOX or TP53 activation by nutlin-3a treatment (Wilcox test, p = 3.11 × 10) and augmented apoptosis induced with DOX (Wilcox test, p = 0.02). Induction of apoptosis by LIF6 was almost completely blocked by co-treatment with the irreversible broad-spectrum caspase inhibitor Z-VAD-FMK (Wilcox test, p = 1.55 × 10) but not cyclosporine A (Wilcox test, p = 0.23), which inhibits opening of the opening of the mitochondrial permeability transition pore ( Figures 5 B and S4 ). These data suggest that LIF6 contributes to the enhanced apoptotic response that evolved in the elephant lineage, likely through a mechanism that induces caspase-dependent apoptosis.

We have previously shown that elephant cells evolved to be extremely sensitive to genotoxic stress and induce apoptosis at lower levels of DNA damage than their closest living relatives, including the African rock hyrax (Procavia capensis capensis), East African aardvark (Orycteropus afer lademanni), and southern three-banded armadillo (Tolypeutes matacus) (). To test the contribution of LIF6 to this derived sensitivity, we designed a set of three siRNAs that specifically target LIF6 and reduce LIF6 transcript abundance ∼88% ( Figure S2 ). Next, we treated African elephant dermal fibroblasts with DOX or nutlin-3a and either LIF6-targeting siRNAs or a control siRNA and assayed cell viability, cytotoxicity, and apoptosis using an ApoTox-Glo assay 24 hr after treatment. Both DOX (Wilcox test, p = 3.33 × 10) and nutlin-3a (Wilcox test, p = 3.33 × 10) reduced cell viability ∼85%, which was attenuated 5%–15% by LIF6 knockdown in DOX (Wilcox test, p = 1.33 × 10)- or nutlin-3a (Wilcox test, p = 3.33 × 10)-treated cells ( Figure 5 A). While neither DOX nor nutlin-3a induced cytotoxicity ( Figure 5 A), both DOX (4.05-fold, Wilcox test, p = 3.33 × 10) and nutlin-3a (2.64-fold, Wilcox test, p = 3.33 × 10) induced apoptosis ( Figure 5 A).

(B) African elephant fibroblasts were transiently transfected with either an empty expression vector (Ctl) or a LIF6 encoding expression vector (LIF6), and treated with DOX, the caspase inhibitor Z-VAD-FMK, or cyclosporine A (CsA), which inhibits opening of the opening of the MPTP. N = 8; Wilcox test.

(A) African elephant fibroblasts were treated with either DOX or nutlin-3a (N3a), or an equimolar mixture of three siRNAs targeting LIF6 and DOX (DOX/LIF6 siRNA) or nutlin-3a treatment (N3a/LIF6 siRNA). Cell viability, cytoxicity, and the induction of apoptosis was assayed using an ApoTox-Glo assay 24 hr after treatment. NT, no treatment. Ctl siRNA, negative control siRNA. DMSO, carrier for nutlin-3a. N = 16; Wilcox test.

Finally, we transiently transfected elephant fibroblasts with either a negative control siRNA or siRNAs targeting TP53 and a LIF6 expression vector and assayed cell viability, cytotoxicity, and apoptosis using an ApoTox-Glo assay 18 hr after treatment with DOX or control media. We found that LIF6 expression with negative control siRNAs augmented the induction of apoptosis by DOX (Wilcox test, p = 0.033; Figures 4 E and S3 ). Knockdown of TP53 did not inhibit the induction of apoptosis (Wilcox test, p = 0.033; Figures 4 E and S3 ), suggesting TP53 knockdown was insufficient to alter the induction of apoptosis; note that while siRNA-mediated knockdown significantly reduced TP53 transcript levels ( Figure S2 ), we were unable to validate knockdown of the TP53 protein because the FL-393 antibody that recognizes elephant TP53 is no longer available. Interestingly, however, LIF6 transfection induced apoptosis in elephant fibroblasts treated with control media and negative control siRNAs (Wilcox test, p = 0.008), suggesting that LIF6 can induce apoptosis in the absence of DNA damage similar to LIF-T ( Figures 4 E and S3 ). Thus, we conclude that elephant LIF6 is transcriptionally upregulated by TP53 in response to DNA damage and may have pro-apoptotic functions.

To test whether the putative TP53 binding site upstream of elephant LIF6 was a functional TP53 response element, we cloned the –1,100- to +30-bp region of the African elephant LIF6 gene into the pGL3-Basic[minP] luciferase (Luc.) reporter vector and tested its regulatory ability in dual Luc. reporter assays. We found that the African elephant LIF6 upstream region had no effect on basal Luc. expression in transiently transfected African elephant fibroblasts (Wilcox test, p = 0.53). In contrast, both DOX (Wilcox test, p = 1.37 × 10) and nutlin-3a (Wilcox test, p = 1.37 × 10) strongly increased Luc. expression ( Figure 4 C), which was almost completely abrogated by deletion of the putative TP53 binding site in DOX (Wilcox test, p = 1.37 × 10)- and N3a (Wilcox test, p = 1.37 × 10)-treated cells ( Figure 4 C). Next, we performed chromatin immunoprecipitation (ChIP)-qPCR to determine whether the TP53 binding site upstream of LIF6 is bound by TP53 in African elephant fibroblasts treated with DOX or nutlin-3a using a rabbit polyclonal TP53 antibody (FL-393) that we previously demonstrated recognizes elephant TP53 (). DOX treatment increased TP53 binding 14.26-fold (unequal variance t test, p = 0.039) and nutlin-3a increased TP53 binding 10.75-fold (unequal variance t test, p = 0.058) relative to ChIP-qPCR with normal mouse IgG control antibody. This increased binding was almost completely attenuated by siRNA-mediated TP53 knockdown ( Figure 4 D).

