Fig. 6: FTO regulated PLCβ3 expression in a targeted, m6A-dependent manner.
a Colorimetric quantification of m6A in control, WT-FTO-overexpressing and mutant MUT-FTO-overexpressing GT1-7 cells (n = 3). b FTO, PLCβ3, CAM and GnRH mRNA expression as determined by qPCR in control, WT- and MUT-FTO-overexpressing GT1-7 cells (n = 3). c FTO, PLCβ3, CAM, CAMKII and phosphorylated CAMKII proteins detected by western blotting in control, WT- and MUT-FTO-overexpressing GT1-7 cells (n = 3). The bars represent the means ± SEMs (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ns, P > 0.05 versus the WT-FTO-overexpressing group by one-way ANOVA. d Integrative genomics viewer (IGV) plots of m6A peaks at PLCβ3 mRNA. The y-axis shows the sequence read number, blue boxes represent exons, and blue lines represent introns. e MeRIP-qPCR analysis of m6A levels of PLCβ3 mRNA in OE-FTO GT1-7 cells (n = 3). The bars represent the means ± SEMs (n = 3). **P < 0.01, versus OE-control group by Student’s t test. f Top panel: schematic diagram of dual-luciferase reporter constructs. Bottom panel: Relative luciferase activity of the WT or MUT (A- to T- mutation) PLCβ3-exon luciferase reporter in control, WT-FTO-overexpressing and MUT-FTO-overexpressing 293 T cells. Firefly luciferase activity was measured and normalised to Renilla luciferase activity. The bars represent the means ± SEMs (n = 3). **P < 0.01, ***P < 0.001 versus the WT-FTO-overexpressing group by one-way ANOVA. g, h PLCβ3 mRNA stability assay after transfection of GT1-7 cells with FTO overexpression or knockdown lentivirus. The bars represent the means ± SEMs (n = 3). *P < 0.05 versus the control group by two-way ANOVA.
