Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Anthony Oro ( oro@stanford.edu ).

All mice were housed under standard conditions, and animal care was in compliance with the protocols approved by the Institutional Animal Care and Use Committee (IACUC) at Stanford University. Ptch, Gli1-CreER, Trp53mice were generated and used to generate BCC tumors as described previously (). For these studies we used only female mice due to the ability to house multiple per cage. Female mice aged 7.5 weeks were induced with 0.25mg of 4-hydroxytamoxifen per day for three consecutive days and exposed to 4 GY irradiation using an X-Ray irradiator on the fourth day. After an average of 3-5 months post-irradiation, tumors were passaged into NOD SCID gamma mice. 44LLD or scramble (50μg) was administered to passaged tumors by intra-tumoral injection once daily for eight consecutive days. Tumor volumes were measured by calipers, and Hh pathway output was asssesed by immunostaining for GLI1.

This study was approved by the Stanford University Institutional Review Board #18325, with a waiver of consent from participants. Since the samples are de-identified for privacy reasons, gender identity of the specimens cannot be reported. Central biopsies of clinically diagnosed BCC tumor specimens from Mohs Surgery patients at the Stanford Dermatology Clinic were quatered and cultured in EpiLife Media (Life Technologies) +CaCl 2 with or without 20μM vorinostat for 3 hours. Specimen were flash frozen in OCT freezing media for subseqent assays.

Mammalian cell transfection performed using FuGENE 6 Transfection Reagent (Promega), Lipofectamine LTX with Plus Reagent (Thermo Fisher), and MEF 1 Nucleofector Kit (Lonza) per manufacturer protocol. Transient transfection mammalian expression vectors are included below. Stable expression produced by piggybac transposition.

NIH 3T3 (gender: male) and HEK293T (gender: female) cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS). Experiments assaying Hh signaling were carried out in 0%–0.2% FBS supplemented with Smoothened Agonist (SAG, Sigma).

MB21 cells (gender: male) were grown in DMEM:F12 (Thermo Fisher) supplemented with B27 (Thermo Fisher). ASZ001 (ASZ, gender: female) BCC cells were cultured in 154CF media (Life Technologies) supplemented with 2% chelated fetal bovine serum, Human Kerytinocyte Growth Supplement (Thermo Fisher), Penn-strep, and 0.05mM CaCl2. Experiments assaying Hh signaling carried out in serum-free conditions.

Method Details

Fractionation To prepare nuclear extracts, cells were resuspended in hypotonic lysis buffer (10mM HEPES pH7.9, 1.5mM MgCl2, and 10mM KCl) and dounce homogenized. Isolated nuclei were pelleted and resuspended in RIPA buffer. Proteasome inhibitor (MG132, 10μM) was added to observe GLI1 export due to the short half-life of GLI1 in the cytoplasm. Kay et al., 1972 Kay R.R.

Fraser D.

Johnston I.R. A method for the rapid isolation of nuclear membranes from rat liver. Characterisation of the membrane preparation and its associated DNA polymerase. The protocol for nuclear envelope fractionation was adapted from previous reports(). Isolated nuclei were resuspended in Nuclear Envelope Buffer 1 (10mM Tris 8.5, 0.MgCl2, 5mM β-ME, 10% sucrose, DNase I) and incubated at room temperature for 10 minutes to lyse the nuclear membrane and gently remove the nucleoplasmic fraction, characterized by the initial release of lamin A. The crude nuclear envelope, containing non-intergral membrane bound INM components, was pelleted at 38k rcf., resuspended in Nuclear Envelope Buffer 2 (10mM Tris 7.4, 0.MgCl2, 5mM β-ME, 10% sucrose, DNase I), and allowed to incubate at room temperature for 15 minutes. This allowed for the release of peripheral chromatin and non-intergral membrane INM proteins, characterized by the release of BANF1. Purified nuclear envelopes were resuspended in RIPA to solubilize the remaining peptides.

Nuclear Envelope Lift Off Assay ASZ cells were serum starved for 24hrs to induce the Hh pathway, then treated with 20μM vorinostat to hyperacetylate GLI1 and drive it onto the nuclear envelope. Crude nuclear envelopes were isolated (see Nuclear Envelope Fractionation protocol above; crude nuclear envelopes correspond to pelleted sample after Nuclear Envelope Buffer 1 lysis) and resuspended in PBS (supplemented with 0.5mM DTT, 5 μM ZnCl2, 5 mM MgCl2 and 1 mM NAD+) or PBS + recombinant CobB deacetylase (a generous gift from the lab of Dr. Peter Jackson) (supplemented with 0.5mM DTT, 5 μM ZnCl2, 5 mM MgCl2 and 1 mM NAD+). CobB is a E. coli derived non-specific deacetylase. The deacetylation reaction was allowed to proceed for 30mins. Nuclear envelopes were pelleted by centrifugation (15k rpm). Supernatant, containing protiens release following deacetylation, was collected in Laemmli sample buffer corresponding to the “Lift off” sample. The pelleted nuclear envelopes were resuspended in RIPA, corresponding to the “output” sample.

