Fig 1: Effect of FOXA2 knockout on iPSC-derived beta cells.A Western blotting of FOXA2 expression in the definitive endoderm (DE) derived from Ctr-iPSCs, FOXA2+/- iPSCs, and FOXA2-/- iPSCs. B Quantification of FOXA2 protein levels in DE and PP2 derived from Ctr-iPSCs, FOXA2+/- iPSCs, and FOXA2-/- iPSCs. Note the complete absence of FOXA2 expression in the DE derived from FOXA2-/- iPSCs. Representative immunofluorescence images showing the expression of PDX1 and NKX6.1 in PP2 (C) and NGN3 and NKX2.2 in EPs (D) derived from WT controls and FOXA2-/- iPSCs. E Real-time PCR analysis for the expression of FOXA2, NEUROD1, NKX2.2, and NGN3 in EPs derived from FOXA2-/- iPSCs and their WT controls. F Representative immunofluorescence images showing the expression of INS and GCG in stage 7 derived from FOXA2-/- iPSCs and Ctr1-iPSCs. G qPCR quantification for the expression of FOXA2, NEUROD1, NKX2.2, INS, GCG, ABCC8, KCNJ11, PDX1, and HHEX in stage 7 derived from FOXA2-/- iPSC line and its WT control, Ctr1-iPSCs. The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 2: FOXA2 overexpression in PP2 reverse the phenotype of FOXA2 deficiency.A Representative western blotting showing the overexpression of FOXA2 in the PP2 derived from FOXA2+/- iPSCs, and FOXA2-/- iPSCs, 48 h after transfection with FOXA2 plasmid or empty vector (EV). B Real-time PCR analysis for the expression of PDX1, NKX6.1, SOX9, GATA6, PAX4, PTF1A, AMYLASE, TCF7L2, INSR, NOTCH, HES1, MNX1, ISL1, PROX1, PTPRN1, ADRA2A, and CDX2 in PP2 derived from FOXA2+/- iPSCs and FOXA2-/- iPSCs 48 h after transfection. C Real-time PCR analysis for the expression of BMP4, NOG, MSX1, MSX2, DKK1, DLX2, DLX5, EMX2, ERG2, OTX1, OTX2, and RFX6 in PP2 derived from FOXA2+/- iPSCs and FOXA2-/- iPSCs 48 h after transfection. D Real-time PCR analysis for the expression of INS, GCG, SST, PDX1, NKX6.1, ABCC8, KCNJ11, HHEX, and NEUROD1 in stage 7 derived from FOXA2+/- iPSCs and FOXA2-/- iPSCs transfected with FOXA2 plasmid or EV at stage 4. E Representative immunofluorescence images showing the expression of INS and GCG in stage 7 derived from FOXA2+/- iPSCs and FOXA2-/- iPSCs transfected with FOXA2 plasmid or EV at stage 4. F C-PEPTIDE (C-PEP) secretion (% of content) after stimulation with or without 30 mM KCl in the presence of low glucose (LG) in FOXA2+/- iPSC-derived beta cells transfected with FOXA2 plasmid or EV at stage 4. The data are presented as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.
Fig 3: Effect of FOXA2 haploinsufficiency on iPSC-derived beta cells.Representative immunofluorescent images (A) and flow cytometry histograms (B) showing the expression of INSULIN (INS), NKX6.1, C-PEPTIDE (C-PEP), and GLUCAGON (GCG) in stage 7 derived from Ctr-iPSCs and FOXA2+/- iPSCs. C Flow cytometry quantification for the data shown in B. D qPCR quantification for the expression of the pancreatic markers, FOXA2, INS, GCG, ABCC8, KCNJ11, NKX6.1, NKX2.2, and PDX1 in beta cells derived from Ctr-iPSCs and FOXA2+/- iPSCs. GSIS assay: fold change of C-PEPTIDE secretion in iPSC-derived beta cells challenged with low (LG; 2.8 mM) and high glucose (HG; 20 mM) (E) or stimulated with or without 30 mM KCl in the presence of LG (F). Data are represented as mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001.
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