Fig 2: 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.

Fig 3: P4HB maintains GSCs stemness via the Wnt/β-Catenin signaling pathway.A Relative mRNA expression levels of the indicated signaling pathways in 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentiviral vectors. Data are presented as means ± SD, n = 3, t-test. B–D Western blot analyses of P4HB, β-catenin, and Cyclin D1 protein levels in X01, 448, 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, with α-tubulin used as a loading control. E Western blot analysis of P4HB and β-catenin in isolated nuclear or cytosolic lysates from 83 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus. Lamin B1 and β-actin were used as markers for the nucleus and cytoplasm, respectively. F Heatmap analysis of different gene expression clusters in X01 shCtrl and shP4HB GSCs by RNAseq analysis. G Differentially expressed genes were mapped by fold change between the shCtrl and shP4HB groups. H Western blot analysis of P4HB and LRP6 protein levels in X01 GSCs infected with shCtrl or shP4HB-1 and shP4HB-2 lentivirus, Vinculin was used as a loading control. I Western blot analysis of β-catenin, CyclinD1, and LRP6, P-LRP6 protein levels 3 in X01 and 83 GSCs treated with 10 μM ICG001, 25 ng/ml recombinant P4HB protein (rP4HB). GAPDH was used as a loading control.

Fig 4: P4HB enrichment in GSCs is associated with stemness and poor prognosis.A–C Kaplan–Meier survival curves for patients with glioma from the TCGA, CGGA, and REMBRANDT datasets, stratified by high and low expression of P4HB. P-values were calculated using the log-rank test and Wilcoxon test. D UMAP plot showing the clustering of different cell types within the GBM patients’ tissues, and the expression levels of P4HB across different cell clusters. E–G Relative mRNA expression levels of P4HB in X01, 448, and 83 GSCs, respectively, were measured by qRT-PCR after shRNA-mediated knockdown of P4HB. Data are presented as means ± SD, n = 3, ***P < 0.001, t-test. H Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in X01 GSCs after shRNA-mediated knockdown of P4HB, α-tubulin was used as a loading control. I Limit-dilution assay for sphere-forming capacity in X01 GSCs following shRNA-mediated knockdown of P4HB. Log fraction without spheres is plotted against the number of initial cells per well. **P < 0.01, ***P < 0.001, t-test. J Western blot analysis of P4HB, NESTIN, and SOX2 protein levels in 448 GSCs after shRNA-mediated knockdown of P4HB, α-tubulin was used as a loading control. K Limiting dilution assay for sphere-forming capacity in 448 GSCs following shRNA-mediated knockdown of P4HB. **P < 0.01, ***P < 0.001, t-test. L Western blot analysis of P4HB, NESTIN, and CD44 protein levels in 83 GSCs after shRNA-mediated knockdown of P4HB, α-tubulin was used as a loading control. M Limiting dilution assay for sphere-forming capacity in 83 GSCs following shRNA-mediated knockdown of P4HB. **P < 0.01, ***P < 0.001, t-test.

Fig 5: P4HB inhibition impedes GSCs progression and oncogenicity by inducing apoptosis.A–C Cell proliferation assay for X01, 448, and 83 GSCs infected with shCtrl, shP4HB-1, and shP4HB-2 lentiviral vectors. Data are presented as means ± SD, n = 3, *P < 0.05, ***P < 0.001, t-test. D Kaplan–Meier survival curves of mice implanted with 83 GSCs infected with shCtrl or shP4HB lentivirus (n = 6 in each group, 1 × 104 GSCs/mouse), P < 0.0001, log-rank test. E H&E staining of the whole brain slides derived from the mice implanted with 83 GSCs infected with shCtrl or shP4HB lentivirus. F–H Flow cytometry analysis of Annexin V/PI staining for cellular apoptosis analysis of X01, 448, 83 GSCs infected with shCtrl or shP4HB lentivirus. I–K Western blot analysis of PARP, C-PARP, P4HB, Caspase-3, CC3, and BCL2 protein levels in X01, 448, 83 GSCs infected with shCtrl or shP4HB lentivirus, α-tubulin was used as a loading control.