Fig. 2. Dislocated cap cells exhibit altered cell cycle progression and are apoptotic.

(A) s-ship-eGFP marks cap cells. Phalloidin (F-actin marker, red) was used as a contrast stain. Inset depicts a dislocated cap-in-body cell cluster still attached to the outer cap cell layer. DAPI (blue)=nuclei. Quantification shows TEB composition. Scale bar=50μm. n=5 mice, 25 TEBs. (B) Lineage-traced TEBs at t1 used to assess cap-in-body cell clonality. Arrowheads=α-SMA⁺, GFP⁺ single cap-in-body cells. Scale bar=50μm. (C) BrdU⁺ cells (green) indicate replicative cells. Basal/cap and cap-in-body cells are marked with α-SMA (red). DAPI (blue)=nuclei. Arrowhead=BrdU⁺ cap-in-body cell still attached to the outer cap cell layer. Scale bar=50μm. The right panel shows quantification. n=5 mice, 12 TEBs. (D) pH3⁺ cells (green) indicate mitotic cells. Basal/cap and cap-in-body cells are marked with α-SMA (red). Almost no cap-in-body cells were mitotic (inset, arrowhead). DAPI (blue)=nuclei. Scale bar=50μm. The right panel shows quantification. n=5 mice, 18 TEBs. (E) cc3⁺ cells (red) are apoptotic. Cap and cap-in-body cells are marked with s-ship-eGFP. Inset, arrowhead=magnified view of apoptotic cap-in-body cell clusters. DAPI (blue)=nuclei. Scale bar=50μm. The right panel shows quantification. n=5 mice, 25 TEBs. (F) TEBs from mice treated with z-VAD-FMK. cc3⁺ cells (red) indicate apoptotic cells. Cap and cap-in-body cells are marked with s-ship-eGFP. DAPI (blue)=nuclei. Scale bar=50μm. The right panel shows quantification. n=3 mice, 10 TEBs each (vehicle, treated).

Error bars=mean ± SD. See also Fig. S2.