Identification by HTS of ABMA, a ricin inhibitor active in vitro and in vivo

As previously described14, we performed a cell-based HTS to identify small chemical compounds active against ricin-mediated cell intoxication. We screened a library of 16,500 small molecules for those endowed with the capacity to prevent the inhibition of protein biosynthesis induced by ricin treatment. Four hits were confirmed; two hits named Retro-1 and Retro-2 were reported as inhibitors of ricin and Shiga-like toxins (Stx) by blocking their retrograde transport inside host cells14. ChemBridgeTM compound 1-Adamantyl (5-Bromo-2-Methoxybenzyl) Amine (ABMA, Fig. 1A) discussed in this article, was one of the other hits from that screen, which bears a hydrophobic adamantane and a substituted aromatic moiety.

Figure 1 Hit compound ABMA identified as an inhibitor of ricin by HTS. (A) Chemical structure of ABMA. (B) Intoxication of pulmonary A549 cells by increasing concentrations of ricin in the presence of 3, 10 and 30 µM of ABMA. A549 cells were incubated 4 h in DMEM with ABMA (open and half-filled circles), or solvent only as control (DMSO, black circles) before addition of increasing concentrations of ricin for 20 h. Media was removed and replaced with DMEM containing [14C]-leucine at 0.5 µCi/mL for 6 h. Protein synthesis was measured by scintillation counting as the amount of [14C]-leucine incorporated in cells. Each data point represents the mean of duplicate ± SD of a representative experiment. (C) ABMA protects mice against ricin challenge. The survival of mice treated once with the indicated doses of ABMA and then exposed to an LD 90 of ricin via nasal instillation was monitored. In each experiment, treated animals received a single ip dose of ABMA (2 mg/kg, open circles; 20 mg/kg, circles with right half black; and 200 mg/kg, circles with left half black) 1 h prior to toxin exposure (2 µg/kg by nasal instillation), while control animals (black circles) received vehicle only prior to ricin administration. The curves for treated animals are statistically different from control as measured by the log rank test (p < 0.01 for 2 mg/kg of ABMA; p < 0.001 for 20 mg/kg,; p < 0.001 for 200 mg/kg). Full size image

Inhalation is considered as a major risk factor for ricin exposure18. Thus, ABMA protective activity was first tested in vitro by challenging human pulmonary alveolar basal epithelial A549 cells with increasing concentrations of ricin (Fig. 1B). In five independent experiments, ABMA treatment induced a decrease in ricin cytotoxicity with an EC 50 of 3.8 µM, and a protection factor (R) at 30 µM ranging from 5 to 10. ABMA retained almost 100% of its biological activity against ricin-induced cytotoxicity up to six days after incubation in culture medium at 37 °C (Fig. S1), indicating a robust stability. As expected, ABMA had no observed inhibitory effect on cell protein synthesis up to at least 90 µM as measured by [14C]-leucine incorporation (Fig. S2). AlamarBlue® cell viability assay also confirmed its low toxicity on human cells, with a CC 50 (50% cytotoxicity concentration) on cultured (HeLa) and primary human cells (Human Umbilical Vein Endothelial Cells, HUVECs) at more than 200 μM (Fig. S3).

Based on these in vitro results, we investigated whether ABMA could protect mice against a lethal ricin challenge. ABMA was non-toxic to animals after one intra-peritoneal (ip) administration up to 200 mg/kg. A model of ricin intoxication by nasal instillation14 was used to mimic exposure by aerosols, as would occur during an intentional release. Briefly, mice were challenged by an LD 90 of ricin (2 μg/kg) at day 0 (Fig. 1C, closed circles). The first clinical signs of intoxication appeared within 24 h, all mice displaying bristly and greasy hairs. From day 2, weight loss was observed. At later time points other signs were noticed such as prostration, shaking and respiratory distress, with animals needing to be euthanized starting from day 7 post exposure. A statistically significant protection according to survival curves was observed with a single ip dose of 2 mg/kg of ABMA 1 h prior toxin challenge (p < 0.01 versus control, Fig. 1C, open circles). Forty eight % (n = 25; 3 independent experiments) of ABMA-treated mice survived, while in the control group, survival was 11.5% (n = 130, from 10 independent experiments). Based on this result, additional experiments were performed with escalating doses of ABMA. Administration of a single ip dose of 20 mg/kg and 200 mg/kg of ABMA prior to ricin intoxication gave improved, statistically significant, levels of protection as compared to the control group (p < 0.001, n = 5 for both groups). The 20 mg/kg dose fully protected animals through to day 21 (Fig. 1C). The 200 mg/kg dose resulted in 80% of protection of mice against ricin challenge with a single animal succumbing on day 15. The lower protection seen with the higher dose may be due to solubility issues of ABMA in aqueous solution resulting in uncertain biodistribution at the highest dose.

