Anti oct4

The protein expression was determined by western blot analysis. In brief, the sample cells were lysed by radio-immunoprecipitation assay buffer (RIPA; Roche, Basel, Switzerland) containing protease and phosphatase inhibitors, then resolved by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and blotted onto 0.45 ​μm polyvinylidene fluoride membranes (Merck Millipore, Burlington, MA, USA). The membranes were subsequently blocked in 5% BSA and probed with primary antibodies: anti-Nanog (#4903; Cell Signaling Technology), anti-Oct4 (#2750S; Cell Signaling Technology), anti-Sox2 (#14962S; Cell Signaling Technology), anti-CXCR4 (ab124824; Abcam), and anti-Glyceraldehyde 3-phosphate dehydrogenase (GAPDH, ab181603; Abcam) at 4 ​°C overnight. After extensive washing, the membranes were incubated with horseradish peroxide-conjugated secondary antibodies (HRP-conjugated 2nd antibodies): goat anti-rabbit IgG (ab6721; Abcam) and goat anti-mouse IgG (ab6789; Abcam) for 1 ​h at room temperature, then developed using enhanced chemiluminescence (ECL; Merck Millipore). Immuno-reactive bands were detected by UVP BioSpectrum 810 imaging system (Thermo Fisher Scientific) and analyzed by VisionWorks LS software (UVP, CA, USA). The protein expression levels were normalized to GAPDH as an internal loading control.

Capsazepine, PI, imatinib mesylate, ionomycin and DCFDA were from Sigma Aldrich. BAF, Fluo‐3 AM, JC‐1, and DAPI were from ThermoFisher Scientific. CBD was from ENECTA. Tranilast was from Bio‐Techne SRL. CBD, tranilast, Capsazepine and BAF were dissolved in DMSO used as vehicle (maximum 0.05%, considered no toxic).¹⁶ Abs used according to datasheet: anti‐LC3, anti‐COX IV, anti‐OCT4, anti‐ATG16L1, anti‐ATG5‐ATG12, anti‐pink1, anti‐parkin, anti‐caspase 3, anti‐GAPDH, anti‐optineurin and anti‐PU.1 were from Cell Signaling Technology (1:1000); rabbit anti‐human TRPV1 (1:1000) was from Invitrogen and goat anti‐human TRPV2 (1:50) was from Santa Cruz Biotechnology).
Secondary Abs used: HRP‐conjugated anti‐rabbit IgG (1:5000; Jackson ImmunoResearch Europe Ltd), HRP‐conjugated anti‐mouse IgG (1:2000; Cell Signaling Technology), PE‐conjugated goat anti‐rabbit Ab (1:40; BD Biosciences), FITC‐conjugated goat anti‐mouse Ab (1:40; BD Biosciences), Alexa Fluor‐594‐conjugated goat anti‐rabbit Ab (1:100; Cell Signaling Technology), and Alexa Fluor‐488‐conjugated donkey anti‐goat Ab (1:100; Invitrogen).

Western blot, immunofluorescence (IF),⁵¹ (link) and immunohistochemistry (IHC)⁵² (link) were performed as previously described. Anti-Notch1, anti-β-catenin, anti-p84, anti-APC, anti-GSK3β, anti-CK1α, and anti-β-TrCP antibodies were purchased from Abcam. Anti-Nanog, anti-BMI1, anti-Oct4, and anti-HES1 antibodies were obtained from Cell Signaling Technologies. Anti-TET3 and anti-actin antibodies were purchased from GeneTex and Sigma, respectively. Western band intensity was quantified using ImageJ software (NIH).

Anti-PRMT7 antibodies were purchased from Epicypher (#13-1009), Santa Cruz Biotechnology (SC9882) and Millipore (#07-639). Anti-Nanog (#61419), anti-Sox2 (#39823) and anti-H4R3me2s (#61187) antibodies were from Active Motif. Anti-Oct4 (#2840), anti-c-Myc (#5605) and anti-Klf4 (#4038) antibodies were from Cell Signaling. Anti-β-actin antibody (A5441) was from Sigma-Aldrich. Anti-H4R3me1 antibody (PA5-27065) was from Thermo Scientific. Anti-H3 antibody (ab1791) was from Abcam. Anti-mouse Argonaute 2 (Ago2) antibody was from Wako Chemicals (292-67301) and Cell Signaling (2897P). The cDNA constructs of human (OpenBiosystem) and mouse PRMT7 (OpenBiosystem) were cloned into pCDH1-EF1-IRES-GFP vector (System Biosciences) using the cloning sites EcoRI and NotI. Human PRMT7's catalytic mutant (m. PRMT7) was generated from pCDH1-EF1-PRMT7-IRES-GFP by mutating several residues (E144A, E153A, E478A and H644A) using the QuickChange Site-Directed Mutagenesis Kit (Stratagene). These residues are likely located in arginine binding pockets according to crystal structures of PRMT1 and Caenorhabditis elegans PRMT7 (18 (link),19 (link)). Oligonucleotides used for cloning, ChIP-PCR, reverse-transcription (RT)-PCR, site-directed mutagenesis are listed in Supplementary Table S1.