Triton Extraction Assay ASZ cells were washed with PBS prior to incubation in 1% Triton X-100 in PBS supplemented with protease inhibitor cocktail for 10 minutes. Supernatant was collected and analyzed by dot blot with three technical replicates per experiment. Each condition was assayed independently three times as well.

BASU Vicinal Labeling BASU fusions were stably expressed via piggybac transposition. 50μM d-biotin supplemented media was added to cells for 5 hours to allow labeling of proximal interactors. Cells were collected in RIPA lysis buffer supplemented with protease inhibitor and Pierce Universal Nuclease. Whole cell extract was homogenized by sonication and clarified by centrifugation. Free biotin was removed by iterative spin concentrate-dilution steps using spin concentration units (Millipore, MWCO:3kDa) to lower free biotin concentration by 125 fold. Purified lysate bound-biotin was quantied by dot blot and input bound-biotin was normalized prior to streptavidin pulldown (Dynabeads MyOne Streptavidin C1). Following stringent washing (3X 10mins RIPA, 2X 5min 2%SDS in PBS, 1X 1min RIPA), biotinylated proteins were eluted by boiling (95°C, 800rpm) in Laemmli sample buffer supplemented with 10mM biotin.

APEX2 Vicinal Labeling Lam et al., 2015 Lam S.S.

Martell J.D.

Kamer K.J.

Deerinck T.J.

Ellisman M.H.

Mootha V.K.

Ting A.Y. Directed evolution of APEX2 for electron microscopy and proximity labeling. 2 O 2 was added to induce labeling and was quenched following 1 minute of labeling. Biotinylated species were purified as described above for BASU and immunoblotted. An APEX2-GLI1 fusion was stably expressed in ASZ by lentiviral transduction. APEX labeling was performed as previously described (). Briefly, biotin-phenol supplemented media was introduced 30 minutes prior to labeling. Hwas added to induce labeling and was quenched following 1 minute of labeling. Biotinylated species were purified as described above for BASU and immunoblotted.

Immunoprecipitation All immunoprecipitation were in the absence of crosslinking. For the majority of experiments cell lysates were prepared in 1% Triton X-100 in TBS supplemented with protease and phosphatase inhibitors (Roche). Immunoblot of whole cell extract under these conditions confirmed solubilization of INM proteins. Anti-FLAG M2 Sepharose Beads (Sigma) and Pierce Anti-HA Magnetic Beads (Thermo Fisher) were pre-blocked in 5% BSA prior to incubation overnight. Washes were carried out at room temperature using lysis buffer (3X 3mins). Samples were eluted by boiling in Laemmli sample buffer. For graded high salt washes, large scale co-IP experiments were carried out and subsequently aliquoted and washed seperately. For in vitro translated protein IP’s, the indicated components were mixed and allowed to bind at 4°C for 1 hour prior to the addition of beads. Whole cell extracts for these experiments were generated by the lysis of HEK293T pellets in 1% Triton X-100 in TBS supplemneted with protease inhibitor. For experiments comparing LAP2 isoforms, the pan-LAP2 antibody was used thereby reducing error associated with different antibodies.

In vitro translation Proteins of interest were generated in vitro using Promega’s TnT Quick Coupled Rabbit Reticulocyte or Wheat Germ Cell Lysate Transcription/Translation System according to manufacturer’s protocol. For in vitro translation, pCS2 vectors harboring SP6 promoters were utilized. In general, all in vitro translations utilized the rabbit reticulocyte lysate system. Where specifically indicated, wheat germ cell lysate was used to test for “direct” binding in a context without endogenous mammalian cofactors.

Vorinostat Pulse-Chase ASZ cells were serum starved for 24hrs and vorinostat (20μM) treated for 1hr. Vorinostat media was replaced with fresh prewarmed media to allow deacetylation to take place. Cells were collected in RIPA buffer supplemented with protease inhibitor and 1mM Sodium Butyrate to hault deacetyation. Cell extracts were spotted onto nitrocellulose and immunoblotted. Each time point represents three biological replicates with three technical replicates each.

