Fig 2: BBR increases the expression of SETD2 and H3K36me3 in HepG2 cells exposed to high-lipid nutrition. Western blot analysis (A,D) and PCR analysis (B,E) of SETD2 and H3K36me3 in HepG2 cells treated with or without BBR (10 or 20 µM) for 24 hours in serum-free media containing 100 µM palmitate. (C) Luciferase activity of SETD2 in HepG2 cells treated with BBR (20 µM). The data are presented as mean ± SEM, n=3 each group. **, P<0.01. BBR, berberine; SEM, standard error of the mean; SETD2, SET domain-containing protein 2.

Fig 3: BBR attenuates the accumulation of triglycerides and increases the expression of SETD2 and H3K36me3 in HFHS diet-induced steatotic livers of mice. Male mice at 6 weeks old were fed by a HFHS diet for 12 weeks and then treated with high-dose BBR (10 mg/kg/day), low-dose BBR (5 mg/kg/day), or vehicle (PBS) by i.p. injection once daily for 5 weeks. (A) Representative photomicrographs of Oil Red O staining of the liver sections of mice (magnification, ×200). The levels of liver (B) and plasma (C) triglycerides were assessed in mice. Western blot analysis (D) and PCR analysis (E) of SETD2, H3K36me3, and GAPDH in mice. (F) Immunohistochemical staining of SETD2 in the livers of mice (magnification, ×200). The data are presented as mean ± SEM, n=3 each group. *, P<0.05; **, P<0.01. BBR, berberine; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; HFHS, high-fat, high-sucrose; H3K36me3, trimethylation of lysine 36 on histone 3; NS, not significant; PBS, phosphate buffered saline; SEM, standard error of the mean; SETD2, SET domain-containing protein 2.

Fig 4: SETD2 overexpression promoted the proliferation and migration of HCC cell lines. A. Schematic diagram showing the major domains of SETD2 and the truncated fragments. Fragment 2 (F2) was used in the subsequent experiments. B. The effect of SETD2 overexpression on the proliferation of HCC cell lines was determined via a CCK-8 assay. C. The effect of SETD2 overexpression on the proliferation of HCC cell lines was evaluated by colony formation assays. D. The effect of SETD2 overexpression on the migration of HCC cell lines was detected by a scratch wound healing (cell migration) assay, the pictures were screenshots from the same position of origin picture as shown in supplement material (Figure S7) using Photoshop (data are given as the mean ± SD, n = 3, *P <0.05, **P < 0.01, ***P < 0.005, and ****P < 0.001).

Fig 5: Validation of the function of SETD2 in tumor tissues, 786‐O, and ACHN ccRCC cell lines. (A) SETD2 and PLXNA2 expression was analyzed by immunohistochemistry in 60 ccRCC patients' cancer tissues. Scale bars, 300 μm. (B) Correlation of SETD2 and PLXNA2 IHC score. (C) Knock‐down of SETD2 inhibited PLXNA2 expression. (D) Cell proliferation of 786‐O and ACHN after being transfected with negative control and sh‐SETD2. (E) Wound healing assay of 786‐O and ACHN cells expressing a vector control or sh‐SETD2. Scale bars, 200 μm. (F) Comparison of the number of cell invasion and migration in the SETD2 control and knock‐down of 786‐0 and ACHN cells. Scale bars, 100 μm. ns, not significant; *p < 0.05; **p < 0.01; ***p < 0.001; NC, negative control.