Skip to main content
. 2021 Mar 8;6(5):e141553. doi: 10.1172/jci.insight.141553

Figure 2. Inhibiting replication fork progression promotes endocrine differentiation independent of apoptosis.

Figure 2

(A) Quantification of NKX6.1 or C-peptide–positive cells on day 15 (n = 3) and on day 27 (B) with different concentrations of APH. (C) Quantification of C-peptide–positive cells in control and 1 μM APH on day 27 (n = 12). Two-tailed paired t test with ****P < 0.0001. (D) Quantification of C-peptide/glucagon-positive cells (n = 6) or C-peptide/somatostatin-positive cells (n = 5). (E) Immunostaining of APH-treated clusters for C-peptide, NKX6.1, glucagon, and somatostatin on day 27. Scale bar: 100 μm. Insets: 6.25× higher magnification. (F) A schematic diagram indicates APH duration. (G) Flow cytometry quantification of C-peptide–positive and C-peptide/NKX6.1-positive cells in indicated conditions on day 27 (n = 3). One-way ANOVA test, *P < 0.05; **P < 0.01; ***P < 0.001. (H) Flow cytometry profile of cell cycle progression on days 15, 18, 20, and 27 without and with APH, indicated by the percentage of cells positive for C-peptide and EdU. (I) Quantification of total C-peptide–positive cells on days 20 and 27. One-way ANOVA test, *P < 0.05; **P < 0.01. (J) Total cell numbers on days 20 and 27. (K) Immunostaining on day 27 clusters for TUNEL and C-peptide (6.25× higher magnification in inset). Scale bar: 100 μm. Quantification of TUNEL-positive and C-peptide–negative cells on day 27. (L) Quantification of C-peptide– and TUNEL-positive cells on days 17, 20, and 27 (n = 3). (M) Timing of insulin-GFP expression. (N) NGN3 expression determined by quantitative real-time PCR (RT-PCR). (O) Schematic diagram of experimental conditions. (P) Flow cytometry analysis of day 20 cells for NKX6.1 and C-peptide when day 13 pancreatic progenitors were cultured in progenitor medium with and without APH (day 20 control no APH: 41.5% ± 13.44%; day 20 progenitor medium no APH: 13% ± 1.4%; day 20 progenitor medium plus APH: 52% ± 9.9%, n = 2). (Q) Quantitative RT-PCR for NGN3 expression of day 15 cells cultured in basal endocrine medium with and without APH (n = 3). Two-tailed unpaired t test, *P < 0.05. DBZ, γ-secretase inhibitor; T3, thyroid hormone; LDN, LDN193189; KGF, keratinocyte growth factor.