Fig. 6

Temporal requirement of JNK signaling for finfold regeneration. (A) Experimental scheme of inhibitor shift assay to determine the temporal requirement of JNK signaling during regeneration. The JNK inhibitor (5 μM) or DMSO (0.05 %) was applied at different time windows, and proliferating cells were labeled by BrdU at 12–18 hpa. (B) Representative results of BrdU labeling after temporal inhibition of JNK signaling. The regeneration-dependent cell proliferation (bracketed regions) was not significantly reduced by the early inhibitor treatment during 0–3 hpa. (C) Quantification of cell proliferation. In all cases except the inhibitor application at 0–3 hpa, the inhibitor treatment significantly reduced the regeneration-dependent cell proliferation. Error bars depict SEM. *P < 0.05; **P < 0.01; N.S., not significant. (D) Insensitivity of early Jun phosphorylation to the MAPK inhibitors. The early Jun phosphorylation at 0.5 hpa was examined after treatment with inhibitors of JNK, p38, MEK1/2, or their combination. All inhibitors were used at 10 μM, starting the treatment 1 h before amputation. The control DMSO was added at 0.1%. None of the inhibitors had a significant inhibitory effect on early Jun phosphorylation. We also tested another p38 inhibitor, SB203580, and obtained the same result. Note that the epithelial tissue contraction by the actin cables around the stump was neither disrupted by these inhibitors. The scale bar represents 50 μm in (B) and (D).