Previous studies have shown that TP53 regulates basal and inducible transcription of LIF in response to DNA damage through a binding site located in LIF intron 1 (), suggesting that duplicate LIF genes may be regulated by TP53. Therefore, we computationally predicted TP53 binding sites within a 3-kb window around Atlantogenatan LIF genes and identified binding site motifs in the first intron of African elephant, hyrax, manatee, tenrec, and armadillo LIF1 genes, whereas the only duplicate LIF gene with a putative TP53 binding site was elephant LIF6; note that the putative TP53 binding sites around LIF1 and LIF6 are not homologous ( Figure S1 ). Next, we treated African elephant primary dermal fibroblasts with the DNA-damaging agent doxorubicin (DOX) or the MDM2 antagonist nutlin-3a and quantified the transcription of canonical LIF1, duplicate LIF genes, and the TP53 target gene BAX by qRT-PCR. DOX treatment induced LIF6 expression 8.18-fold (Wilcox test, p = 1.54 × 10) and nutlin-3a induced LIF6 expression 16.06-fold (Wilcox test, p = 1.00 × 10), which was almost completely attenuated by small interfering RNA (siRNA)-mediated TP53 knockdown ( Figures S2 and 4 B). Treatment with DOX (Wilcox test, p = 1.55 × 10) or nutlin-3a (Wilcox test, p = 1.55 × 10) also upregulated the TP53 target gene BAX ( Figure 4 B), which again was almost blocked by knockdown of TP53 ( Figure 4 B). In contrast, neither treatment upregulated LIF1 ( Figure 4 B), and we observed no expression of the other duplicate LIF genes in African elephant fibroblasts or any LIF duplicate in hyrax fibroblasts treated with DOX or nutlin-3a. These data suggest that while LIF6 encodes a transcribed gene in elephants, transcription of the other LIF duplicates is either induced by different signals or they are pseudogenes.

If expansion of the LIF gene repertoire plays a role in the evolution of enhanced cancer resistance, then one or more of the LIF genes should be transcribed. To determine whether duplicate LIF genes were transcribed, we assembled and quantified elephant LIF transcripts with HISAT2 () and StringTie () using deep 100-bp paired-end RNA-sequencing (RNA-seq) data (>138 million reads) we previously generated from Asian elephant dermal fibroblasts (), as well as more shallow (∼30 million reads) single-end sequencing from Asian elephant peripheral blood mononuclear cells (PBMCs) (), African elephant dermal fibroblasts (), and placenta (). We identified transcripts corresponding to the LIF-D, LIF-M, and LIF-T isoforms of the canonical LIF1 gene, and one transcript of a duplicate LIF gene (LIF6) in Asian elephant dermal fibroblasts ( Figure 4 A). The LIF6 transcript initiates just downstream of canonical exon 2 and expression was extremely low (0.33 transcripts per million), as might be expected for a pro-apoptotic gene. No other RNA-seq dataset identified duplicate LIF transcripts.

(E) Knockdown of TP53 inhibits DOX-induced apoptosis in African elephant fibroblasts. Fibroblasts were transiently transfected with either a negative control siRNA (siCtl) or three siRNAs targeting TP53, and either a empty vector control or a LIF6 expression vector. Apoptosis was assayed using an ApoTox-Glo 18 hr after treatment with DOX or control media. N = 8. ∗∗∗ Wilcox test, p < 0.001.

(D) ChIP-qPCR indicates that the putative TP53 binding site is bound by TP53 in response to DOX (DOX−) or nutlin-3a treatment (N3a−), and is significantly attenuated by siRNA-mediated TP53 knockdown (DOX+ or N3a−). Data are shown as fold change relative to carrier controls (water or DMSO) and standardized to IgG control. N = 3. ∗ Unequal variance t test, p < 0.06.

(C) Dual Luc. reporter assay indicates that the LIF6 upstream region (p53RE) activates Luc. expression in African elephant fibroblasts treated in response to DOX or nutlin-3a treatment (N3a) and is significantly attenuated by deletion of the putative TP53 binding site (Δp53). Data shown as fold change relative to controls (water for DOX, DMSO for N3a). NT, no DOX or nutlin-3a treatment. N = 8. ∗∗∗ Wilcox test, p < 0.001.

