H3k4me3

Cells were grown on 6-well plates at a seeding density of 2.5 × 10⁵ cells per well. These cells were starved in serum-free RPMI-1640 for 24 h. They were then treated with 5 ng/mL TGFβ and 20 µM GSK-J4, both individually and in combination. After 48 h of treatment, the cells were scraped in RIPA lysis buffer for protein extraction. Protein concentration was estimated using the Bradford assay. SDS-PAGE and Western blot were carried out as per standard protocol. Primary antibodies used were p-SMAD3 (Santa Cruz, Dallas, TX, USA), N-cadherin (Novus, St. Louis, MO, USA), vimentin (Novus, St. Louis, MO, USA), H3 (Invitrogen, Waltham, MA, USA), H3K9me3 (Santa Cruz, Dallas, TX, USA), H3K4me3 (Invitrogen, Waltham, MA, USA), H2K27me3 (Abcam, Cambridge, UK), and actin (Invitrogen, Waltham, MA, USA), and the secondary antibodies were Chicken Anti-Mouse IgG H&L (HRP) (Abcam, Cambridge, UK) and Chicken Anti-Rabbit IgG H&L (HRP) (Abcam, Cambridge, UK). The blot was developed using an ECL reagent (Thermo Scientific SuperSignal West Femto Maximum Sensitivity Substrate, Waltham, MA, USA). The experiment was repeated thrice. The statistical analysis was carried out using ImageJ 1.52a software. The student’s t-test was carried out to calculate significance using GraphPad Prism 9.5.0 software.

A673 cells were treated with vehicle (DMSO) or 2 μM JIB-04 for 36 hours. Chromatin immunoprecipitation analysis followed by qPCR (ChIP-qPCR), to compare H3K4me3 levels at the CDKN1A promoter in vehicle and drug-treated cells was performed essentially as previously described [18 (link)], with the following modifications: 4 x 10⁶ cells were resuspended in 500ul of lysis buffer, and sonicated in the Diagenode Bioruptor for 25 cycles (30sec on/90sec off on High setting); lysate was diluted up to 4x the volume before clearing with Protein A/G beads; 5 μg of control (rabbit IgG; Santa Cruz, sc-2027) and specific (H3K4me3; Invitrogen, #49-1005) antibodies were used for immunoprecipitation. Primers used for qPCR are listed in Supplementary Materials (Supplementary Table 2).

The protein precipitated from the samples used to measure α-KG was used to analyze the histone methylation status through western blot to better understand the correlation of nuclear α-KG and its effect on histone methylation. The TCA-precipitated protein was washed with acetone and dissolved in 4x sample loading dye. The samples were boiled at 90 °C for 10 min. The blots were probed for histone H3 (Cell Signaling 96C10), H3K79me2 (Invitrogen 710802), H3K4me3 (Invitrogen MA5-11199), and H3K36me3 (Invitrogen PA5-17109).