ABMA, a broad-spectrum inhibitor active in vitro against various bacterial toxins

The mechanism of action of ricin toxin shares common general principles with those of intracellular-acting bacterial protein toxins: binding to a cell-surface receptor, internalization in endocytic compartments, trafficking through intracellular transport pathways, translocation from transport vesicles or compartments into the cytosol and catalytic modification of a cellular target. Thus, investigating the effect of ABMA on bacterial toxins may lead to identify other sensitive toxins and hence, get some insights into ABMA mechanism of action4,5. Table S1 summarizes the features of the tested toxins.

Appropriate model cell lines were pretreated with solvent alone (DMSO) or various concentrations of ABMA, then respectively incubated with increasing concentrations of diphtheria toxin from Corynebacterium diphtheriae (DT), lethal toxin from Bacillus anthracis (LT), toxin B from Clostridium difficile (TcdB), lethal toxin from Clostridium sordellii (TcsL), Shiga-like toxin 2 from Escherichia coli (Stx2) or Botulinum neurotoxin A (BoNT/A) from Clostridium botulinum (Table S1 and Fig. 2). The inhibitory effect of DT on protein biosynthesis was measured by the incorporation of [14C]-leucine into newly synthesized proteins. We observed higher levels of protein biosynthesis on A549 cells exposed to DT in the presence of ABMA than in its absence (Fig. 2A) with an EC 50 of 62.5 ± 0.3 µM (n = 3). At 90 µM of ABMA, DT toxicity was reduced more than 100-fold in the assay conditions. We also found that ABMA protected other cell lines (e.g. Vero, PC3, A431 and DLD1) as well as HUVEC primary cells against DT (data not shown). This indicates that the inhibitory effect of ABMA on DT is not cell type-specific. Anthrax LT cleaves the mitogen-activated protein kinase MEK219. Figure 2B shows that MEK2 cleavage by LT in HUVECs was partially inhibited in the presence of ABMA at 30 µM. TcdB and TcsL inactivate small GTPases by their glucosyltransferase activity. This disrupts the actin cytoskeleton and induces cell rounding20. ABMA reduced Vero cells rounding 4 and 8 folds, respectively, following a challenge by TcdB for 4 h and by TcsL for 18 h (Fig. 2C,D). The EC 50 s were 73.3 ± 9.1 µM for TcdB and 86.7 ± 6.8 µM for TcsL (n = 3).

Figure 2 ABMA inhibits cytotoxicity of several bacterial toxins. Cells were incubated with the indicated concentrations of ABMA and then challenged with increasing concentrations of the indicated toxins. (A) A549 cells were exposed to DT for 18 h. Culture media was removed and replaced with DMEM containing [14C]-leucine at 0.5 µCi/mL for 3 h before protein biosynthesis determination. (B) Immunoblots showing the levels of MEK2 in HUVEC cells left untreated (line 1) or treated with Anthrax LT (lines 2–3, LT = PA 3 µg/mL + LF 1 µg/mL) in the absence and presence of 30 µM of ABMA. Immmunoblot of anti-actin show equal protein loading. (C,D) Vero cells were intoxicated with TcdB for 4 h or TcsL for 18 h and morphological changes of intoxicated cells were imaged and analyzed. (E) HeLa cells were exposed to Stx2 for 16 h before protein biosynthesis determination as for DT. (F) ABMA or DMSO were added to rat cerebellar granule neurons (CGNs) 1 h prior to BoNT/A exposure (500 pM) in the presence of compounds for 24 h. Immunoblots showing the levels of SNAP-25 and its cleaved form in the absence and presence of ABMA. Immunoblots images from single experiment (B and E) were spliced to rearrange the order of samples. Full-length blots are presented in Supplementary Figure S8. Full size image

Stx2 blocks cell protein biosynthesis by cleaving adenine 4324 of the 28 S ribosomal RNA with the same N-adenine glycohydrolase activity as ricin14. ABMA at 30 and 60 µM had barely any protective effect on intoxication of HeLa cells by Stx2 and a very weak protection at 90 µM (Fig. 2E). Botulinum neurotoxin A (BoNT/A) cleaves the SNARE protein SNAP25 that is essential for the fusion of neuromediator vesicles to the presynaptic membrane of nerve termini, inducing paralysis of the neuromuscular junction21. Figure 2F shows that ABMA at 30 µM was unable to prevent SNAP25 cleavage by BoNT/A in rat cerebellar granule primary cultured neurons, a model for BoNT/A activity.

