Fig 2: Synergistic effect of SP and BIX01294 in the 3D spheroid model.A 3D spheroid formation assay with HCT116 cell lines. Cells treated with SP (0 and 10 mM) were loaded onto ULA plates and incubated for 4 days. The cells were photographed under a microscope each day. Scale bar, 500 μm (left). Enlargement of spheroids at 4 days (right). Red line: dissociated cells. White line: aggregated cells. B qRT-PCR analysis of HECTD2, TNFAIP1, and CDH1 at 4 days. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). C Western blot analysis after SP treatment using anti-EHMT2 and anti-PARP antibodies. ACTB was used as the internal control. D 3D spheroid formation assay with HCT116 cell lines. Cells cotreated with SP and BIX01294 were loaded onto ULA plates and incubated for 2 days. The cells were photographed under a microscope each day. Scale bar, 500 μm. Enlargement of spheroids in 2 days. Red line: dissociated cells. White line: aggregated cells (left). The size of spheroids was measured using ImageJ software (right). Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001). E qRT-PCR analysis of HECTD2, TNFAIP1, and CDH1 at 2 days. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (*p < 0.05, **p < 0.01, ***p < 0.001). F Western blot analysis after SP treatment using anti-EHMT2 and anti-PARP antibodies. ACTB was used as the internal control.

Fig 3: 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 4: The supernatant of BT culture medium suppresses the growth of HCT116 and LS174T cells via propionate-induced apoptosis.A The concentration of metabolites was determined using a high-performance liquid chromatography (HPLC) system (1200 series, Agilent, USA) with a refractive index detector (RID) and an ion-exchange column (300 × 78 mm; Aminex HPX-87H; Bio-Rad, USA). The mobile phase was 2.5 mM H2SO4, the flow rate was 0.6 ml/min, the column temperature was 65 °C, and the RID was maintained at 45 °C. B, C Cell growth assay (crystal violet staining and CCK-8 assay) after BT Sup treatment for 48 h. HCT116 cells were fixed in 100% methanol and stained with crystal violet solution. Scale bar, 200 μm. (B). 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) (C). D Western blot analysis after BT Sup treatment using anti-PARP. ACTB was used as the internal control in HCT116 cells. The signal intensities were quantified using ImageJ software. E qRT-PCR analysis of EHMT2 expression after BT Sup treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (**p < 0.01). F Western blot analysis after BT sup treatment using anti-EHMT2. ACTB was used as the internal control. The signal intensities were quantified using ImageJ software. G Immunocytochemical analysis of EHMT2 and TNFAIP1. HCT116 cells treated with BT Sup were fixed with 100% methanol and stained with anti-EHMT2 and anti-TNFAIP1 (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 50 μm. H qRT-PCR analysis of HECTD2 and TNFAIP1 after BT Sup treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (***p < 0.001, **p < 0.01). I The concentration of metabolites was determined using a high-performance liquid chromatography (HPLC) system. J Cell growth assay (crystal violet staining) after CA Sup treatment for 48 h. HCT116 cells were fixed in 100% methanol and stained with crystal violet solution. Scale bar, 200 μm. K qRT-PCR analysis of EHMT2, HECTD2, and TNFAIP1 expression after CA Sup treatment. Mean ± SD of three independent experiments. p values were calculated using Student’s t-test (*p < 0.05, n.s. nonsignificant). L Western blot analysis after CA Sup treatment using anti-EHMT2 and anti-PARP antibodies. ACTB was used as the internal control. The signal intensities were quantified using ImageJ software. M Immunocytochemical analysis of EHMT2 and TNFAIP1. HCT116 cells treated with CA Sup were fixed with 100% methanol and stained with anti-EHMT2, anti-TNFAIP1 (Alexa Fluor 488, green) and DAPI (blue). Scale bar, 50 μm.