Journal: bioRxiv

Article Title: Identification and Characterization of Adult Islet Pancridia Cells Capable of Differentiating into Islet Organoids

doi: 10.1101/2025.07.28.667223

Figure Lengend Snippet: (A) Induction of NFAT family isoform genes in IPC clusters treated with ISX9 for 24 hours in the presence of CN inhibitor FK506. (B) RFX6 and NEUROD1 promoter activation by ISX9 in IPCs in the presence of FK506 or transfected with gene vectors overexpressing dominant negative NFAT (dnNFAT) and mutated control (dnNFATm). (C) ChIP assay of association of NFATC2, p300, HDAC1, HDAC2, and HDAC3 with RFX6, NEUROD1, and NEUROG3 promoters upon 6-hour treatment of IPCs with ISX9 with and without 24-hour pretreatment with ITF2357. (D) Schematic of mechanism of CN/NFATC2 induction of RFX6 and NEUROD1 genes by ISX9 to stimulate islet organoid differentiation. Graphed values are expressed as mean ± SD. Asterisks above bars indicate statistically significant differences (*p < 0.05, ***p < 0.001) in mean values for treatments based on a two-way ANOVA and Sidak’s multiple comparison test. Data shown are results from at least three independent experiments using MSCs derived from three individual donors.

Article Snippet: Gluc-ON reporters for RFX6 (HPRM53326-PG04), NEUROD1 (HPRM69533-PG04), and INS (HPRM30189-PG04) promoters were obtained from GeneCopoeia.

Techniques: Activation Assay, Transfection, Dominant Negative Mutation, Control, Comparison, Derivative Assay

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Volcano plots of bulk RNA-seq analysis of NGN3-GFP-derived pancreatic progenitors reseeded on LN411 or FN at the indicated time points. Expression of YAP target genes ( CCN1 / CYR61 , CCN2/CTGF ), early endocrine genes ( NEUROG3, NEUROD1, INSM1, NKX2-2) , PDX1 and PDK4 are indicated. Differential expression analysis was performed using DESeq2 ( n = 3, biological replicates). ( B ) Venn diagram of the top 100 upregulated genes by significance at 6 h after reseeding on LN411 (compared to reseeding on FN) or treatment with latrunculin B (LatB) (compared to before treatment). ( C ) qPCR analysis of PDK4 , and endocrine marker genes ( NEUROG3 and NEUROD1 ) 2 days after transfection with siRNA targeting PDK4 . The data were analyzed using two-tailed paired t -tests and are shown as mean fold change in expression ± SEM ( n = 6, biological replicates). ( D ) Brightfield (top panels) and epifluorescence (bottom panels) images of NGN3-GFP derived pancreatic progenitor cells 2 days after reseeding on LN411 with or without treatment with 10 mM DCA. High-density regions form a web-like structure rather than discrete circular aggregates. Scale bar = 100 μm. ( E ) qPCR analysis of endocrine marker genes NEUROG3 and NEUROD1 in pancreatic progenitors 2 days after reseeding on LN411 with or without 10 mM DCA treatment. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6, biological replicates). .

Article Snippet: NEUROD1 TaqMan probe , Thermo Fisher , Hs00159598_m1.

Techniques: RNA Sequencing, Derivative Assay, Expressing, Quantitative Proteomics, Marker, Transfection, Two Tailed Test

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Volcano plots of bulk RNA-seq analysis of NGN3-GFP-derived pancreatic progenitors treated with latrunculin B or DMSO at indicated time points. Expression of YAP target genes ( CCN1 / CYR61 , CCN2/CTGF ), early endocrine genes ( NEUROG3, NEUROD1, INSM1, NKX2-2) and PDK4 are indicated. Differential expression analysis was performed using DESeq2 ( n = 3). ( B ) qPCR analysis of PDK4 expression 24 h after reseeding on FN or LN411. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 4). ( C ) Western blot of PDK4 expression 24 h after reseeding on FN or LN411 (LN). Vinculin is included as a loading control. ( D ) Quantification of ( C ). The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 7). ( E ) Brightfield (top panels) and epifluorescence (bottom panels) images of pancreatic progenitors after 2 days of no treatment or treatment with 10 mM DCA. Scale bar = 100 μm. ( F ) qPCR analysis of endocrine marker genes NEUROG3 and NEUROD1 in pancreatic progenitors after 2 days of no treatment or treatment with 10 mM DCA. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6). ( G ) qPCR analysis of YAP target genes CYR61 and CTGF in pancreatic progenitors after 2 days of no treatment or treatment with 10 mM DCA. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6).

