Fig. 6: circGlis3 prevents β-cell apoptosis by directly binding to SCOTIN.
From: Circular RNA circGlis3 protects against islet β-cell dysfunction and apoptosis in obesity
a The mass spectrometry assay revealing the proteins (score > 20 by Mascot Software) pulled down by biotin-labeled circGlis3 from the MIN6 cells lysate. b The endogenous circGlis3 interacting with SCOTIN in MIN6 cells were detected by RIP assay and RT-PCR (n = 6 biological replicates). c Biotin-labeled sense or anti-sense circGlis3 were used for circGlis3-protein pulldown against MIN6 cells lysate, and Western Blotting showing that SCOTIN was pulled down. d Colocalization between circGlis3 and SCOTIN in MIN6 cell (Red represents circGlis3, Green represents SCOTIN, Scale bars represent 50 μm, n = 5 biological replicates) and pancreas sections (Scale bars represent 100 μm, n = 5 biological subjects) by circRNA fluorescent in situ hybridization (circFISH) and Immunofluorescence assays. e, f RT-PCR analysis and Western Blotting results of Scotin mRNA and protein level in the islets from the HFD-fed mice and Leprdb/db mice (n = 6 biological subjects). g Annexin V/PI staining and flow cytometry analysis of cell apoptosis in MIN6 cells with oe-circGlis3 or/and oe-SCOTIN treatment (n = 5 biological replicates). h TUNEL assays in MIN6 cell (Red represents positive TUNEL cells, Green represents Insulin; Scale bars represent 100 μm; n = 5 biological replicates) and mice islets (Red represents Insulin, Green represents positive TUNEL cells; Scale bars represent 50 μm; n = 5 biological subjects) with oe-circGlis3 or/and oe-SCOTIN treatment. i. TUNEL assays in human islets (Red represents positive TUNEL cells, Green represents Insulin; Scale bars represent 100 μm) with oe-circGlis3 or/and oe-SCOTIN treatment (n = 5 biological subjects). j β-cell mass in oe-circGlis3 or/and oe-SCOTIN treated HFD-fed mice (n = 6 biological animals). k TUNEL assays in pancreatic sections of oe-circGlis3 or/and oe-SCOTIN treated mice with HFD-fed for 20 weeks (Red represents positive TUNEL cells, Green represents Insulin; Scale bars represent 100 μm; n = 5 biological animals). l, m Western Blotting showing Caspase 3, cleaved-Caspase 3, BAX, and BCL-2 in palmitate-stimulated MIN6 cell and islets of the HFD-fed mice, both with oe-circGlis3 or/and oe-SCOTIN treatment, respectively. b, c, e–m For bar graphs, data represents mean ± SEM. b, c, g–m One-way ANOVA with Tukey’s post-test. e, f Unpaired two-tailed Student’s t-test. *p < 0.05, **p < 0.01, ***p < 0.001. Source data are provided as a Source data file.
