Fig. 4: FTO positively regulated GnRH expression by affecting the intracellular levels of free Ca²⁺.

a, b Overexpression of FTO (OE-FTO) in GT1-7 cells as confirmed by qPCR and western blotting (n = 3). c GnRH mRNA levels detected by qPCR in OE-FTO cells (n = 3). d GnRH abundance in OE-FTO cells as determined by ELISA (n = 3). e, f Knockdown of FTO (KD-FTO) in GT1-7 cells as determined by qPCR and western blotting (n = 3). g Expression of GnRH mRNA as determined by qPCR (n = 3). h Protein expression of GnRH in KD-FTO cells as determined by ELISA (n = 3). i Free Ca²⁺ concentrations of OE-Control and OE-FTO GT1-7 cells as determined by fluorescence of Calbryte-630 with flow cytometry as described in the Methods (n = 3). Left: flow cytometric histograms of Calbryte-630 and gating with no Calbryte-630 probe as background values. Right: quantitative analysis of cell fluorescence for Calbryte-630. j Levels of intracellular free Ca²⁺ (red colour) between OF-FTO and OE-Control cells as determined by IF (n = 6). The concentration of fluorescence-labelled Calbryte 630 was 5 μM. Scale bars, 100 μm. The bars represent the means ± SEMs. *P < 0.05, **P < 0.01, ***P < 0.001, and ns, P > 0.05 versus OE-control or KD-control group by Student’s t test.