Fluoresence Recovery After Photobleaching (FRAP) and Live Cell Imaging Live cell imaging and FRAP experiments were carried out in cells plated onto Nunc Lab-Tek Chambered Coverglass coated with poly-D lysine. Prior to imaging, cells were placed in Flourobright media supplemented with Hoescht HCl. Live floresence imaging on Leica SP8 White Light Confocal (100x objective) equiped with Hybrid detectors housed in a temperature, humidity, and CO 2 regulated imaging chamber. Bleaching area (5 μm in diameter) were constant and bleaching intensity was determined empirically to produce even bleach points. Recovery was observed in 2 s intervals using Leica LAX software and analyzed in Prism.

Immunofluorescence and Fluorescence Imaging Cells were fixed in 4% formaldehyde in phosphate buffered saline and prepared per a general immunofluorescence protocol (abcam). Primary antibodies were used at the manufacturers’ recommended concentration. Fluorescent-labeled secondary antibodies utilized: Alexa Fluor 488, Alexa Fluor 555, Alexa Fluor 594, and Alexa Fluor 647 (1:500, Life Technologies). Confocal imaging was carried out using a Leica SP8 microscope equipped with adjustable white light laser and hybrid detectors. Images were quantified using ImageJ or ICY image analysis software, and when making comparisons between images laser intensities and imaging settings were held constant. Occasionally an imaging control, such as LAP2β, whose expression is independent of the experimental variable was utilized to normalize for staining variability across a specimen. To draw scale bars, Lieca LAX software was used. Radial GLI1/AcGLI1 distributions were calculated by line averaging on unique nuclei with the line starting in the nucleoplasm, ending in the cytoplasm, and centering on the edge of the DAPI signal. The 90% reduction point of the DAPI signal was indicated as the nuclear envelope and marked with a dotted line. Standard error is displayed as thin dotted radial tracings.

Proximity Ligation Assay Proximity Ligation Assays were conducted per manufacturer’s protocol (Duolink/ Sigma) to detect interaction between endogenous proteins in a cellular context. Notably, PLA experiments all involve the change in signal between an antibody pairs under experimental perturbation. In our experience, it is inappropriate to compare different antibody pairs and single antibody controls inadequately assess specificy. PLA signal was measured using constant imaging setting and averaged across fields. Puncta were quantified for cell line PLA experiments while total PLA floresence signal was quantified for tissues due to the lack of clear puncta separation.

3D Structure Illumination Microcopy ASZ cells were cultured on poly-D lysine coated high performance 0.17mm coverglass (Zeiss). OMX BLAZE 3D-Structured Illumination Super Resolution Microscope (Stanford University Cell Sciences Imaging Facility). System specifications: OMX V4 microscope platform with X, Y, Z Nanomotion stage movers with 25 mm travel; OMX BLAZE SI patterns generated using an electro-optical high-speed SI diffraction grating engine; Piezo controlled Fast Z axis system - 100 micron range; 60X, N.A. 1.30silicon immersion objective for matching of refractive indexes during live cell imaging; Offline workstation with SoftWoRx software for generating SI image reconstructions.

Acetyl-GLI1 Antibody Generation Antibodies and peptides were generated by 21st Century Biochemicals. Animals were injected with both human (Acetyl-IGTRGL[K-ac]LPPLPHT-Ahx-C-amide) and mouse (Acetyl-IGSRGL[K-ac]LPSLTHA-Ahx-C-amide) HPLC-purified immunogenic Acetyl-K518 GLI1 peptides. Affinity purified antibodies were generated by the column purification.

Electrophoretic Mobility Shift Assay (EMSA) Recombinant zinc-finger GLI1-GST fusion protein and wheat germ cell in vitro translated LAP2α was incubated with a LICOR IR-dye labeled oligonucleotide harboring GLI1 binding motif (Genescript, ACGTGGACCACCCAAGACGAA) were run on a 6% DNA Retardation Gel (ThermoFischer), and imaged using Odyssey CLx (Li-Cor). Mock lane is empty GST vector transfected bacterial lysate. We note that LAP2α did not induce a clean supershifted band, but a supershifted smear. This is indicative of a micromolar interaction which would struggle to remain stable during an EMSA which requires interactions in the nanomolar range.

DamID Tabled 1 Step-Target Forward Reverse 2-ptch1 acagggtggtggatagtgcg tacgaggcgttgttccctgc 2-GLI1 cgccggcttcctaagggtta Cggacagacgtctccgactc 3-nkx2-9 CTACCAAGCGTGCCTAAAGT TTTATCCCAGGGAGCTAAAG 3-ACTB GGCACCACACCTTCTACAAT GTTACCCGGGATACTGACCT 3-JUNB CAGGAAGGCTGCAGTTACTCT AGCGGCTTTCAACAGACC 3-NEUROG2 TTAACTGGAGTGCCTTGG CTCGTGTGTTGTCGTTC 3-SMAD7 TTTTAAAGCGACAGGGTGTCTAGA TCT GCT CGG CTG GTT CCA

Quantitative Reverse Transcription PCR RNA isolated using QIAGEN RNeasy Mini Kit according to manufacturer protocol. TaqMan RNA-to-CT 1-Step Kit used for reverse transcription and quantitative PCR. TaqMan Gene Expression Assay Probes Life Technologies: GLI1 (FAM-MGB probe Mm00494654_m1); GAPDH (Mm99999915_g1); Hprt (Mm01545399_m1) used in an assay dependent concentration.