(B) qPCR showing that LIF6 is upregulated in African elephant fibroblasts treated with doxorubicin (DOX) or nutlin-3a (N3a) and either a negative control siRNA (−) or an siRNA to knockdown TP53 expression (+); TP53 knockdown prevents LIF6 upregulation in response to DOX or N3a. Data are shown as fold change relative to control (water) or DMSO (a carrier for nutlin-3a). N = 8. ∗∗∗ Wilcox test, p < 0.001.

(A) Structure of the African elephant LIF/LIF6 locus (loxAfr3). The ENSEMBL LIF and geneID gene models are shown in blue and cyan. Transcripts assembled by StringTie (option “do not use GFF/GTF”) are shown in black. The region upstream of LIF6 used in TFBS prediction and Luc. assays is shown in red; the location of the putative p53 binding site is shown in dark red.

Barring transcription initiation from cryptic upstream sites encoding in frame start codons, all duplicate LIF genes encode N-terminally truncated variants that are missing exon 1, lack the propeptide sequence, and are similar in primary structures to LIF-T ( Figure 3 A). While some duplicates lack the N-terminal LIFR interaction site ( Figure 3 A), all include the leucine and/or isoleucine repeat required for inducing apoptosis ( Figure 3 A) (). Crucial residues that mediate the interaction between LIF and LIFR ( Figure 3 B) () are relatively well conserved in duplicate LIF proteins, as are specific leucine and/or isoleucine residues that are required for the pro-apoptotic functions of LIF-T ( Figure 3 C) ().suggested that the leucine and/or isoleucine residues of LIF-T are located on a single face of helix B and may form an amphipathic α-helix. Similar to LIF-T, leucine and/or isoleucine residues of duplicate LIF proteins are located on a single face of helix B ( Figure 3 D). These data suggest that at least some of the structural features that mediate LIF functions, in particular the pro-apoptotic function(s) of LIF-T, are conserved in duplicate LIFs.

(C) Sequence logo showing conservation of the leucine and/or isoleucine repeat region in duplicate LIF proteins. Leucine and/or isoleucine residues required for pro-apoptotic functions of LIF-T are indicated with red arrows. Amino acids are colored according to physicochemical properties. Column height indicates overall conservation at that site (4, most conserved).

(B) Sequence logo showing conservation of LIFR interaction sites in duplicate LIF proteins. Residues in LIF that make physical contacts with LIFR are indicated with black arrows. Amino acids are colored according to physicochemical properties. Column height indicates overall conservation at that site (4, most conserved).

(A) Domain structure of the LIF-D and LIF-T isoforms and of duplicate elephant, hyrax, and manatee LIF duplicates with coding potential. Locations of the propeptide, interactions sites with the LIF receptor (LIFR), and L/I repeat are shown.

LIF duplicates may result from independent duplication events in the elephant, hyrax, and manatee lineages, ancestral duplications that occurred in the paenungulate stem lineage followed by lineage-specific duplication and loss events, or some combination of these processes. We used Bayesian phylogenetic methods to reconstruct the LIF gene tree and gene tree reconciliation to reconstruct the pattern of LIF duplication and loss events in paenungulates. Consistent with a combination of ancestral and lineage-specific duplications, our phylogenetic analyses of paenungulate LIF genes identified well-supported clades containing loci from multiple species as well as clades containing loci from only a single species ( Figure 2 B). The reconciled tree identified 17 duplication and 14 loss events ( Figure 2 C). These data indicate that the additional LIF genes result from repeated rounds of segmental duplication, perhaps mediated by recombination between repeat elements.

We characterized LIF copy number in 53 mammalian genomes, including large, long-lived mammals such as the African elephant (Loxodonta africana), bowhead (Balaena mysticetus) and minke (Balaenoptera acutorostrata scammoni) whales, as well as small, long-lived mammals such bats and the naked mole rat. We found that most mammalian genomes encoded a single LIF gene; however, the manatee (Trichechus manatus), rock hyrax (Procavia capensis), and African elephant genomes contained 7–11 additional copies of LIF ( Figure 1 ). None of the duplicate LIF genes includes the 5′-UTR, coding exon 1, or a paired low complexity (CGAG)n/CT-rich repeat common to the canonical LIF genes in elephant, hyrax, manatee, tenrec, and armadillo ( Figure 2 A). Most of the duplicates include complex transposable element insertions composed of tandem tRNA-Asn-AAC/AFROSINE and AFROSINE3/tRNA-RTE/MIRc elements within introns 1 and 2 ( Figure 2 A). Fine mapping of the duplicate ends by reciprocal best BLAST-like alignment tool (BLAT) indicates that there is no region of homology upstream of the tRNA-Asn-AAC/AFROSINE elements for duplicates that include exon 2, whereas duplicate LIF genes that lack exon 2 have ∼150- to 300-bp regions of homology just upstream of the paired AFROSINE3/tRNA-RTE/MIRc elements in intron 2. The LIF encoding loci in the hyrax and manatee genomes have not been assembled into large-scale scaffolds, but the African elephant LIF loci are located within a 3.5-Mb block of chromosome 25 (loxAfr4).

(C) Reconciled LIF gene trees African elephant (loxAfr), hyrax (ProCap), and manatee (triMan). Duplication events are indicated with red squares, and gene loss events are indicated in blue and noted with “ ∗ LOST.” Cannonical LIF genes (LIF1) are shown in red.

