Journal: PLoS ONE

Article Title: Metabolic regulation of GLP-1 and PC1/3 in pancreatic α-cell line

doi: 10.1371/journal.pone.0187836

Figure Lengend Snippet: Cells were incubated with 16.7mM glucose and hormone concentration in the medium determined by ELISA method. Panel A: Total GLP-1 secretion; Panel B: Active GLP-1 secretion and Panel C: Glucagon secretion. Results are % of control and expressed as MEAN±SEM, n = 4. *p<0.05 compared to control.

Article Snippet: Glucagon quantikine ELISA Kit was from R&D Systems (Adbingdon, UK).

Techniques: Incubation, Concentration Assay, Enzyme-linked Immunosorbent Assay, Control

Journal: PLoS ONE

Article Title: Metabolic regulation of GLP-1 and PC1/3 in pancreatic α-cell line

doi: 10.1371/journal.pone.0187836

Figure Lengend Snippet: Cells were incubated for 16 h in the presence of 5.5mM or 16.7mM, in the additional presence of glucagon, FFA and IL-6; hormone secretion at medium was determined by ELISA method using specific kits. Panel A: GLP-1 Active, Panel B: GLP-1 Total and Panel C: Glucagon. Results are % of control (cells incubated in 5.5mM glucose) and expressed as MEAN±SEM, n = 4. *p<0.05 compared to 5.5mM glucose control; $p<0.05 compare to FFA (5.5mM glucose).

Article Snippet: Glucagon quantikine ELISA Kit was from R&D Systems (Adbingdon, UK).

Techniques: Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Science Advances

Article Title: The expression order determines the pioneer functions of NGN3 and NEUROD1 in pancreatic endocrine differentiation

doi: 10.1126/sciadv.adt4770

Figure Lengend Snippet: ( A ) Generation of the NeuroD1-Venus-flox/flox strain by replacing the CDS of NeuroD1 with the Venus-P2A-Loxp- NeuroD1 CDS-Loxp sequence. Excision of the NeuroD1 CDS is mediated by Pdx1-Cre ER . ( B ) UMAP plots of 10x scRNA-seq data displaying the distribution of cell types (left) from the E15.5 control (Ctrl: WT, Pdx1-Cre ER , or NeuroD1-flox/flox ) and Pdx1-Cre ER ; NeuroD1-flox/flox (CKO) pancreata (right). The control and CKO samples each had two replicates. The cell counts are labeled in brackets. ( C ) Bar plots quantifying the proportions of different cell types across Ctrl and CKO scRNA-seq samples, with two replicates for each condition. Black dots represent the proportion of each cell type in individual replicates. ( D ) NeuroD1 -Venus expression and IF staining of INS and GCG in the E16.5 pancreata. Scale bars, 50 μm. ( E ) Statistical analysis of the proportions of INS + and GCG + cells in E16.5 pancreata from control (Ctrl) and Pdx1-Cre ER ; NeuroD1 -CKO (CKO) embryos. The data are presented as the means ± SEMs. n denotes the number of embryos analyzed. P values were calculated via the Wilcoxon rank-sum test. ( F ) LDA plot showing differences in chromatin accessibility among the Ngn3 -KO, Ngn3 high , NeuroD1 -CKO, and NeuroD1 -Venus + control cells. ( G ) UMAP plots showing snATAC-seq data of the control (Ctrl) and Pdx1-Cre ER ; NeuroD1 -CKO (CKO) cells projected onto the UMAP plot in fig. S2A. The cell counts are labeled in brackets. ( H ) Bar plots displaying the quantified proportions of different cell types across Ctrl and CKO snATAC-seq samples.

Article Snippet: Sections were stained with primary antibodies against FLAG (1:100; CST, 14793S), NGN3 (1:100; DSHB, F25A1B3), INS (1:500; CST, 3014S), GCG (1:200; R&D Systems, MAB1249), MUC1 (1:500; Abcam, ab15481), mCherry (1:100; SICGEN, AB8181-200), and NEUROD1 (1:100; Invitrogen, PA5-47381).

Techniques: Sequencing, Control, Labeling, Expressing, Staining

Journal: Science Advances

Article Title: The expression order determines the pioneer functions of NGN3 and NEUROD1 in pancreatic endocrine differentiation

doi: 10.1126/sciadv.adt4770

Figure Lengend Snippet: ( A ) Generation of the NeuroD1-EGFP-OE strain via the insertion of the CAG-LSL-Flag- NeuroD1 CDS-P2A-EGFP-PolyA sequence at the H11 site. Upon Ngn3-Cre- or Pdx1-Cre ER –mediated excision of the STOP sequence, the CDS of NeuroD1 is expressed under the CAG promoter. ( B ) UMAP plots of Smart-seq3 scRNA-seq data displaying the distribution of cell types from the control (Ctrl: WT, Ngn3-Cre , Ngn3-GFP/+ , or NeuroD1-OE/+ ) and Ngn3-Cre ; NeuroD1 -COE; Ngn3 -KO pancreata. The contribution of the cell source is color-coded. The cell counts are labeled in brackets. ( C ) IF staining of INS and GCG in the E15.5 pancreata (top). Scale bars: 50 μm. Statistical analysis of the proportions of GCG + and INS + cells and the GCG + /INS + cell ratio in the pancreata at E15.5 (bottom). The data are presented as the means ± SEMs. n denotes the number of embryos analyzed. P values were calculated via the Wilcoxon rank-sum test. ( D ) Metaplots and heatmaps illustrating chromatin accessibility in the E13.5 Ngn3 -KO cells, E15.5 NeuroD1 -Venus + control (Ctrl) cells, and GFP high cells from the Ngn3-Cre ; NeuroD1 -COE; Ngn3 -KO pancreata. Each line represents a peak, with colors ranging from blue to red indicating low to high levels of chromatin accessibility at ±2 kb from the center. ( E ) Boxplots showing the intensity levels of the chromatin accessibility of group 1 sites in the E13.5 Ngn3 -KO cells, E15.5 NeuroD1 -Venus + control (Ctrl) cells, and GFP high cells from the Ngn3-Cre ; NeuroD1 -COE; Ngn3 -KO pancreata. P values were calculated via the Wilcoxon rank-sum test. ( F ) Venn diagram showing the overlap of NEUROD1-binding sites in the control (Ctrl) pancreata and NEUROD1-binding sites in the Ngn3-Cre ; NeuroD1 -COE; Ngn3 -KO pancreata.

Article Snippet: Sections were stained with primary antibodies against FLAG (1:100; CST, 14793S), NGN3 (1:100; DSHB, F25A1B3), INS (1:500; CST, 3014S), GCG (1:200; R&D Systems, MAB1249), MUC1 (1:500; Abcam, ab15481), mCherry (1:100; SICGEN, AB8181-200), and NEUROD1 (1:100; Invitrogen, PA5-47381).

Techniques: Sequencing, Control, Labeling, Staining, Binding Assay