Fig. 7: FUS sequestrates circGlis3 to reduce its abundance in diabetes by assembling cytoplasmic SG.
From: Circular RNA circGlis3 protects against islet β-cell dysfunction and apoptosis in obesity
a Expression of circGlis3 and Qki mRNA in patients with IGT or type 2 diabetes mellitus (T2DM) (n = 98 human individuals). b Expression of circGlis3 and Qki mRNA in HFD-fed mice with obesity or T2DM (n = 40 biological animals). c RT-PCR analysis of FUS mRNA expression in islets of the NCD- and HFD-fed mice (n = 6 biological subjects). d Western Blotting showing FUS protein expression in islets of the NCD- and HFD-fed mice. e Western Blotting showing FUS shuttle between the nucleus and cytoplasm in responses to palmitate stress in MIN6 cells. f Biotin-labeled sense or anti-sense circGlis3 were used for RNA-protein pulldown against MIN6 cells lysate, and Western Blotting showing that FUS was pulled down. g The endogenous circGlis3 interacting with FUS in MIN6 cells were detected by RIP assay and RT-PCR (n = 6 biological replicates). h RT-PCR analysis of circGlis3 expression in MIN6 cells (n = 6 biological replicates) and islets with FUS overexpression (n = 5 biological subjects). i RT-PCR analysis of miR-124-3p, Ins1, Ins2, NeuroD1 and Creb1 mRNA in MIN6 cells with FUS and circGlis3 overexpression (n = 6 biological replicates). j RIP and RT-PCR assay analyzed fold enrichment of endogenous circGlis3 by SCOTIN in palmitate-stimulated and oe-FUS-treated MIN6 cells (n = 6 biological replicates). k RIP and RT-PCR assay analyzed fold enrichment of endogenous circGlis3 by FUS in palmitate-stimulated and oe-SCOTIN-treated MIN6 cells (n = 6 biological replicates). l Colocalization between circGlis3 and FUS-stress granules (FUS-SG) in the palmitate-stimulated MIN6 cells with si-Fus and Emetine treatment (Red represents circGlis3, Green represents FUS; Scale bars represent 20 μm, n = 5 biological replicates). m. Colocalization between circGlis3 and FUS-SG in islets of the HFD-fed mice (Red represents circGlis3, Green represents FUS; Scale bars represent 100 μm, n = 6 biological animals). n. RT-PCR analysis of circGlis3 level in the palmitate-stimulated MIN6 cells with si-FUS and Emetine treatment (n = 6 biological replicates). c–k, n For bar graphs, data represents mean ± SEM. a, b LOESS Curve Fitting, 35% of points to fit. c–e Unpaired two-tailed Student’s t-test. f, n One-way ANOVA with Tukey’s post-test. g–k Two-way ANOVA with Bonferroni’s post-test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source data file.