(A) Organization of the LIF loci in African elephant (loxAfr), hyrax (ProCap), and manatee (triMan), tenrec (echTel), and armadillo (dasNov) genomes. The location of homologous transposable elements around LIF genes and TP53 TFBSs are shown.

LIF copy number in mammalian genomes. Clade names are shown for lineages in which the genome encodes more than one LIF gene or pseudogene.

The precise mechanisms by which mitochondrial dysfunction leads to apoptosis are uncertain, however, during early stages of apoptosis the pro-death Bcl-2 family members Bax and Bak hetero- and homo-oligomerize within the mitochondrial outer membrane leading to permeabilization (MOMP) and the release of pro-apoptotic protein such as cytochrome c (). In contrast, during necrosis, the collapse of the MMP and the opening of the MPTP leads to mitochondrial swelling, rupture, and cell death (). Our observations that CsA did not inhibit LIF6-induced apoptosis, and that LIF6 overexpression did not induce apoptosis in Bax/Bak-null MEFs suggests that LIF6 functions in a manner analogous to the pro-apoptotic Bcl-2 family members by inducing the opening of the outer mitochondrial membrane pore. Furthermore, our observation that LIF6 overexpression includes apoptosis in elephant dermal fibroblasts, Chinese hamster ovary cells, and MEFs indicates the LIF6 mechanism of action is neither of cell type nor species specific. The molecular mechanisms by which LIF6 induces apoptosis, however, are unclear and the focus of continued studies.

While we are unable to do the kinds of reverse and forward genetic experiments that traditionally establish causal associations between genotypes and phenotypes, we were able to use primary African elephant and hyrax dermal fibroblasts to functionally characterize LIF duplicates. We found, for example, that the elephant LIF6 gene is transcribed at very low levels under basal conditions but is upregulated by TP53 in response to DNA damage. One of the constraints on the refunctionalization of pseudogenes is that they must evolve new cis-regulatory elements to direct their expression, but random DNA sequences can evolve into promoters with only a few substitutions suggesting de novo origination of regulatory elements may be common (). There should be strong selection against the origin of constitutively active enhancers and/or promoters for pro-apoptotic pseudogenes, however, because their expression will be toxic. These results imply refunctionalizing LIF pseudogenes may impose a potential evolutionary cost. One of the ways to avoid that cost is through the gain of inducible regulatory elements that appropriately respond to specific stimuli, such as a TP53 signaling. Indeed, our phylogenetic analysis indicates that a TP53 response element upstream of LIF6 evolved before the divergence of mastodons and the modern elephant lineage, suggesting that LIF6 refunctionalized in the stem lineage of proboscideans coincident with the origin of large body sizes and thus may have been permissive for the large bodies.

A comprehensive analyses of genetic changes associated with the resolution of Peto’s paradox in the elephant lineage has yet to be performed, but candidate gene studies have identified functional duplicates of the master tumor suppressor TP53 as well as putative duplicates of other tumor suppressor genes (). Caulin et al., for example, characterized the copy number of 830 tumor-suppressor genes () across 36 mammals and identified 382 putative duplicates, including five copies of LIF in African elephants, seven in hyrax, and three in tenrec. Here, we show that an incomplete duplication of the LIF gene in the paenungulate stem lineage generated a duplicate missing the proximal promoter and exon 1, generating a gene with similar structure to the LIF-T isoform (), which functions as an intra-cellular pro-apoptotic protein independently from the LIFR-mediated signaling. Additional duplications of this original duplicate increased LIF copy number in paenungulates; however, most LIF duplicates lack regulatory elements, are not expressed in elephant or hyrax fibroblasts (manatee cells or tissues are unavailable), and, with the exception of elephant LIF6, are likely pseudogenes.

