Fig. 3.

Membrane cholesterol promoted the proliferation of GH3 cells by activating the Hh signaling pathway. a A schematic showing the process via which cholesterol promotes cell proliferation by activating the Hh signaling pathway. The Mβ-CD/CHO complex (CHO) is an agonist and vismodegib is an antagonist that binds and modulates the activity of SMO. SMO RNAi was used to knock down SMO expression. Forskolin blocked signaling by elevating cAMP levels, which increased PKA activity. b Vector GH3 cells were treated with Mβ-CD (20 μg/ml) or the Mβ-CD/CHO complex (20 μg/ml) for 48 h. Protein expression levels of PKA, SUFU, GLI1, BCL2 and CCND1 in the four groups (Vector, SCP2-OE, Vector + Mβ-CD and Vector + Mβ-CD/CHO) were assessed by Western blotting. c SCP2-OE GH3 cells were treated with vismodegib (VIS, 50 μM) and Vector GH3 cells were treated with the Mβ-CD/CHO complex (20 μg/ml) in the presence or absence of vismodegib (50 μM) for 0, 24, 48 or 72 h. Cell proliferation in the four groups (SCP2-OE, SCP2-OE + VIS, Vector + Mβ-CD/CHO and Vector + Mβ-CD/CHO + VIS) at different time points was assessed by a CCK-8 assay (n = 6, ± SEM). Protein expression levels of PKA, SUFU, GLI1, BCL2 and CCND1 at 48 h were assessed by Western blotting. Cell apoptosis and the cell cycle at 48 h were analyzed using flow cytometry (n = 3, ± SEM). d SCP2-OE and wild-type GH3 cells were infected with negative control vector (NC) or SMO shRNA vector (SMO-2 and SMO-3) for 48 h, and the transfected wild-type GH3 cells were incubated with the Mβ-CD/CHO complex (CHO, 20 μg/ml) for another 0, 24, 48 or 72 h. Cell proliferation in the six groups (SCP2-OE + NC, SCP2-OE + SMO-2, SCP2-OE + SMO-3, NC+ CHO, SMO-2 + CHO and SMO-3 + CHO) at different time points were assessed by a CCK-8 assay (n = 6, ± SEM). Protein expression levels of PKA, SUFU, GLI1, BCL2 and CCND1 at 48 h were examined by Western blotting. Cell apoptosis and the cell cycle at 48 h were analyzed using flow cytometry (n = 3, ± SEM). e SCP2-OE GH3 cells were treated with forskolin (FOR, 25 μM) and Vector GH3 cells were treated with the Mβ-CD/CHO complex (20 μg/ml) in the presence or absence of forskolin (25 μM) for 0, 24, 48 or 72 h. Cell proliferation in the four groups (SCP2-OE, SCP2-OE + FOR, Vector + Mβ-CD/CHO and Vector + Mβ-CD/CHO + FOR) at different time points was assessed by a CCK-8 assay (n = 6, ± SEM). Protein expression levels of PKA, SUFU, GLI1, BCL2 and CCND1 at 48 h were assessed by Western blotting. Cell apoptosis and the cell cycle at 48 h were analyzed using flow cytometry (n = 3, ± SEM). An unpaired t-test was used to assess statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001