Fig. 3: PHB specific mediates low levels of mitochondrial peroxide in GSCs.

a–e The peroxide levels, as indicated by DCFDA fluorescence, were measured by flow cytometry in PHB KO GSCs (a), PHB inducible-KD GSCs (b), GSCs and matched NSTCs (c), GSCs, hNP1 and NHA (d), or PHB KD NHA and hNP1 (e) (mean ± SD, n = 3, biologically independent experiments). f Ctrl or PHB KO 4121 GSCs were treated with vehicle control or NAC (5 mM) for 36 h. Flow cytometry analysis of peroxide by DCFDA staining is shown (left). IB of PHB, SOX2, and Olig2 levels are shown (middle). Quantifications of tumorsphere numbers (2000 cells/well) formed by GSCs are shown (right) (mean ± SD, n = 3, biologically independent experiments). g Co-IF staining of PHB (green) and 8-OHdG (red) in primary GBM specimens are shown. Quantifications of 8-OHdG staining intensity in PHB− (n = 139) and PHB+ (n = 127) cells are shown (right). (Boxplots represent the median, 25th, and 75th percentiles). Nuclei were counterstained with Hoechst (blue). Scale bars, 40 μm (up), 20 μm (down). h and i RNA-seq analysis in control and PHB KO 4121 GSCs. In h, the heatmap shows relative expression levels of genes downregulated or upregulated in the indicated cells (p < 0.05, FC > 2). It includes, respectively, 185 and 740 genes downregulated and upregulated in PHB KO compared to control GSCs. Raw data were log₂ transformed. A relative color scheme used the minimum and maximum values in each row to convert values to colors. In i, overrepresented Gene Ontology (GO) terms from RNA-seq analysis of upregulated gene sets (top) and downregulated gene sets (bottom) in PHB KO compared to control GSCs. j Quantitative real-time PCR (Q-PCR) analysis of mRNA levels of indicated genes in Ctrl and PHB KO 4121 GSCs (mean ± SD, n = 3, biologically independent experiments). k Q-PCR analysis of mRNA levels of indicated genes in PHB KO (top) and PHB inducible-KD (bottom) 4121 GSCs treated with vehicle control or NAC (5 mM) (mean ± SD, n = 3, biologically independent experiments). Welch’s two-sided t-test (a–c, e–g, j, k), Unpaired two-sided Student’s t-test (f, right). Source data are provided as Source Data file.