Figure 4

Cell-cycle arrest acts downstream to ERK signaling in PCV ECs

(A–E) Confocal images of 48-hpf Tg(mrc1a:EGFP) embryos treated with DMSO (A), roscovitine (B), SL327 (C), or roscovitine+SL327 (D) showing partial rescue of lymphovenous sprouting following roscovitine + SL327 (D) treatment, and quantified in (E) (n_DMSO = 12, n_rosco = 13, n_SL327 = 17, n_rosco+SL327 = 16). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Blue arrowheads in (A)–(D) point to PCV sprouts.

(F–L) Confocal images of Tg(fli1:dsRed2) embryos treated with DMSO (F and H) or SL327 (G, I, J, and K) stably expressing lyve1:p21-EGFP (H and I) or injected with lyve1:p53-EGFP (J) or lyve1:p27-EGFP (K), showing partially restored lymphovenous sprouting, and quantified in (L) (n_DMSO = 7, n_SL327 = 26, n_{lyve1:p21-EGFP+DMSO} = 7, n_{lyve1:p21-EGFP+SL327} = 15, n_{lyve1:p27-EGFP+SL327} = 17, n_{lyve1:p53-EGFP+SL327} = 25). Statistical analysis was performed using one-way ANOVA followed by Tukey’s multiple comparisons test. Light-blue arrowheads in (H)–(K) point to GFP+ ECs in the dorsal PCV, white arrowheads denote GFP+ lymphovenous sprouts, and co-localization channel is shown in yellow.

(M–P) Confocal images of 34-hpf Tg(mrc1a:EGFP) embryos showing reduced expression of p27 (M and N) and p53 (O and P) following SL327 treatment, and quantified in (Q and R) (n_{DMSO p27} = 7, n_{SL327 p27} = 8, n_{DMSO p53} = 9, n_{SL327 p53} = 6). Data show mean ± SEM (unpaired t test). Co-localization channel is shown in yellow. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; ns, not significant. Scale bars: 30 μm