Immunobloting Whole cell extracts were harvested using radioimmunoprecipitation (RIPA) buffer supplemented with protease and phosphatase inhibitors (Roche) and run on gradient SDS-PAGE gels (Life Technologies) followed by wet transferred onto nitrocellulose membranes (0.45microns, BioRad). Primary antibodies used in an assay dependent concentration in 5% Bovine Serum Albumin in TBST. Fluorescent secondary antibodies compatible with Odyssey CLx (Li-Cor) used for 2-color imaging of membranes.

Cloning and Vectors pCS2FLAG (Addgene Plasmid #16331) and pCS2HA (Addgene Plasmid #16330) used to express epitope tagged protein in in vitro translation and mammalian cell culture. pGex and pQE-80L used to generate recombinant peptides. Piggybac transposition entry vector pbCAG (modifed from Addgene Plasmid #40973) and lentiviral transfecer vector pLex (modified from Addgene Plasmid #41390) utilized to generate stable lines. pEGFP-N3 used to generate GFP fusions for mammalian expression. To generate vicinal labeling constructs the follow plasmid were used: BASU (a generous gift from Dr. Paul Khavari), APEX2 (Addgene Plasmid #79056), DamID (Addgene #59201). GLI1 source cDNA was derived from our lab’s previous projects on GLI1. HDAC1 (Addgene Plasmid #13820), p300 (Addgene Plasmid #23252), HDAC3 (Addgene Plasmid #13819), LAP2α (synthesized by Genecopia) and LAP2β (Addgene Plasmid #21047). NEB Q5 PCR mix used per manufacturers protocol to generate PCR inserts with primers synthesized by Elim BioPharm (Hayward, CA). In-Fusion (Clontech) recombination based cloning performed according to manufacturers protocol.

Mass Spectrometry Mass spectrometry was performed by the Stanford University Mass Spectrometry Core. Samples were run on a 4%–12% SDS-PAGE gel and resolved by Coomassie staining. Gel bands were excised out in a 1.5 mL Eppendorf tubes and then cut in 1x1 mm squares. The excised gel pieces were then reduced with 5 mM DTT, 50 mM ammonium bicarbonate at 55°C for 30 min. Residual solvent was removed and alkylation was performed using 10 mM acrylamide in 50 mM ammonium bicarbonate for 30 min at room temperature. The gel pieces were rinsed 2 times with 50% acetonitrile, 50 mM ammonium bicarbonate and placed in a speed vac for 5 min. Digestion was performed with Trypsin/LysC (Promega) in the presence of 0.02% protease max (Promega) in a standard overnight digest at 37°C. Samples were then centrifuged and the solvent including peptides were collected and further peptide extraction was performed by the addition of 60% acetonitrile, 39.9% water, 0.1% formic acid and incubation for 10-15 min. The peptide pools were dried in a speed vac. Samples were reconstituted in 12.5μl reconstitution buffer (2% acetonitrile with 0.1% Formic acid) and 3μl (100ng) of it was injected on the instrument. Mass spectrometry experiments were performed using an Orbitrap Q-Exactive HFX mass spectrometer (Thermo Scientific, San Jose, CA) with liquid chromatography using a Nanoacquity UPLC (Waters Corporation, Milford, MA). For each LCMS experiment a flow rate of 250 nL/min was used, where mobile phase A was 0.2% formic acid in water and mobile phase B was 0.2% formic acid in acetonitrile. EASY-spray nanoLC analytical columns (Thermo Scientific) were used, measuring 50 cm in length and heated to 50 C. Peptides were directly injected onto the analytical column using a gradient (3%–45% B, followed by a high-B wash) of 80min. The mass spectrometer was operated in a data dependent fashion using HCD fragmentation for MS/MS spectra generation. For data analysis, the .RAW data files were processed using Byonic v 2.14.27 (Protein Metrics, San Carlos, CA) to identify peptides and infer proteins. Proteolysis was assumed to be tryptic allowing for N-ragged cleavage with up to two missed cleavage sites. Precursor and fragment mass accuracies were held within 12 ppm. Proteins were held to a false discovery rate of 1%, using standard approaches.