HNF1a-Q125ter impaired β-cell function in vitro. (A) Western blot analysis of HNF1a in INS-1 cells after transfection with control, HNF1a-OE, or HNF1a-Q125ter plasmids for 24 h. (B) The cell-proliferation analysis of control, HNF1a-WT, or HNF1a-Q125ter plasmid transfected Ins-1 cells at 0, 24, and 48 h. (C,D) RT-qPCR analysis of the expression levels of insulin genes Ins1 (C) and Ins2 (D) in Ins-1 cells after plasmid transfection for 24 h. (E) RT-qPCR quantification of insulin-secretion markers in control, HNF1a-WT, and HNF1a-Q125ter after plasmid transfection for 24 h: Slc2a2, GCK, Abcc8, Kcnj11, and Kcnh6. (F,G) Representative immunofluorescence images (F) and quantification of insulin fluorescence (G) of insulin content from control, HNF1a-WT, and HNF1a-Q125ter transfected Ins-1 cell. Insulin was stained with red, and HNF1a was stained with Magenta; GFP indicates the plasmid-transfected positive cells and DAPI-stained nuclei. Scale bar indicates 20 μm. (H) RT-qPCR quantification of β-cell maturation and differentiation markers (Mafa, Pdx1, and Pax6) in control, HNF1a-WT, and HNF1a-Q125ter after transfection for plasmids at 24 h. (I,J) Immunofluorescence analysis of insulin granules in different plasmid-transfected Ins-1 cells. The representative images (I) and quantification (J) of insulin granules are displayed. Insulin stained with red, HNF1a stained with magenta, GFP indicates the plasmid-transfected positive cells, and DAPI is stained nuclei. Scale bar indicates 40 μm. (K) Glucose-stimulated insulin secretion analysis in Ins-1 cells transfected with different plasmids. Results are represented as means with standard errors: * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. One-way ANOVA. All experiments were performed at least three times, unless otherwise indicated. WT, wild type.
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