Fig. 2
Knock-down of YTHDF2 inhibits the tumor progression of PCa in vitro. a The knock-down efficiency of YTHDF2 shRNAs (shYTHDF2–1 and shYTHDF2–2) with lentivirus constructs in DU-145 and PC-3 cell lines was confirmed by western blot. GAPDH was the internal reference. b m6A RNA dot-blot assay. The m6A level alterations at different total RNA concentrations (50 ng, 100 ng, 200 ng, 400 ng) in DU-145 and PC-3 cell lines were detected. Methylene blue staining was loading control. c The m6A levels detected by IF in DU-145 cell line after knocking down YTHDF2 (shYTHDF2–1), scale bar = 100 μm. d The proliferation ability after knocking down YTHDF2 was evaluated by colony formation assay (representative wells were presented) and (e) statistically analyzed by Mann-Whitney test. f Flow cytometry assay (representative images were presented) and western blot assay were used to confirm the apoptosis analysis induced by knock-down of YTHDF2. GAPDH was the internal reference. Student’s t test was used for the statistical analysis. g and h Trans-well assay and wound-healing assay (representative wells were presented) were assessed for the cell migration in YTHDF2 knock-down cell lines. Mann-Whitney test was used for the statistical analysis. i Western blot assay was used to detect the alterations of EMT-associated proteins and AKT phosphorylation level in YTHDF2 knock-down PCa cell lines. GAPDH was the internal reference. j The overexpression efficiency of pYTHDF2 plasmid (transient transfection with FuGENE HD Transfection Reagent, 0.5 μg/ml) compared with control pNull in DU-145 and PC-3 cell lines was detected by western blot assay. GAPDH was the internal reference. k and l m6A RNA dot-blot assay and IF assay were used to determine the m6A levels after overexpression of YTHDF2. Methylene blue staining was loading control. m Proliferation ability was evaluated by colony formation assay (representative wells were presented) in YTHDF2-overexpressed cell lines, and (n) statistically analyzed with Mann-Whitney test. o Trans-well assay (representative wells were presented) was used to detect the cell migration. Mann-Whitney test was used for the statistical analysis. p The alterations of EMT-associated proteins and AKT phosphorylation level were all detected by western blot assay after overexpression of YTHDF2 in DU-145 and PC-3 cell lines. GAPDH was the internal reference. Error bars represent the SD obtained from at least three independent experiments; *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001