Article Snippet: NEUROD1 TaqMan probe , Thermo Fisher , Hs00159598_m1.

Techniques: RNA Sequencing, Derivative Assay, Expressing, Quantitative Proteomics, Two Tailed Test, Western Blot, Control, Marker

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Brightfield (top panels) and epifluorescence (bottom panels) images of pancreatic progenitors 2 days after reseeding on LN411 with or without 10 mM sodium acetate treatment. ( B ) qPCR analysis of endocrine marker genes NEUROG3 and NEUROD1 in pancreatic progenitors 2 days after reseeding on LN411 with or without 10 mM sodium acetate treatment. The data are shown as mean expression ± SEM ( n = 6, biological replicates). ( C ) Representative maximum intensity projections of NGN3-GFP derived pancreatic progenitors 1 day after reseeding on LN411 with or without 10 mM sodium acetate treatment. A mask of NKX6-1 + nuclei generated using Ilastik is shown in the first panel. Immunofluorescence for NKX6-1 (red), YAP (white), and DAPI (blue). Scale bar = 50 μm. ( D ) Quantification of nuclear YAP expression in 253 NKX6-1 + nuclei from three independent replicates (127 without sodium acetate and 126 with sodium acetate treatment). The data were analyzed using a two-tailed Welch’s t -test. ( E ) qPCR analysis of the YAP target genes CYR61 and CTGF in pancreatic progenitor cells 2 days after reseeding on LN411 with or without 10 mM sodium acetate treatment. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6, biological replicates). ( F ) qPCR analysis of PDX1 expression in pancreatic progenitors 2 days after reseeding on LN411 with or without 10 mM acetate or DCA. The data were analyzed by Dunnett’s test compared to LN411 alone and are shown as mean expression ± SEM ( n = 6, biological replicates). ( G ) Model depicting the role of ECM, integrins, and PDK4 in endocrine fate choice. Exposure to LN411 induces downregulation of integrins on both the protein and RNA levels. This is turn reduces the level of actin polymerization and leads to disinhibition of PDK4 expression. PDK4 inhibits the generation of acetyl-CoA and YAP activity. Reduced levels of acetyl-CoA promote maintenance of PDX1 expression. The combination of YAP inhibition and high PDX1 expression permits the expression of NEUROG3 and endocrine fate choice. .

Article Snippet: NEUROD1 TaqMan probe , Thermo Fisher , Hs00159598_m1.

Techniques: Marker, Expressing, Derivative Assay, Generated, Immunofluorescence, Two Tailed Test, Activity Assay, Inhibition

Journal: EMBO Reports

Article Title: Extracellular matrix-driven metabolic control of pancreatic endocrine lineage allocation

doi: 10.1038/s44319-025-00610-6

Figure Lengend Snippet: ( A ) Brightfield (top panels) and epifluorescence (bottom panels) images of NGN3-GFP-derived pancreatic progenitors after 2 days of no treatment or treatment with 20 mM sodium acetate. Scale bar = 100 μm. ( B ) qPCR analysis of endocrine marker genes NEUROG3 and NEUROD1 in pancreatic progenitors after 2 days of no treatment or treatment with 20 mM sodium acetate. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6). ( C ) qPCR analysis of YAP target genes CYR61 and CTGF in pancreatic progenitors after 2 days of no treatment or treatment with 20 mM sodium acetate. The data were analyzed using two-tailed paired t -tests and are shown as mean expression ± SEM ( n = 6). ( D ) qPCR analysis of PDX1 in pancreatic progenitors after 2 days of no treatment or treatment with 10 mM DCA or 20 mM sodium acetate. The data were analyzed by Dunnett’s test with comparison to the untreated condition and are shown as mean expression ± SEM ( n = 6). ( E ) ELISA analysis of intracellular acetyl-CoA concentrations in pancreatic progenitor cells at 3, 6, and 24 h after reseeding on FN, LN411, or LN411 with 10 mM DCA or 20 mM sodium acetate. The data were analyzed by two-way repeated measures ANOVA testing for treatment differences across the entire time course (3–24 h), followed by Dunnett’s test with comparison to LN411 alone and are shown as mean expression ± SEM ( n = 5).

Article Snippet: NEUROD1 TaqMan probe , Thermo Fisher , Hs00159598_m1.

Techniques: Derivative Assay, Marker, Two Tailed Test, Expressing, Comparison, Enzyme-linked Immunosorbent Assay