Experimental Procedures

Identification of LIF Genes in Mammalian Genomes We used BLAT to search for LIF genes in 53 sarcopterygian genomes using the human LIF protein sequences as an initial query. After identifying the canonical LIF gene from each species, we used the nucleotide sequences corresponding to this LIF coding DNA sequence (CDS) as the query sequence for additional BLAT searches within that species genome. To further confirm the orthology of each LIF gene, we used a reciprocal best BLAT approach, sequentially using the putative CDS of each LIF gene as a query against the human genome; in each case, the query gene was identified as LIF. Finally, we used the putative amino acid sequence of the LIF protein as a query sequence in a BLAT search. We thus used BLAT to characterize the LIF copy number in human (Homo sapiens; GRCh37/hg19), chimp (Pan troglodytes; CSAC 2.1.4/panTro4), gorilla (Gorilla gorilla gorilla; gorGor3.1/gorGor3), orangutan (Pongo pygmaeus abelii; WUGSC 2.0.2/ponAbe2), gibbon (Nomascus leucogenys; GGSC Nleu3.0/nomLeu3), rhesus (Macaca mulatta; BGI CR_1.0/rheMac3), baboon (Papio hamadryas; Baylor Pham_1.0/papHam1), marmoset (Callithrix jacchus; WUGSC 3.2/calJac3), squirrel monkey (Saimiri boliviensis; Broad/saiBol1), tarsier (Tarsius syrichta; Tarsius_syrichta2.0.1/tarSyr2), bushbaby (Otolemur garnettii; Broad/otoGar3), mouse lemur (Microcebus murinus; Broad/micMur1), Chinese tree shrew (Tupaia chinensis; TupChi_1.0/tupChi1), squirrel (Spermophilus tridecemlineatus; Broad/speTri2), mouse (Mus musculus; GRCm38/mm10), rat (Rattus norvegicus; RGSC 5.0/rn5), naked mole rat (Heterocephalus glaber; Broad HetGla_female_1.0/hetGla2), guinea pig (Cavia porcellus; Broad/cavPor3), rabbit (Oryctolagus cuniculus; Broad/oryCun2), pika (Ochotona princeps; OchPri3.0/ochPri3), kangaroo rat (Dipodomys ordii; Broad/dipOrd1), Chinese hamster (Cricetulus griseus; C_griseus_v1.0/criGri1), pig (Sus scrofa; SGSC Sscrofa10.2/susScr3), alpaca (Vicugna pacos; Vicugna_pacos-2.0.1/vicPac2), dolphin (Tursiops truncatus; Baylor Ttru_1.4/turTru2), cow (Bos taurus; Baylor Btau_4.6.1/bosTau7), sheep (Ovis aries; ISGC Oar_v3.1/oviAri3), horse (Equus caballus; Broad/equCab2), white rhinoceros (Ceratotherium simum; CerSimSim1.0/cerSim1), cat (Felis catus; ICGSC Felis_catus 6.2/felCat5), dog (Canis lupus familiaris; Broad CanFam3.1/canFam3), ferret (Mustela putorius furo; MusPutFur1.0/musFur1), panda (Ailuropoda melanoleuca; BGI-Shenzhen 1.0/ailMel1), megabat (Pteropus vampyrus; Broad/pteVam1), microbat (Myotis lucifugus; Broad Institute Myoluc2.0/myoLuc2), hedgehog (Erinaceus europaeus; EriEur2.0/eriEur2), shrew (Sorex araneus; Broad/sorAra2), minke whale (Balaenoptera acutorostrata scammoni; balAcu1), bowhead whale (Balaena mysticetus; v1.0), rock hyrax (Procavia capensis; Broad/proCap1), sloth (Choloepus hoffmanni; Broad/choHof1), elephant (Loxodonta africana; Broad/loxAfr3), Cape elephant shrew (Elephantulus edwardii; EleEdw1.0/eleEdw1), manatee (Trichechus manatus latirostris; Broad v1.0/triMan1), tenrec (Echinops telfairi; Broad/echTel2), aardvark (Orycteropus afer afer; OryAfe1.0/oryAfe1), armadillo (Dasypus novemcinctus; Baylor/dasNov3), opossum (Monodelphis domestica; Broad/monDom5), Tasmanian devil (Sarcophilus harrisii; WTSI Devil_ref v7.0/sarHar1), wallaby (Macropus eugenii; TWGS Meug_1.1/macEug2), and platypus (Ornithorhynchus anatinus; WUGSC 5.0.1/ornAna1).

Phylogenetic Analyses and Gene Tree Reconciliation of Paenungulate LIF Genes Rohland et al., 2010 Rohland N.

Reich D.

Mallick S.

Meyer M.

Green R.E.

Georgiadis N.J.

Roca A.L.

Hofreiter M. Genomic DNA sequences from mastodon and woolly mammoth reveal deep speciation of forest and savanna elephants. Chen et al., 2000 Chen K.

Durand D.

Farach-Colton M. NOTUNG: a program for dating gene duplications and optimizing gene family trees. The phylogeny of LIF genes was estimated using an alignment of the LIF loci from the African elephant, hyrax, manatee, tenrec, and armadillo genomes and BEAST (v1.8.3) (). We used the HKY85 substitution, which was chosen as the best model using HyPhy, empirical nucleotide frequencies (+F), a proportion of invariable sites estimated from the data (+I), four gamma-distributed rate categories (+G), an uncorrelated random local clock to model substitution rate variation across lineages, a Yule speciation tree prior, uniform priors for the GTR substitution parameters, gamma shape parameter, proportion of invariant sites parameter, and nucleotide frequency parameter. We used an unweighted pair group arithmetic mean (UPGMA) starting tree. The analysis was run for 10 million generations and sampled every 1,000 generations with a burn-in of 1,000 sampled trees; convergence was assessed using Tracer, which indicated convergence was reached rapidly (within 100,000 generations). We used Notung v2.6 () to reconcile the gene and species trees.

Gene Expression Data (Analyses of RNA-Seq Data and RT-PCR) Kim et al., 2015 Kim D.

Langmead B.

Salzberg S.L. HISAT: a fast spliced aligner with low memory requirements. Pertea et al., 2015 Pertea M.

Pertea G.M.

Antonescu C.M.

Chang T.-C.

Mendell J.T.

Salzberg S.L. StringTie enables improved reconstruction of a transcriptome from RNA-seq reads. Sulak et al., 2016 Sulak M.

Fong L.

Mika K.

