Fig. 5

METTL14 decreases stability and expression of ITGB4 mRNA by m6A-modifying its 3′UTR. a Protein expression of METTL14 and ITGB4 in ACHN cells with or without METTL14 depletion detected by western blot. b Protein expression of METTL14 and ITGB4 in Caki-1 cells with or without METTL14 overexpression detected by western blot. c, d Alterations of ITGB4 mRNA level by METTL14 depletion (c) and METTL14 overexpression (d) detected by qRT-PCR. e–h After treatment of cells with transcription inhibitor (TI) actinomycin D at 5 μg/ml for indicated times, ITGB4 mRNA expression in the cells with indicated transfections detected by qRT-PCR. i An m6A motif identified in ITGB4 3’UTR. j, K MeRIP assay performed to detect the level of m6A-modification on ITGB4 3’UTR in sh-NC or sh-METTL14 ACHN cells (j) and METTL14-overexpressing or empty vector-overexpressing Caki-1 cells (k) by respectively using m6A-specific antibody and IgG antibody as a negative control. l Dual-luciferase reporter established with wild-type ITGB4 3′UTR or mutant ITGB4 3’UTR. m, n Luciferase activities of sh-METTL14 or sh-NC ACHN cells (m) and METTL14-overexpressing or empty vector-overexpressing Caki-1 cells (n) co-transfected with either the wild-type ITGB4 3’UTR or the mutant ITGB4 3’UTR. o Transcript correlations of ITGB4 to indicated genes in TCGA database. Each experiment was conducted independently for three times. Bar graphs: means ± SDs. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ns, not significant