Fig. 4: Pharmacological inhibition of P4HB triggers apoptosis in GSCs with Wnt/β-Catenin signaling response.

A Schematic illustration of CCF642 inhibiting P4HB reductase enzyme activity by sheltering the active enzyme site. B Cell proliferation assays for X01 GSCs treated with different doses of CCF642 after 24-h treatments. Data are presented as means ± SD, n = 3, ***P < 0.001, t-test. C Western blot analysis of stemness markers NESTIN and SOX2 protein level in X01 GSCs after treatment with 1 μM CCF642 for 48 h, Vinculin was used as a loading control. D Limit-dilution assay for sphere-forming capacity in X01 GSCs treated with 0.1 μM CCF642. ***P < 0.001, t-test. E Flow cytometry analysis of cellular apoptosis of X01 GSCs treated with different doses of CCF642 by Annexin V/PI staining. F Western blot analysis of PARP, C-PARP, P4HB, Caspase-3, CC3, and BCL2 protein levels in X01 GSCs treated with 1 μM CCF642 under different time points, α-tubulin was used as a loading control. G Western blot analysis of PARP, C-PARP, P4HB, Caspase-3, and CC3 protein levels in X01 GSCs treated with 1 μM CCF642 in the presence of 25 μM V-ZAD-FMK, α-tubulin was used as a loading control. H Cell proliferation assays for X01 GSCs treated with 1 μM CCF642 along with apoptosis inhibitor V-ZAD-FMK (25 μM). Data are presented as means ± SD, n = 3, ***P < 0.001, t-test. I–K Western blot analysis of P4HB, β-catenin, CyclinD1, and LRP6 protein levels in X01, 448, 83 GSCs treated with 1 μM CCF642, α-tubulin was used as a loading control. L Western blot analysis of β-catenin protein level in fractionated nuclear or cytosolic lysates from 83 GSCs treated with 1 μM CCF642, α-tubulin, and LaminB1 was used as loading control, respectively.