Fig. 4: STAT3 is pivotal in the YTHDF3-mediated negative regulation of HPV carcinogenesis by type I IFNs.

A qPCR analysis was used to measure HPV DNA levels in SiHa cells transfected with either siSTAT3 (STAT3 siRNA) or siNC (negative control siRNA). The data are presented as the means ± SEMs of two independent experiments. *p < 0.05. B qPCR analysis of the mRNA levels of the HPV E1, E2, and E6 genes in siSTAT3-transfected cervical cancer cells compared with those in negative control cells. The data are presented as the means ± SEMs of two independent experiments. *p < 0.05, **p < 0.01. C Western blot analysis showing E6 protein expression in SiHa cells transfected with either a control vector or siSTAT3 for 48 h. D Western blot analysis of STAT3 and IRF7 protein levels in SiHa and CaSki cells transfected with either a control vector or siSTAT3 for 48 h. STAT3 and IRF7 expression was normalized with GAPDH. Bar graphs exhibited normalized IRF7 expressions from 3 independent experiments. Data were presented as mean ± SD from 3 independent experiments. P values were calculated by Student’s t test, indicating *p < 0.05, **p < 0.01. GAPDH was used as loading control. E Western blot analysis was used to assess E6 protein expression in CCa cells following transfection with a control vector, a YTHDF3 knockdown (siRNA), a STAT3 overexpression plasmid, or cotransfection with a YTHDF3 knockdown and STAT3 overexpression plasmid. The cells were incubated for 48 h after transfection. STAT3 and E6 expression was normalized with GAPDH. Bar graphs exhibited normalized E6 expressions from 3 independent experiments. Data were presented as mean ± SD from 3 independent experiments. P values were calculated by Student’s t-test, indicating *p < 0.05, **p < 0.01. GAPDH was used as loading control. F Western blot analysis of STAT3 and IRF7 protein levels in YTHDF3 knockout (YTHDF3^−/−) CCa cells with STAT3 overexpression (OE-STAT3) compared with control YTHDF3^−/− cells. STAT3 and IRF7 expression was normalized with GAPDH. Bar graphs exhibited normalized IRF7 expressions from 3 or 4 independent experiments. Data were presented as mean ± SD from 3 or 4 independent experiments. P values were calculated by Student’s t-test, indicating *p < 0.05. GAPDH was used as loading control. G Immunofluorescence assays were used to visualize the location and expression of YTHDF3 (green fluorescence) and HPV DsRed (red fluorescence) in YTHDF3^−/− SiHa cells and YTHDF3^−/− SiHa cells overexpressing STAT3 (YTHDF3^−/− OE-STAT3). Original magnification, 60×; scale bar = 100 µm.