Chigurupati S.

Yon L.

Mongan N.P.

Emes R.D.

Lynch V.J. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants. Cortez et al., 2014 Cortez D.

Marin R.

Toledo-Flores D.

Froidevaux L.

Liechti A.

Waters P.D.

Grützner F.

Kaessmann H. Origins and functional evolution of Y chromosomes across mammals. Sulak et al., 2016 Sulak M.

Fong L.

Mika K.

Chigurupati S.

Yon L.

Mongan N.P.

Emes R.D.

Lynch V.J. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants. Reddy et al., 2015 Reddy P.C.

Sinha I.

Kelkar A.

Habib F.

Pradhan S.J.

Sukumar R.

Galande S. Comparative sequence analyses of genome and transcriptome reveal novel transcripts and variants in the Asian elephant Elephas maximus. Afgan et al., 2016 Afgan E.

Baker D.

van den Beek M.

Blankenberg D.

Bouvier D.

Čech M.

Chilton J.

Clements D.

Coraor N.

Eberhard C.

et al. The Galaxy platform for accessible, reproducible and collaborative biomedical analyses: 2016 update. To determine whether duplicate LIF genes were basally transcribed, we assembled and quantified elephant LIF transcripts with HISAT2 () and StringTie () using deep 100-bp paired-end RNA-seq data (>138 million reads) we previously generated from Asian elephant dermal fibroblasts (), as well as more shallow (∼30 million reads) single-end sequencing from African elephant dermal fibroblasts () and placenta (), and Asian elephant PBMCs (). HISAT2 and StringTie were run on the Galaxy web-based platform ( https://usegalaxy.org ) () using default settings, and without a guide GTF and/or GFF file. We determined whether LIF transcription was induced by DNA damage and p53 activation in African elephant primary fibroblasts (San Diego Frozen Zoo) using RT-PCR and primers designed to amplify elephant duplicate LIF genes, including the following: LIF1-F, 5′-GCACAGAGAAGGACAAGCTG-3′; LIF1-R, 5′-CACGTGGTACTTGTTGCACA-3′; LIF6-F, 5′-CAGCTAGACTTCGTGGCAAC-3′; LIF6-R, 5′-AGCTCAGTGATGACCTGCTT-3′; LIF3-R, 5′-TCTTTGGCTGAGGTGTAGGG-3′; LIF4-F, 5′-GGCACGGAAAAGGACAAGTT-3′; LIF4-R, 5′-GCCGTGCGTACTTTATCAGG-3′; LIF5-F, 5′-CTCCACAGCAAGCTCAAGTC-3′; and LIF5-R, 5′-GGGGATGAGCTGTGTGTACT-3′. We also used primers to elephant BAX to determine whether it was upregulated by TP53: BAX-F, 5′-CATCCAGGATCGAGCAAAGC-3′; BAX-R, 5′-CCACAGCTGCAATCATCCTC-3′. African elephant primary fibroblasts were grown to 80% confluency in T75 culture flasks at 37°C/5% CO 2 in a culture medium consisting of fibroblast growth medium (FGM)/Eagle’s minimum essential medium (EMEM) (1:1) supplemented with insulin, fibroblast growth factor (FGF), 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). At 80% confluency, cells were harvested and seeded into six-well culture plates at ∼10,000 cells/well. Once cells recovered to 80% confluency, they were treated with either vehicle control, 50 μM DOX, or 50 μM Nutlin-3a. Total RNA was extracted using the RNAeasy Plus Mini kit (QIAGEN), then DNase treated (Turbo DNA-free kit, Ambion) and reverse-transcribed using an olgio-dT primer for cDNA synthesis (Maxima H Minus First Strand cDNA Synthesis kit; Thermo Scientific). Control RT reactions were otherwise processed identically, except for the omission of reverse transcriptase from the reaction mixture. RT products were PCR-amplified for 45 cycles of 94°/20 s, 56°/30 s, 72°/30 s using a Bio-Rad CFX96 real-time qPCR detection system and SYBR Green master mix (QuantiTect; QIAGEN). PCR products were electrophoresed on 3% agarose gels for 1 hr at 100 V, stained with SYBR safe, and imaged in a digital gel box (ChemiDoc MP; Bio-Rad) to visualize relative amplicon sizes.

Statistical Methods We used a Wilcox or t test test implanted in R for all statistical comparisons, with at least four biological replicates. The specific statistical test used and number replicates for each experiment are indicated in figure legends.

Luc. Assay and Cell Culture Mathelier et al., 2016 Mathelier A.

Fornes O.

Arenillas D.J.