Taken together, our results show that ABMA had no effect against Stx2 and BoNT/A cell intoxication. Nevertheless, it displays a broad-spectrum antitoxin activity against potent plant and bacterial toxins acting inside cells: ricin, DT, LT, TcdB and TcsL.

Various steps of the toxins’ mechanism of action may be considered as the target of ABMA: receptor binding, internalization, intracellular trafficking, translocation into the cell cytosol and catalytic modification of a cell substrate. The toxins sensitive to ABMA have different catalytic activities: N-adenine glycohydrolase for ricin and Stx2, ADP-ribosyltransferase for DT, Zn2+ metalloprotease for anthrax LT and BoNT/A and glucosyltransferase for TcdB and TcsL. Thus, it is very unlikely that ABMA inhibits the toxins’ catalytic activities. Each of those toxins uses a different cell-surface component as a receptor for cell binding and internalization (see Table S1). Thus, ABMA probably doesn’t inhibit the binding of the toxins to their receptors. Nevertheless, we investigated whether ABMA could inhibit the binding of DT receptor binding domain, named DTR822, to its receptor pro-HB-EGF (Precursor of heparin binding epidermal growth factor analog) as a model for toxin-receptor interaction23. A fluorescent DTR8 was made by chemical coupling with Alexa488 (DTR A488 ). FACS analysis showed that ABMA did not affect binding of Alexa488 labeled-DTR8 (DTR A488 ) with its receptor on Vero cells (Fig. S4), suggests that the inhibition of DT cytotoxicity induced by ABMA is not due to a reduced binding of DT to its receptor.

Since ABMA can inhibit several toxins with different catalytic activities and different receptors, we hypothesized that the inhibitor is not acting directly on the toxin itself but rather on a common host target, necessary for the toxins to reach the host cell’s cytosol and exert their toxicity. Our results demonstrate that ABMA has a pronounced inhibitory effect on DT, LT, TcdB and TcsL, which are all well-characterized acidic endosome-dependent toxins. They require low-pH endosomes (early endosomes (EE) and LE) where they undergo a conformational change, leading to the interaction of their transmembrane and catalytic domains with the compartments’ membrane and translocation of their catalytic domain into the cytosol8. In contrast, ABMA had barely any protective effect on the intoxication of HeLa cells by Stx2. Stx2 follows exclusively the retrograde route from the EE to the endoplasmic reticulum (ER) via the Golgi apparatus after internalization into cells8. Finally, ABMA is not able to inhibit BoNT/A, which relies on peculiar synaptic vesicle recycling and endocytosis processes to enter into neurons24,25. Thus, we hypothesized that ABMA is targeting host’s endosomal trafficking pathway between EE and the lysosomes.

ABMA inhibits cell infection by viruses that enter the host cytosol from acidified endosomes

Knowing that ABMA protects cells from multiple toxins that rely on acidic endosomes to translocate into the cytosol, we investigated whether ABMA was able to inhibit cell infection by viruses that have a pH-dependent mechanism of capsid release from these compartments. We tested Ebola virus (EBOV), Rabies virus (RABV), Dengue-4 virus (DENV4) and Chikungunya virus (CHIKV) (Table S2), which bear a surface glycoprotein that mediates fusion of the virus membrane with that of the endosome after which the capsid is released to the cell cytosol to initiate infection26. HeLa cells were incubated 1 h with increasing concentrations of ABMA before infection with a recombinant EBOV Mayinga strain carrying an enhanced green fluorescent protein (eGFP). ABMA treatment inhibited EBOV-eGFP infection with an EC 50 of 3.3 µM (Fig. 3A). Baby hamster kidney (BSR) cells were incubated 4 h with ABMA before infection with the Pasteur vaccins/PV strain of RABV, ABMA inhibited the infection with an EC 50 of 19.4 µM (Fig. 3B). Ribavirin, an antiviral drug inhibiting viral RNA synthesis and viral mRNA capping was used as a reference molecule and exhibited a similar EC 50 . Finally, Vero cells were incubated 1 h with ABMA before infection with DENV4, ABMA inhibited infection with an EC 50 of 8.2 µM, while Ribavirin was about four fold less efficient in protecting cells from the infection (Fig. 3C). ABMA reduced cell infection by the three viruses up to at least 90% at 20 µM for EBOV and DENV4 and at 100 µM for RABV. In contrast, ABMA up to 100 µM did not inhibit infection of HEK293 cells by Chikungunya virus (CHIKV) (Fig. S5, see discussion section). In summary, we observed that ABMA inhibited three endosomal acidification–dependent viruses.

