Fig. 3.

Dose-dependent effects of the JNK inhibitor SP600125 in Hydra. (A) Tissue lysates from SP600125-treated Hydra show a reduced level of c-Jun phosphorylation in a JNK assay kit (Cell Signaling Technology), whereas the level of phosphorylated Hydra ERK is unaffected in these lysates. (B) SP600125 treatment decreases JNK phosphorylation in Hydra as visualized by using an anti-phospho-JNK immunoblot. (C) Tissue lysates from SP600125-treated Hydra show a constant level of ATF2 phosphorylation in a p38 MAP kinase assay kit (Cell Signaling Technology). Anti-actin immunoblotting was the loading control in all Western blottings. (D and F) Continuous SP600125 treatment of freshly detached and daily fed Hydra and head regenerates leads to dose-dependent inhibition of bud formation and tentacle evagination during regeneration. Data points represent the mean ± SEM from 3 independent experiments; 12 polyps per time point were assayed in each experiment. (E) Changes in the diameters of initially circular patches of carbon-labeled ectodermal epithelial cells of SP600125-treated buds compared with DMSO-treated control buds; oral-aboral refers to the mouth-foot axis of the mother polyp, distal-proximal to the perpendicular axis of evaginating buds. Bars represents mean ± SD from 5 to 14 samples. (G) Treatment with 25 μM SP600125 during Hydra head regeneration did not affect expression of hvwnt5, hvwnt8, and hydsh mRNA as revealed by in situ hybridizations.