Molecular mechanism of modified Yigong San formula against colorectal cancer via EZH2/METTL3/SOX4 pathway-mediated apoptosis

Figure 1 The mRNA expression levels of enhancer of zeste homolog 2 and methyltransferase-like 3 in cancerous tissues and adjacent tissues. A: Comparison of enhancer of zeste homolog 2 mRNA expression levels in cancer tissues and adjacent tissues; B: Comparison of methyltransferase-like 3 mRNA expression levels in cancer tissues and adjacent tissues; compared with cancer tissues, ^aP < 0.0001; ^bP < 0.0001. EZH2: Enhancer of zeste homolog 2; METTL3: Methyltransferase-like 3.

Figure 2 Determination of optimal drug concentration using Cell Counting Kit-8 assay and cell migration experiment. A: Light microscopic observation of the effects of different treatment groups on SW480 cell migration ability; B: Effects of different treatment groups on SW480 cell migration ability at 48 hours; C: Inhibitory effects of different drug concentrations on SW480 cells as detected by Cell Counting Kit-8 assay. ^aP < 0.01; ^bP < 0.01; ^cP < 0.0001; ^dP < 0.0001; ^eP < 0.0001; ^fP < 0.0001; ^gP < 0.0001. YGS: Yigong San.

Figure 3 Expression levels of enhancer of zeste homolog 2, methyltransferase-like 3, SRY-box transcription factor 4, B-cell lymphoma 2, and Bax proteins in different groups. A: SRY-box transcription factor 4 protein expression level; B: Methyltransferase-like 3 protein expression level; C: Enhancer of zeste homolog 2 protein expression level; D: B-cell lymphoma 2 protein expression level; E: Bax protein expression level; F: Protein expression images of GAPDH, enhancer of zeste homolog 2, methyltransferase-like 3, SRY-box transcription factor 4, Bax, and B-cell lymphoma 2. ^aP < 0.01; ^bP < 0.01; ^cP < 0.01; ^dP < 0.01; ^eP < 0.0001; ^fP < 0.0001; ^gP < 0.0001. SOX4: SRY-box transcription factor 4; METTL3: Methyltransferase-like 3; EZH2: Enhancer of zeste homolog 2; Bcl2: B-cell lymphoma 2; NC: Negative control; YGS: Yigong San; OE: Overexpressing.

Figure 4 The mRNA expression levels of enhancer of zeste homolog 2, methyltransferase-like 3, SRY-box transcription factor 4, B-cell lymphoma 2, and Bax in different cell groups. A: Enhancer of zeste homolog 2 mRNA expression level; B: Methyltransferase-like 3 mRNA expression level; C: SRY-box transcription factor 4 mRNA expression level; D: B-cell lymphoma 2 mRNA expression level; E: Bax mRNA expression level. ^aP < 0.0001; ^cP < 0.0001; ^bP < 0.05; ^dP < 0.0001; ^eP < 0.0001. SOX4: SRY-box transcription factor 4; METTL3: Methyltransferase-like 3; EZH2: Enhancer of zeste homolog 2; Bcl2: B-cell lymphoma 2; NC: Negative control; YGS: Yigong San; OE: Overexpressing.

Figure 5 Migration ability of cells in different groups. A: Effects of different groups on SW480 cell migration ability observed under light microscopy; B: Effects of different groups on SW480 cell migration ability at 48 hours. ^aP < 0.001; ^bP < 0.01; ^cP < 0.01; ^dP < 0.0001. NC: Negative control; YGS: Yigong San; OE: Overexpressing; EZH2: Enhancer of zeste homolog 2.

Figure 6 Apoptosis rates of cells in different groups. A: Flow cytometry (FCM) analysis results of the control group; B: FCM analysis results of the control + Chinese medicine group; C: FCM analysis results of the control + negative control group; D: FCM analysis results of the control + overexpressing enhancer of zeste homolog 2 group; E: FCM analysis results of the control + overexpressing enhancer of zeste homolog 2 + Chinese medicine group; F: Quantitative analysis of FCM results. ^aP < 0.0001; ^bP < 0.0001; ^cP < 0.0001. NC: Negative control; YGS: Yigong San; OE: Overexpressing; EZH2: Enhancer of zeste homolog 2.

Figure 7 Enrichment level of enhancer of zeste homolog 2 in the promoter region of methyltransferase-like 3. A: Enrichment of enhancer of zeste homolog 2 at methyltransferase-like 3 site 1; B: Enrichment of enhancer of zeste homolog 2 at methyltransferase-like 3 site 2. ^aP < 0.001. METTL3: Methyltransferase-like 3; IgG: Immunoglobulin G; EZH2: Enhancer of zeste homolog 2.