Fig. 6: Generation of FXYD2High HILOs.
From: FXYD2 marks and regulates maturity of β cells via ion channel-mediated signal transduction
a Drug screening (1 μM, 24 h stimulation) in FXYD2 promoter driven luciferase and mCherry expressing EndoC-βH1 cells. Dex and T3 upregulated FXYD2 promoter activity. The heatmap is shown by three independent screening data with fold change to control (DMSO, n = 18). b qPCR analyses of FXYD2 gene expression in EndoC-βH1 cells. Dex (10 μM for 24 h), T3 (1 μM for 24 h) and retinoic acids (RA, 2 μM for 24 h) synergistically enhanced FXYD2 gene expression in EndoC-βH1 cells. Brain-derived neurotrophic factor (BDNF, 10 μg/ml for 24 h) and ciliary neurotrophic factor (CNTF, 10 μg/ml for 24 h) are used as negative control. n = 3. Unpaired two-tailed t-tests. c qPCR analyses of indicated gene expression in EndoC-βH1 cells and hiPSC-derived β-like cells. NaCl stimulation enhanced FXYD2 and ATP1B1 gene expression in EndoC-βH1 cells and hiPSC-derived β-like cells (dispersed HILOs in 2D). n = 3. Unpaired two-tailed t-tests. d qPCR analyses of FXYD2 gene expression in EndoC-βH1 cells. Dex and NaCl synergistically stimulate FXYD2 expression in EndoC-βH1 cells. n = 3. Unpaired two-tailed t-tests. e Cell survival was measured by cellular ATP level (ratio). dFXYD2OE (DOX +) showed resistance to NaCl (osmotic pressure)-induced cell death in EndoC-βH1 cells. In contrast, FXYD2KD shows increased sensitivity to NaCl induced cell death in EndoC-βH1 cells. n = 3. Two-way ANOVA. f Schematics of generation of FXYD2High and FXYD2Low HILOs. Transient Dex 10 μM and NaCl 180 mM stimulation was used to improve visualization of FXYD2. g Representative fluorescence images of FXYD2-driven mCherry (red) and corresponding optical image. FXYD2High and FXYD2Low HILOs can be identified by FXYD2-fLuc-mCherry expression. FXYD2High and FXYD2Low enriched insulin positive cells were isolated from HILOs. Size-matched pseudo-HILOs (pHILOs) were generated from FXYD2High and FXYD2Low expressing HILOs. h Human c-peptide secretion response (pM/10,000 cells) in FXYD2High and FXYD2Low pHILOs. n = 3. Unpaired two-tailed t-tests. Scale bars = 100 μm. G3 = Glucose 3 mM, G20 = Glucose 20 mM, KCl20 = KCl 20 mM. Error bars indicate SEM.