Figure 3 ABMA inhibits EBOV, RABV and DENV4. (A) HeLa cells were pre-incubated with increasing concentrations of ABMA solubilized in DMSO, or DMSO only, for 1 h and then challenged with EBOV-eGFP in the presence of the drug for 24 h. Cells were fixed, stained with DAPI, and numbers of nuclei and eGFP-positive (infected) cells were counted using the CellProfiler software. The relative infection efficiencies were calculated by dividing the number of infected cells by the number of nuclei. The percentages of infected cells in DMSO- and ABMA-treated samples were reported relative to the infection efficiency in non-treated cell. Data are representative of three independent experiments. (B) BSR cells were pretreated for 4 h with increasing concentrations of ABMA or ribavirin solubilized in DMSO, then challenged with the PV strain of RABV (MOI = 14) for 1 h. Cells were washed to remove the non-fixed virus, then incubated again in the presence of the same concentrations of the compounds for 24 h. Cells were fixed, nuclei were stained with Hoechst and infected cells were detected by immunostaining of the RABV ribonucleocapsid. RABV-positive cells were counted and their number was reported to that of non-treated cell, allowing calculating a percentage of inhibition. The average of three independent experiments and standard deviations are shown. (C) Vero cells were treated with ABMA or ribavirin solubilized in DMSO and then challenged with 125 TCID 50 of a DENV4 serotype virus for 7 days. Viral replication was detected by ELISA using specific serum from DENV4-infected non-human primate. Full size image

ABMA inhibits cell infection by Simkania negevensis and Chlamydia trachomatis

Simkaniaceae and Chlamydiaceae from the order Chlamydiales are obligate intracellular Gram-negative pathogenic bacteria. They use host-cell materials to form a distinct, degradation-resistant but replication-permissive membranous compartment, the vacuole or inclusion. Despite differences, their intracellular life-styles share several common features. Proteomic characterization of the Simkania negevensis (Sn) containing vacuole (SnCV) has shown that it contains proteins from several main host transport pathways including the endosomal pathway13. Chlamydia trachomatis (Ctr) recruits multiple Rab proteins from the endosomes to the inclusion membrane and avoids travelling to the phago-lysosome as a final destination27. Besides the Golgi apparatus, multi-vesicular bodies (MVBs), also known as LE, are another essential source of cholesterol and sphingomyelin for the development of Ctr inclusions28,29. Thus, we tested whether ABMA could inhibit the infection of cells by Sn and Ctr.

Figure 4A shows that 75 µM of ABMA sharply reduced the amount of Sn in infected cells as revealed by immunoblotting of Sn heat-shock protein 60 (snHSP60). In parallel, inclusion sizes were smaller as revealed by immunofluorescence (Fig. 4B). The Sn progeny harvested from ABMA-treated cells reduced the amount of Sn (Fig. 4C) and the number of inclusions upon infection of fresh, untreated cells (Fig. 4D,E). ABMA at 75 µM slightly reduced the cellular load of GFP-expressing Ctr strain in the primary infection as seen by Ctr HSP60 (ctrHSP60) immunoblotting (Fig. 5A). However, ABMA treatment dramatically reduced chlamydial progeny infectivity indicated by the reduced inclusion number and bacterial load (Fig. 5A,B). Together, our results show that ABMA inhibits the capacity of Sn and Ctr to develop properly during cell infection and leads to a progeny with reduced infectivity.

