Fig 2: SP treatment induces HECTD2 expression for EHMT2 degradation.A Heat map of RNA-seq data (E3 ubiquitin ligase) in the control and SP treatments. B qRT-PCR analysis of HECTD2 after SP treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (**p < 0.01, ***p < 0.001) (upper). Western blot analysis after sodium propionate treatment using anti-HECTD2. ACTB was used as the internal control in HCT116 cells (below). The signal intensities were quantified using ImageJ software. C Western blot analysis of histone H3K27 acetylation (left) and H3K9 acetylation (right) after SP treatment for 24 h in HCT116 cells. Histone H3 was used as the internal control. The signal intensities were quantified using ImageJ software. D The ChIP assay was performed using anti-H3K27ac antibody. The result is shown as an enrichment fold of input chromatin compared with the control in HCT116 cells after SP treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001). E Western blot analysis of EHMT2 after treatment with siHECTD2 and SP for 24 h in HCT116 cells. ACTB was used as the internal control. The signal intensities were quantified using ImageJ software. F Western blot analysis of EHMT2 after treatment with siHECTD2 and SP for 24 h and CHX for 12 h in HCT116 cells. ACTB was used as the internal control. The signal intensities were quantified using ImageJ software. G Western blot analysis after transfection with a FLAG-tagged EHMT2 expression vector together with HA-tagged ubiquitin and treatment with siHECTD2 and SP for 24 h and MG132 for 6 h using HA and FALG antibodies in HCT116 cells. H FACS analysis using Muse Caspase 3/7 working solution was performed after cotreatment with HECTD2 knockdown and SP in HCT116 cells. The upper right panel indicates the apoptotic and dead cell proportions (upper). Quantification of caspase 3/7 activity. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001) (below).

Fig 3: Effects of EZH2 and G9a inhibitors on the cell cycle and apoptosis in MM cells.A Cell cycle analysis in MM cell lines treated with the indicated drugs (1 μM, 6 days). Representative data are shown on the left. Summarized data from three biological replications are shown on the right; error bars represent SEMs. B Apoptosis assays in MM cells treated with the indicated drugs (1 μM, 6 days). Representative data are shown on the left. Summarized data from three biological replications are shown on the right; error bars represent SEMs. *P < 0.05, **P < 0.01.

Fig 4: Upregulation of ISGs and suppression of IRF4-MYC axis genes by EZH2 and G9a inhibition in MM cells.A Heat maps for expression of ISGs and IRF4-MYC axis genes in MM cell lines treated with the indicated drugs (1 μM each, 6 days). B qRT-PCR analysis of ISGs. Results are normalized to cells treated with DMSO. Shown are means of three technical replications; error bars represent SEMs. C Western blot analysis of Stat1 and phosphorylated Stat1. D qRT-PCR analysis of ISGs in RPMI-8226 cells treated with EPZ-6438 and/or UNC0642. Results are normalized to cells treated with DMSO. Shown are means of three technical replications; error bars represent SEMs. E qRT-PCR analysis of IRF4-MYC axis genes. Results are normalized to cells treated with DMSO. Shown are means of three technical replications; error bars represent SEMs. *P < 0.05, **P < 0.01.

Fig 5: Negative regulation of TNFAIP1 by EHMT2 induces CRC apoptosis.A Expression level of TNFAIP1 in RNA-seq results after treatment with SP or siEHMT2 (upper). qRT-PCR analysis of TNFAIP1 after treatment with SP or siEHMT2. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001) (below). B Immunocytochemical analysis of TNFAIP1. HCT116 cells treated with SP or siEHMT2 were fixed with 100% methanol and stained with anti-TNFAIP1 (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 50 μm. C, D Cell growth assay after cotransfection with siTNFAIP1 and siEHMT2 for 48 h. HCT116 cells were fixed in 100% methanol and stained with crystal violet solution. Scale bar, 200 μm (C). CCK-8 solution was added to the culture medium, and the cells were incubated for 5 min at 37 °C. The intensity of cell growth was measured using a microplate reader (450 nm). Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001) (D). E Western blot analysis after cotransfection of siEHMT2 and siTNFAIP1 using anti-PARP, anti-EHMT2, and anti-TNFAIP1 antibodies. ACTB was used as the internal control in HCT116 cells. The signal intensities were quantified using ImageJ software. F FACS analysis using Muse Caspase 3/7 working solution was performed after cotransfection of siEHMT2 and siTNFAIP1. The upper right panel indicates the apoptotic and dead cell proportions (upper). Quantification of caspase 3/7 activity. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001) (below). G Scatter plots of EHMT2 and TNFAIP1 in the GTEx portal. Each dotted line indicates a linear regression line of the expression of EHMT2 and TNFAIP1. p values and correlation coefficients (r) between two genes were obtained using the Pearson correlation method.