Chen C. JASPAR 2016: a major expansion and update of the open-access database of transcription factor binding profiles. We used the JASPAR database of transcription factor binding site (TFBS) motifs () to computationally predict putative TFBSs within a 3-kb window around atlantogenatan LIF genes and identified matches for the TP53 motif (MA0106.3), including a match (sequence, CACATGTCCTGGCAACCT; score, 8.22; relative score, 0.82) ∼1 kb upstream of the African elephant LIF6 start codon. To test whether the putative p53 binding site upstream of elephant LIF6 was a functional p53 response element, we synthesized (GeneScript) and cloned the –1,100- to +30-bp region of the African elephant LIF6 gene (loxAfr3_dna range = scaffold_68:4294134-4295330 strand = + repeatMasking = none) and a mutant lacking the CACATGTCCTGGCAACCT sequence into the pGL3-Basic[minP] Luc. reporter vector. African elephant primary fibroblasts (San Diego Frozen Zoo) were grown to 80% confluency in T75 culture flasks at 37°C/5% CO 2 in a culture medium consisting of FGM and EMEM (1:1) supplemented with insulin, FGF, 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). At 80% confluency, 104 cells were harvested and seeded into 96-well white culture plates. 24 hr later, cells were transfected using Lipofectamine LTX and either 100 g of the pGL3-Basic[minP], pGL3-Basic[minP] –1,100 to +30 bp, pGL3-Basic[minP] −1,100 to +30bp Δp53TFBS Luc. reporter vectors and 1 ng of the pGL4.74[hRluc/TK] Renilla control reporter vector according the standard protocol with 0.5 μL/well of Lipofectamine LTX reagent and 0.1 μL/well of PLUS reagent. 24 hr after transfection, cells were treated with either vehicle control, 50 μM DOX, or 50 μM Nutlin-3a. Luc. expression was assayed 48 hr after drug treatment, using the Dual-Luciferase Reporter Assay System (Promega) in a GloMax-Multi+ Reader (Promega). For all experiments, Luc. expression was standardized to Renilla expression to control for differences transfection efficiency across samples; Luc./Renilla data are standardized to (Luc./Renilla) expression in untreated control cells. Each Luc. experiment was replicated three independent times, with 8–16 biological replicates per treatment and control group.

ChIP-qPCR and Cell Culture African elephant primary fibroblasts were grown to 80% confluency in T75 culture flasks at 37°C/5% CO 2 in a culture medium consisting of FGM and EMEM (1:1) supplemented with insulin, FGF, 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). 104 cells were seeded into each well of six-well plate and grown to ∼80% confluency. Cells were then treated with either a negative control siRNA or equimolar amounts of a combination of three siRNAs that specifically target the canonical TP53 transcript using Lipofectamine LTX according to the suggested standard protocol. The next day, cells were treated with either water, DMSO, 50 μM DOX, or 50 μM Nutlin-3a in three biological replicates for each condition. After 18 hr of incubation with each drug, wells were washed three times with ice-cold PBS and PBS replaced with fresh media, and chromatin cross-linked with 1% fresh formaldehyde for 10 min. We used the MAGnify Chromatin Immunoprecipitation System (Thermo Fisher; #492024) to perform ChIP according to the suggested protocol. However, rather than shearing chromatin by sonication, we used the ChIP-It Express Enzymatic Shearing Kit (Active Motif; #53009) according to the suggested protocol. Specific modifications to the MAGnify Chromatin Immunoprecipitation System included using 3 μg of the polyclonal TP53 antibody (FL-393; lot #DO215; Santa Cruz Biotechnology). We used qPCR to assay for enrichment of TP53 binding from the ChIP-seq using the forward primer 5′-TGGTTTCCAGGAGTCTTGCT-3′ and the reverse primer 5′-CATCCCCTCCTTCCTCTGTC-3′. 100 ng of ChIP DNA was used per PCR, which was amplified for 45 cycles of 94°/20 s, 56°/30 s, and 72°/30 s using a Bio-Rad CFX96 real-time qPCR detection system and SYBR Green master mix (QuantiTect; QIAGEN). Data are shown as fold increase in TP53 ChIP signal relative to the background rabbit IgG ChIP signal and standardized to the control water for DOX or DMSO for nutlin-3a treatments.

