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. 2018 Jul 26;175(17):3548–3562. doi: 10.1111/bph.14423

Figure 2.

Figure 2

Effect of different treatments on the action of Shh on the adherens junction. (A) mESCs were transfected with Gli1 siRNA or NT siRNA (20 nM) for 24 h prior to Shh treatment for 24 h. The levels of MMP isotypes and MT1‐MMP mRNA expressions were measured by real‐time PCR. The data are expressed as mean ± SEM for five experiments. *P < 0.05 versus control of MMP2; # P < 0.05 versus Shh + NT siRNA of MMP2; & P < 0.05 versus control of MMP9; $ P < 0.05 versus Shh + NT siRNA of MMP9. (B) mESCs were treated with Shh for various time (0–24 h), and the secretion of MMP2 and MMP9 into the medium and the expression of MMP2 and MMP9 were detected by Western blotting. β‐Actin was used as the loading control to normalize the levels of total MMP2/9. The data are expressed as mean ± SEM for five independent experiments. *P < 0.05 versus 0 h of secreted MMP2; & P < 0.05 versus 0 h of MMP2; $ P < 0.05 versus 0 h of secreted MMP9; + P < 0.05 versus 0 h of MMP9. (C) mESCs were pretreated with cyclopamine for 30 min and transfected with Gli1 siRNA or NT siRNA for 24 h prior to Shh treatment for 24 h. The secretion of MMP2 and MMP9 into the medium and the expression of MMP2 and MMP9 were detected by Western blotting. β‐Actin was used as the loading control to normalize the levels of total MMP2/9. The data are expressed as mean ± SEM for five independent experiments. *P < 0.05 versus control of secreted MMP2; **P < 0.05 versus Shh of secreted MMP2; & P < 0.05 versus control of MMP2; && P < 0.05 versus Shh of MMP2; $ P < 0.05 versus control of secreted MMP9; $$ P < 0.05 versus Shh of secreted MMP9; + P < 0.05 versus control of MMP9; ++ P < 0.05 versus Shh of MMP9. (D) mESCs were pretreated with Gant 61 (Gli inhibitor; 10 μM) or MMP inhibitor (10−7 M) prior to Shh treatment for 24 h. Complexes were immunoprecipitated from cell lysate with an anti‐E‐cadherin antibody and blotted with antibodies against E‐cadherin and β‐catenin. The data are expressed as mean ± SEM for five independent experiments. *P < 0.05 versus control; # P < 0.05 versus Shh. (E) mESCs were pretreated with Gant 61 or MMP inhibitor prior to Shh treatment for 24 h. In situ PLA assay result demonstrates the interaction between β‐catenin and E‐cadherin in mESCs (Green dot, β‐catenin/E‐cadherin; Red, nuclear). Scale bar = 20 μm. Ctrl, control; NT, non‐targeting; ROD, relative optical density.