H3k36me2

HEK293T cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1% penicillin/streptomycin (Hyclone; GE Healthcare Life Sciences) in an incubator at 37 °C and 5% CO₂. Analysis of H3K36me2 with a titration of SETMAR expression was performed by transfecting HEK293T cells grown to ∼70 to 80% confluence in a 6-well dish with 0.25, 0.5, and 1 μg of pFLAG-cytomegalovirus 4 (CMV4)-SETMAR or pFLAG-CMV4 (empty vector) using XtremeGENE 360 (Roche). After 24 h, cells were harvested by trypsinization, washed with PBS, and resuspended in lysis buffer (10 mM Pipes [pH 7.0], 300 mM sucrose, 100 mM NaCl, 3 mM MgCl₂, 0.1% Triton X-100, 1× Universal Nuclease, and 1× Protease Inhibitor Cocktail from Thermo). Total protein was quantified by Bradford Assay (Bio-Rad). Samples were resolved by SDS-PAGE, transferred to polyvinylidene difluoride membrane (Thermo), and probed with the indicated antibodies (FLAG [Sigma; catalog no.: F1804]; β-tubulin [Proteintech; catalog no.: 66240-1]; SETMAR [Proteintech; catalog no.: 25814-1-AP #05–661]; Histone H3 [Cell Signaling Technologies; catalog no.: 9715]; and H3K36me2 [Cell Signaling Technologies; catalog no.: 2901]).

Nuclear extracts from mouse brains or human postmortem tissues were prepared according to the manufacturer’s instructions (Life Technologies) with modifications. Briefly, PFC punches from mouse slices containing GFP fluorescence or human postmortem tissues were collected, and then homogenized with 500 μl hypotonic buffer (20 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl₂, 0.5% NP-40, 1 mM PMSF, with cocktail protease inhibitor). The homogenate was incubated on ice for 15 min and followed by centrifugation at 3,000 g, 4 °C for 10 min. The nuclear pellet was resuspended in 50 μl nuclear extract buffer (100 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 10% glycerol, 1 mM PMSF, with cocktail protease inhibitor) and incubated on ice for 30 min with periodic vortexing to resuspend the pellet. After centrifugation, the supernatant for nuclear fractions was collected, boiled in 2 × SDS loading buffer for 5 min, and then separated on 6 or 12% SDS-polyacrylamide gels. Western blotting experiments for nuclear proteins were performed with antibodies against Ash1L (1:1000, LSBio, LS-B11718), H3K4me3 (1:1000, Cell Signaling, 9751), H3K36me2 (1:1000, Cell Signaling, 2901), H3K36me3 (1:1000, Cell Signaling, 9763), and Histone 3 (1:500, Cell Signaling, 4499).

Primary antibodies used are CDH1 (Cell Signaling Technology, Beverly, MA, USA; #3195S), MUC1 (Abcam, Boston, MA, USA; #ab109185), SNAI2 (Cell Signaling Technology; #9585S), MMP2 (Proteintech, Rosemont, IL, USA; #10373‐2‐AP), CD44 (GeneTex, Irvine, CA, USA; GTX102111), CD34 (Proteintech; 60180), vimentin (Santa Cruz Biotechnology; B0719), total SMAD2 (Cell Signaling Technology; #3103S), phosphorylated SMAD2 (Cell Signaling Technology; #3108S), TGF‐β (Cell Signaling Technology; #3711S), H3K36me1 (Abcam; #ab9048), H3K36me2 (Cell Signaling Technology; #2901S), H3K36me3 (Active Motif; #61101), histone H3 (Abcam; #ab1791), SOX2 (R&D Systems; #AF2018‐SP), OCT2 (Thermo Fisher; #39‐5400), PRRX1 (Novus Biologicals, St. Charles, MO, USA; #NBP1‐06067), anti‐FLAG (Sigma‐Aldrich; #F1804), GAPDH (Cell Signaling Technology; #2118S), and lamin B1 (Proteintech; #12987‐1‐AP). Secondary antibodies are anti‐rabbit (Invitrogen, Waltham, MA, USA; #SA5‐3557) and anti‐mouse (LI‐COR, Lincoln, NE, USA; #926‐68070). Lentiviral vectors are eGFP (control vector; Genecopoeia; EX‐EGFP‐Lv181), SOX2 (Genecopoeia; EX‐T2547‐Lv181), OCT2 (Genecopoeia; EX‐A2204‐Lv181), and PRRX1 (Genecopoeia; EX‐T1345‐Lv181).

Cells were harvested in lysis buffer and analyzed by SDS-PAGE with the following antibodies: phospho-AKT (S473), AKT, phospho-S6K (T389), S6K, phospho-S6 (S235/236), S6, phospho-GSK3 (S21), GSK3, phospho-Rb, mme-K, dme-K, tme-K, H3K4-me2, H3K4-me3, H3K9-me2, H3K27-me2, H3K27-me3, H3K36-me2, H3K79-me2 (purchased from Cell Signaling Technology, USA), phospho-PDK1 (S241), PDK1, RAS (bought from Abcam, UK), AMD1 (Proteintech, USA), and Actin (HuaBio, China). Immunoblots were imaged and analyzed using an Odyssey system (LI-COR Biosciences, USA), ImageQuant LAS 4000 (GE Healthcare, USA), and ChemiDoc MP (Biorad, USA).

Rabbit antibodies targeting the following proteins were acquired from Bethyl Laboratories (Montgomery, TX, USA): LEDGF/p75 (Cat# A300-848A), menin (Cat# A300-105A), JPO2 (Cat# A300-846A), and HRP2 (Cat# A304-314A). Rabbit antibodies targeting the following proteins were from Cell Signaling Technology (Danvers, MA, USA): MDR1 (Cat# 13342), IWS1 (Cat# 5681), c-MYC (Cat# 18583), GR (Cat# 12041S), H3K36me2 (Cat# 2091T), and GAPDH (Cat# 5174). Other rabbit antibodies used were against Med-1/TRAPP220 (Abcam, Waltham, MA, USA, Cat# ab243893), PogZ (Aviva Systems Biology, San Diego, CA, USA, Cat# RP39173-P050) and histone H3 (GeneTex, Irvine, CA, USA, Cat# GTX122148). Mouse monoclonal antibodies included ASK1 (Abnova, Walnut, CA, USA Cat# H00010926-M01) JPO2 (Novus Biologicals, Centennial, CO, USA, Cat# NBP2-46198); and horseradish peroxidase (HRP)-conjugated anti-β-actin (Cell Signaling Technologies, Cat# 12620). Human sera containing antinuclear autoantibodies (ANAs) displaying the characteristic monospecific dense fine speckled (DFS) nuclear immunofluorescence pattern that defines immunoreactivity to LEDGF/p75 [16 (link),20 (link),42 (link)], or specific to DNA topoisomerase I (TOPO-1/Scl-70), were from the autoimmune serum collections of Werfen (formerly Inova Diagnostics, San Diego, CA, USA) and the Casiano Laboratory.