ApoTox-Glo Viability, Cytotoxicity, and Apoptosis Experiments T75 culture flasks were seeded with 200,000 African elephant primary fibroblasts and grown to 80% confluency at 37°C/5% CO 2 in a culture medium consisting of FGM and EMEM (1:1) supplemented with insulin, FGF, 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). 5,000 cells were seeded into each well of two opaque-bottomed 96-well plates. In each plate, one-half of the columns in the plate were transfected with pcDNA3.1/LIF6/eGFP (GenScript) using Lipofectamine LTX (Thermo Scientific; 15338100); the other half were mock transfected with the same protocol without any DNA. In the plate designated for the 18-hr time point, each column was treated with one of the following: 50 μM (−)-Nutlin-3 (Cayman; 18585), 20 μM Z-VAD-FMK (Cayman; 14463), 2 μM cyclosporin A (Cayman; 12088), 50 μM DOX (Fisher; BP251610), DMSO (Fisher; BP231100), or Dulbecco’s PBS (DPBS) (GIBCO; 14190136). For the 24-hr time point, the same schema for treatment was used, but with half-doses. Each treatment contained eight biological replicates for each condition. After 18 hr of incubation with each drug, cell viability, cytotoxicity, and caspase-3/7 activity were measured using the ApoTox-Glo Triplex Assay (Promega) in a GloMax-Multi+ Reader (Promega). Z-VAD-FMK readings were normalized to the PBS-treated, mock-transfected cells; all others were normalized to the DMSO-treated, mock-transfected cells. T75 culture flasks were seeded with 250,000 wild-type (ATCC; CRL-2907) and Bak/Bax double-knockout (ATCC; CRL-2913) MEFs, or Chinese hamster ovary cells (CHO-K1; Thermo; R75807), and allowed to grow to 80% confluency at 37°C/5% CO 2 in a culture medium consisting of high-glucose DMEM (GIBCO) supplemented with GlutaMax (GIBCO), sodium pyruvate (GIBCO), 10% FBS (GIBCO), and penicillin-streptomycin (GIBCO). 3,000 cells were seeded into each well of an opaque-bottomed, 96-well plate. One-half of the columns in the plate were transfected with pcDNA3.1/LIF6/eGFP (GenScript) using Lipofectamine LTX (Thermo Fisher Scientific; 15338100); the other half were mock transfected with the same protocol without any DNA. 6 hr post-transfection, the transfection reagents and media from each well were replaced: for the 24-hr time point, drug-supplemented media was placed within the wells; for the 48-hr time point, untreated media was placed in the wells, and then replaced with treatment media 24 hr later. Each column was treated with one of the following: 50 μM (−)-Nutlin-3 (Cayman; 18585), 20 μM Z-VAD-FMK (Cayman; 14463), 2 μM cyclosporin A (Cayman; 12088), 50 μM DOX (Fisher; BP251610), DMSO (Fisher; BP231100), or DPBS (GIBCO; 14190136). Each treatment contained eight biological replicates for each condition. After 18 hr of incubation with each drug, cell viability, cytotoxicity, and caspase-3/7 activity were measured using the ApoTox-Glo Triplex Assay (Promega) in a GloMax-Multi+ Reader (Promega). Z-VAD-FMK readings were normalized to the PBS-treated, mock-transfected cells; all others were normalized to the DMSO-treated, mock-transfected cells. 2 in a culture medium consisting of FGM and EMEM (1:1) supplemented with insulin, FGF, 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). 5,000 cells were seeded into each well of two opaque-bottomed, 96-well plates. In each plate, pairs of rows were transfected with either Silencer Select Negative Control No. 1 siRNA (Thermo; 4390843), P53 siRNA (Dharmacon) ( Sulak et al., 2016 Sulak M.

Fong L.

Mika K.

Chigurupati S.

Yon L.

Mongan N.P.

Emes R.D.

Lynch V.J. TP53 copy number expansion is associated with the evolution of increased body size and an enhanced DNA damage response in elephants. For knockdown experiments, T75 culture flasks were seeded with 200,000 African elephant primary fibroblasts and grown to 80% confluency at 37°C/5% COin a culture medium consisting of FGM and EMEM (1:1) supplemented with insulin, FGF, 6% FBS, and gentamicin/amphotericin B (FGM-2; singlequots; Clonetics/Lonza). 5,000 cells were seeded into each well of two opaque-bottomed, 96-well plates. In each plate, pairs of rows were transfected with either Silencer Select Negative Control No. 1 siRNA (Thermo; 4390843), P53 siRNA (Dharmacon) (), and either with or without pcDNA3.1/LIF6/eGFP (GenScript) using Lipofectamine LTX (Thermo Scientific; 15338100). In the plate designated for the 18-hr time point, each column was treated with either 50 uM DOX (Fisher; BP251610) or an equivalent dilution of ethanol (Fisher; BP2818100. For the 24-hr time point, the same schema for treatment was used, but with half-doses. Each treatment contained eight biological replicates for each condition. After 18 hr of incubation with each drug, cell viability, cytotoxicity, and caspase-3/7 activity were measured using the ApoTox-Glo Triplex Assay (Promega) in a GloMax-Multi+ Reader (Promega). All data were normalized to the ethanol-treated scrambled siRNA control samples. siRNAs were designed to specifically target the elephant LIF6 gene. Sequences of the three LIF6-specific siRNAs used are as follows: (1) 5′-GAAUAUACCUGGAGGAAUGUU-3′, (2) 5′-GGAAGGAGGCCAUGAUGAAUU-3′, and (3) 5′-CACAAUAAGACUAGGAUAUUU-3′ (Dharmacon). We also validated efficiency of the knockdown via qRT-PCR using the primer sets described earlier, which specifically the LIF6 gene, and confirmed the combination of all three LIF6 siRNAs was ∼88%. To determine whether LIF6 was sufficient to induce apoptosis, we synthesized and cloned (GeneScript) the African elephant LIF6 gene into the pcDNA3.1+C-DYK expression vector, which adds at DYK epitope tag immediately C-terminal to the LIF6 protein. We transiently transfected CHO cells or MEFs with LIF6_pcDNA3.1+C-DYK expression vector using Lipofectamine LTX according to the manufacturer’s protocol and as described above, and assayed cell viability, cytotoxicity, and the induction of apoptosis using an ApoTox-Glo triplex assay. Mitochondrion membrane potential was assayed in CHO cells using the fluorometric Mitochondrion Membrane Potential Kit (Sigma; MAK147) 48 hr after transfection.