Fig. 3

METTL3 upregulates STAT3 by promoting the translation of STAT3 mRNA. A, B qRT-PCR (A) and Western blotting analysis (B) of METTL3 and STAT3 in MHCC97L cells transfected with the indicated plasmids. The right panel in (B) shows the quantification of the intensity relative to tubulin. C qRT-PCR analysis of METTL3 and STAT3 in MHCC97H cells transfected with the indicated siRNAs. D, E Western blotting analysis of METTL3 and STAT3 in MHCC97H cells transfected with si-Control or si-METTL3. (E) shows the quantification of the intensity relative to tubulin. F MeRIP-qRT-PCR analysis of m6A levels of STAT3 in HCCLM3 and MHCC97H cells treated with the indicated concentration of STM2457 for 48 h. G Western blotting analysis of STAT3 in HCCLM3 and MHCC97H cells treated with the indicated concentration of STM2457 for 48 h. H qRT-PCR analysis of STAT3 mRNA stability in MHCC97L cells upon treatment with transcription inhibitor Actinomycin-D (ActD) for the indicated timepoints. The cells were transfected with empty vector or METTL3. I Western blotting analysis of STAT3 in HEK293T cells time-dependently treated with 100 μg/ml cycloheximide (CHX) after being transiently transfected with the indicated plasmids. The upper panel is the quantification of the intensity of STAT3 relative to tubulin. J qRT-PCR analysis of STAT3 mRNA enrichment in anti-RPL10A immunoprecipitated RNA in MHCC97L cells transfected with the indicated plasmids. K qRT-PCR analysis of STAT3 mRNA enrichment in anti-RPL10A immunoprecipitated RNA in MHCC97H cells transfected with the indicated siRNAs. All experiments were repeated at least three times. Error bars represent mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 by 2-tailed Student’s t-test