Fig. 4

INMT expression is upregulated via the METTL3–m6A modification pathway in PCa cells. A, INMT expression levels in indicated cells were examined via western blot assay. B, The expression levels of m6A regulatory enzymes in corresponding cells were detected via western blot assay. C, Sequence analysis of m6A consensus sequences present on INMT 3’-UTR. D, INMT protein levels were detected via western blot assay after METTL3 knockdown in LNCaP cells. E, INMT protein levels were detected via western blot assay after METTL3 overexpression in LNCaP cells. LNCaP cells were transfected with various doses of METTL3 expression plasmids, followed by immunoblotting. F, Real-time qPCR was performed to detect the relative INMT mRNA levels after METTL3 silencing or overexpression in LNCaP cells. G, m6A meRIP qRT–PCR assays were performed to determine changes in the levels of INMT mRNA undergoing m6A methylation modifications. H, Dual luciferase reporter assays were conducted in LNCaP cells transfected with WT or mutant (MUT) luciferase-INMT 3’-UTR reporter. Luciferase activity of INMT 3′-UTR was tested and normalized to renilla luciferase activity. I, The effects of BHB on the expression of METTL3 were detected via western blot assay in indicated cells. Paired and two-tailed Student’s t-tests were performed, and significant differences were designated as *p < 0.05, **p < 0.01, and ***p < 0.001