Figure 4 Effects of ABMA on SnCV during Sn infection. HeLa 229 cells were infected with Sn (MOI = 0.5) for 3 days in the presence of ABMA or DMSO control at the indicated concentrations. Bacterial were released and transferred to infect fresh cells for 3 days in the absence of compounds. (A) Effect of ABMA on the Sn bacterial load of infected cells measured by snHSP60 immunoblot. Actin was used as loading control. (B) Effect of ABMA on the inclusion sizes of Sn during primary infection. Relative Sn incusion sizes were determined via snHSP60 immunostaining and quantitative analysis using ImageJ. (C) Effect of ABMA on the Sn bacterial load of progeny infected HeLa cells measured by snHSP60 immunoblot. Actin was used as loading control. (D) Effect of ABMA on the number of Sn inclusions during progeny infection. (E) Immunofluorescence images of cells infected by Sn progeny from cells treated with 75 µM ABMA after 3 days of incubation. Nuclei were stained for DAPI (green) and SnCVs were stained for HSP60 (red). Images are representative of 3 independent experiments. Full-length blots (A and C) are presented in Supplementary Figure S9. Full size image

Figure 5 Effects of ABMA on Ctr primary and progeny infections. HeLa 229 cells were pretreated with ABMA or DMSO control at the indicated concentrations for 1 hour prior to infection with Ctr (MOI = 1). Cells were lysed 48 h post infection and lysates were used to infect fresh cells. ABMA was present during primary infection. (A) Immunoblotting analysis of lysed HeLa 229 cells after Ctr primary and progeny infections following ABMA treatment during the primary infection. Bacterial load was detected with antibodies against ctrHSP60 protein and actin was used as a loading control. (B) Immunofluorescence analysis of infectivity with 75 µM ABMA treatment during primary infection. 24 h post progeny infection; cells were fixed and stained for DAPI (blue). Ctr inclusions were detected by their GFP-expression signal (green). Immunoblots image (A) were spliced to rearrange the order of samples. Full-length blots are presented in Supplementary Figure S10. Full size image

ABMA inhibits the development of Leishmania infantum in macrophages

Monocytes and macrophages are important target cells in the pathophysiology of Leishmania parasite infections30. The parasite is internalized and develops into an amastigote form within a parasitophorous vacuole that incorporates endo-lysosomal pathway components30,31. Thus, we investigated whether ABMA could inhibit the infection of RAW 264.7 macrophages by Leishmania infantum amastigotes. Amphotericin B and miltefosine, which are approved drugs for the clinical management of Leishmaniasis, were used as reference drugs. Table 1 shows that all three drugs inhibited L. infantum intramacrophage amastigotes development with various EC 50s . The EC 50 for ABMA was around 7 µM. Interestingly, the two reference drugs were capable of inhibiting axenic amastigotes with an efficacy similar to that found on intramacrophage amastigotes, whereas ABMA had no direct effect on the axenic parasite, up to 100 µM. In addition, the ABMA cytoxicity was lower than those of the reference drugs. These results strongly suggest that ABMA blocks L. infantum intracellular development by an action on host cell while the other drugs are directly toxic to the parasite.

Table 1 Antileishmanial activity and cytotoxicity of ABMA and reference drugs. Full size table

ABMA induces accumulation of late endocytic compartments

We have determined that ABMA can inhibit the intoxication or infection of cells by a variety of toxins, viruses and intracellular microorganisms. The toxins and the viruses rely on endosome acidification to enter the cytosol. The bacteria and parasite build a vacuole that incorporates endosome membranes and proteins to acquire their nutrients and proliferate. This may suggest that ABMA targets and modifies acidic endosomes and their homeostasis. We observed by confocal microscopy that live A549 cells treated with ABMA and stained by LysoTracker® Deep Red exhibited more intensely labeled and enlarged fluorescent puncta than cells treated with DMSO only (Fig. 6A, middle and left panels respectively). In contrast, bafilomycin A1 (Baf A1), a highly specific v-ATPase inhibitor that prevents endosome acidification, decreased fluorescence staining of cells (Fig. 6A, right panel). We obtained similar results with ABMA and Baf A1 on HeLa and RAW 264.7 cells (data not shown). To confirm that ABMA is affecting acidic endosomes, we used acridine orange, another cell-permeant dye for acidic organelles (Fig. 6A, central and lower panels). Similarly, ABMA induced larger and brighter red fluorescent vesicles in A549 cells, in contrast to the effect of Baf A1, which strongly decreased red fluorescence in cytoplasmic vesicles. Altogether, ABMA had an effect on acidic compartments different from that of Baf A1. Baf A1 is known to inhibit DT toxicity by inhibiting endosome acidification. It might be expected that combined effect of both molecules might annihilate the impact of each other: increased endosome acidification for ABMA versus decreased acidification for BafA1. Surprisingly, ABMA combined with Baf A1 had an inhibitory effect on DT cytotoxicity twenty fold higher than for each molecule alone (Fig. S6). This strongly suggests that ABMA and Baf A1 have different mechanisms of action and distinct targets. Moreover, this indicates that the anti-toxin and anti-pathogenic effect of ABMA is not linked to the increase in endosome acidification; otherwise it would be counter balanced by Baf A1.

