Figure 2.

Defining the duration of G₁, S, and G₂/M in β-cells in vivo. A: GKA increases BrdU incorporation in β-cells 13 h after administration. B: Double labeling with thymidine analogs to estimate duration of S phase. Left panels: Representative image from a mouse killed 2 h after IdU injection. CldU was injected 3 h prior to IdU. Arrows point to a CldU⁺ β-cell (left), CldU⁺ IdU⁺ double positive β-cell (middle), and IdU⁺ β-cell (right). Right panel: Graph depicting the percent of CldU⁺ β-cells that costain for IdU, when IdU is injected at different times after CldU. C: BrdU-PH3 staining to estimate duration of G₂ phase. Left panels: Representative immunofluorescence image. BrdU was injected 6 h prior to sacrifice. Arrowheads point to BrdU⁺ PH3⁺ mitotic β-cells. Arrows point to BrdU⁺ PH3⁻ β-cells. Right panel: Percent of BrdU⁺ β-cells that stain for PH3, at different time points after injection of BrdU, reflecting the duration of G₂ as described in text. D: FACS analysis of dissociated islet cells from 3-week-old CyclinB1-GFP mice (n = 4 mice, pooled), costained for GFP and Ki67. A total of 2.8% of islet cells are Ki67⁺, 1.7% of islet cells costain for Ki67 and GFP, marking cells in S-G₂ phases of the cell cycle, and 1.1% are Ki67⁺GFP⁻, representing G₁ cells. *P < 0.05. NS, not significant.