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. 2020 Jul 14;9:e55829. doi: 10.7554/eLife.55829

Figure 6. RUNX2 and GLI1 are downstream target of pERK1/2.

(A–D) Time-dependent effect of U0126 in regulating pERK1/2, RUNX2 and GLI1 expressions has been analyzed by western blotting in the proteins extracted from E15.5 Rab23-/- CD cells. These cells were treated with or without U0126. Relative pERK1/2 levels in Rab23-/- CD cells are drastically reduces upon U0126 exposure at 6 hr and also shows downregulation of pERK1/2 at 24 and 48 hr upon U0126 exposure compare to corresponding untreated Rab23-/- CD cells (A, B). RUNX2 expression significantly reduces in Rab23-/- CD cells only after 48 hr of U0126 exposures (A, C). GLI1 expression in Rab23-/- CD cells sequentially downregulated upon U0126 exposures at 6, 24 and 48 hr (A, D) (n = 3 blots for each time points). (E–H) Time-dependent effect of cyclopamine (10 µM) and combined effect of cyclopamine (10 µM) and U0126 (5 µM) in regulating GLI1, pERK1/2, and RUNX2 expressions has been analyzed by western blotting in the proteins extracted from E15.5 Rab23-/- CD cells. These cells were treated with or without cyclopamine and combined cyclopamine and U0126 for 6, 24 and 48 hr. Relative GLI1 levels are significantly reduces at every time points upon cyclopamine treatment and show further gradual reduction of GLI1 upon combined treatment of cyclopamine with U0126 (E–H). Relative pERK1/2 levels in Rab23-/- CD cells are drastically reduces upon combined cyclopamine and U0126 exposure at 6 hr (E, F) and also shows downregulation of pERK1/2 at 24 and 48 hr upon combined cyclopamine and U0126 exposure compare to corresponding untreated Rab23-/- CD cells (E, G, H). However, pERK1/2 level remain unchanged at every time points upon cyclopamine treatment alone. RUNX2 expression significantly reduces in Rab23-/- CD cells after 24 hr by combined treatment of cyclopamine and U0126 (E, G, H), and only significantly reduces after 48 hr upon cyclopmine treatment alone. (n = 3 blots for each time points). Data represented as mean ± SD, paired Student’s t-test was used. Statistical significance was defined as a p-value <0.05 (*), p-value <0.005 (**), p-value <0.001 (***).

Figure 6.

Figure 6—figure supplement 1. GLI1 is a downstream target of pERK1/2 Immunohistochemical staining shows GLI1 expression in Rab23-/- and U0126-treated Rab23-/- lambdoid sutural sections at 24 and 48 hr of in vitro culture of E18.5 samples.

Figure 6—figure supplement 1.

Blue arrows indicate GLI1 expression. Counter stained with nuclear fast red. pb: parietal bone, ipb: interparietal bone. Dotted lines indicate bone edge. Scale bar: 50 µm.