Figure 6 ABMA induces the accumulation of late endocytic compartments and affects cholesterol transport. (A) LysoTracker® Deep Red (50 nM, 30 min), acridine orange (10 µg/mL, 10 min) staining of A549 cells pretreated with DMSO or ABMA at 60 µM for 2 h. BafA1 at 100 nM was used as a control. (B) DMSO or ABMA 24 h-treated A549 cells were fixed, permeabilized by 0.1% Saponin and stained with antibodies against EEA1, Rab7 or Lamp1. Arrows indicate larger Rab7-positive vesicles. (C) A549 and HeLa cells were treated respectively with ABMA (30 µM), U18666A (10 µg/mL) or DMSO for 18 h, then fixed and stained with the cholesterol-avid fluorophore Filipin III. Nuclei were stained with Hoechst 33342 (blue). Full size image

To further characterize the effect of ABMA on intracellular acidic compartments, immunostaining of protein markers of the EE to lysosomes pathway were performed on A549 cells. Immunostaining of EEA1 (EE marker, Fig. 6B upper panel) and Lamp1 (lysosome marker, Fig. 6B lower panel) were unchanged in ABMA-treated cells and vehicle alone, while Rab7 (LE marker, important GTPase in the late endocytic pathway) staining was visibly enhanced in ABMA-treated cells compared to vehicle alone (Fig. 6B, central panel). Importantly, we did not observe morphological changes on other important cellular organelles such as the Golgi apparatus or ER (stained for Trans-Golgi Network 46 (TGN 46) and Protein disulfide isomerase (PDI), respectively; Fig. S7) as well as endocytosis-related membrane protein (clathrin, adaptin-α, epsin I; data not shown). Altogether, the data show that ABMA targets late endosomal compartments, without affecting the morphology of other organelles: EE, Golgi apparatus, ER and lysosomes.

Besides EE, LE is considered as important and complex sorting stations for proteins and lipids in the endocytic pathway. Cholesteryl esters in LE/Lysosomes are hydrolyzed by lysosomal acid lipase to free cholesterol, before egress from the endo-lysosomal system, allowing for its distribution to other cellular compartments32. We investigated if LE modified by ABMA is consequently accompanied by alterations of cholesterol transport, which may potentially interfere with nutrition of intracellular pathogens12. Filipin III, a fluorescent probe with high affinity for cholesterol was applied to cells treated respectively with DMSO, ABMA and U18666A, an intracellular cholesterol transport inhibitor. Both ABMA and U18666A induced an accumulation of cholesterol inside A549 and HeLa cells as observed by fluorescent microscopy (Fig. 6C). Together, these data show that ABMA affects LE and induces cholesterol accumulation, likely within late endosomal compartments.

The enhanced LysoTracker acidification dye and Rab7 staining led us to further investigate how ABMA affects the number and morphology of LE. We used transmission electron microscopy to examine the ultrastructure morphology of organelles in both DMSO- and ABMA-treated A549 cells. We observed that ABMA induced the accumulation of compartments with a >200 nm size, containing a variable number of smaller intraluminal vesicles (ILVs) but lacking a multilamellar morphology (Fig. 7D–F). These structures are characteristic of multivesicular bodies/LE (MVBs/LE)33. Other organelles appearing as electron-dense multi-lamellar membrane compartments characteristic of lysosomes were visualized in both DMSO- and ABMA-treated cells without observed difference in amount and morphology (Fig. 7). Altogether, the results show that ABMA induces the accumulation of MVBs/LE.