Fig. 2: PHB promotes GSC self-renewal and tumor progression.

a PHB knockout GSCs were generated by CRISPR-Cas9 system. IB showing levels of indicated proteins in Ctrl and PHB KO GSCs. b GSCs stably transduced with Tet-on-inducible-shPHB were treated with DOX (100 ng/ml) or vehicle control. Expression of indicated proteins were assessed by IB. c, d Quantifications of tumorsphere numbers (2000 cells/well) formed by Ctrl or PHB KO GSCs (c, right), or control or PHB inducible-KD GSCs (d) (mean ± SD, n = 4, biologically independent experiments). Representative images of tumorspheres are shown (c, left). Scale bar, 100 μm. e In vitro extreme limiting dilution assays (ELDAs) show that PHB KO decreased the frequency of tumorsphere formation in GSCs. f Inducible-KD of PHB had limit effects on cell growth of NHA (left) and hNP1 (right), as measured by cell viability assay. g` Ctrl or PHB KO 4121 GSCs (5 × 10⁴/mouse) were implanted into the brains of nude mice (nu/nu, n = 6). Kaplan–Meier survival curve of mice is shown (Log-rank Mantel–Cox test). IB showing the efficiency of PHB KO in xenografts (top). h, i GSCs transduced with Tet-on-inducible-shPHB and Luciferase reporter were implanted into the brains of nude mice (nu/nu). Mice were treated with vehicle control or DOX (2 mg/ml in drinking water) to induce expression of shPHB from day 0. GBM xenografts (4121 GSCs) were tracked by bioluminescence (h, left). Bioluminescent quantification of tumor growth is shown (h, right) (mean ± SEM, n = 6, biologically independent mice). Kaplan–Meier survival curves of mice are shown (i) (4121 GSCs, n = 8; 387 GSCs, n = 7; Log-rank Mantel-Cox test). IB showing the efficiency of PHB knockdown in the xenografts (top). j The in vivo serial transplantation assay shows that PHB KO inhibits GSC self-renewal in vivo. Kaplan–Meier survival curves of mice implanted with indicated GSCs (4121) are shown (top) (n = 5; Log-rank Mantel–Cox test). Summary of mice medium survival in the serial transplantation assay is shown (bottom). k Co-IF staining of PHB (green) and SOX2 (red) in GBM xenografts derived from Ctrl or PHB KO GSCs (4121). Quantifications of SOX2+ cells are shown (right) (mean ± SD, images n = 8, from 4 biologically independent samples). Nuclei were counterstained with Hoechst (blue). Scale bars, 40 μm. Unpaired two-sided Student’s t-test (c–e, h), Welch’s two-sided t-test (k). Source data are provided as Source Data file.