Fig 2: Increased VDAC2 in NSTCs couples PFKP on mitochondrion to prevent its cytoplasmic release and inhibits PFKP-mediated glycolysis.a Western blot analyses of VDAC2 expression in GSCs relative to the matched NSTCs derived from human GBMs. COX IV is used as a mitochondrial marker for normalization. b qRT-PCR analysis of VDAC2 expression in GSCs and matched NSTCs (**p < 0.01). c Co-immunoprecipitation analysis showing the interactions between VDAC2 and PFKP. The anti-VDAC2 antibody (upper panel) and anti-PFKP antibody (lower panel) are used for immunoprecipitation, respectively. The input samples of NSTCs are used as positive controls. d Western blot analyses of VDAC2 and PFKP in mitochondrial and cytoplasmic fractions of NSTCs expressing shRNAs against VDAC2 (shVDAC2#1 and #2) or nontargeting shRNA (shNT). COX IV is used as a mitochondrial protein marker and β-tubulin is used as a cytoplasmic protein marker for normalization. Silencing VDAC2 expression reduces the level of PFKP anchored on mitochondrion, but increases PFKP expression in cytoplasm. e PFK enzyme activity in NSTCs expressing shVDAC2 or shNT (***p < 0.001). f Analysis of the relative lactate production in NSTCs expressing shVDAC2 compared to those expressing shNT (***p < 0.001). g Co-immunoprecipitation assay showing the interactions between VDAC2 and PFKP in GSCs expressing VDAC2. The anti-VDAC2 antibody (upper panel) and anti-PFKP antibody (lower panel) are used for immunoprecipitation, respectively. The input samples of GSCs expressing VDAC2 are used as positive controls. h Analysis of PFK enzyme activity in GSCs expressing VDAC2 or control vector (***p < 0.001). i Analysis of the relative lactate production in GSCs expressing VDAC2 or control vector (***p < 0.001)

Fig 3: Disrupting VDAC2 in NSTCs potentiates the acquisition of GSC properties.a Western blot analyses of the GSC markers (CD133, SOX2, and OLIG2) and VDAC2 in NSTCs expressing shVDAC2 or shNT. The levels of GSC markers CD133, SOX2, and OLIG2 are increased in NSTCs expressing shVDAC2 compared with those expressing shNT. b In vitro limiting dilution analysis of the self-renewal capacity of NSTCs expressing shVDAC2 or shNT. Disruption of VDAC2 increases the self-renewal capacity of NSTCs. c, d Representative images of tumor cell clones (c) and quantification of clone formation efficiency (d) in NSTCs expressing shVDAC2 relative to those expressing shNT. Silencing VDAC2 expression promotes the clone formation ability of NSTCs (***p < 0.001). e, f Representative bioluminescent images (e) and the quantification (f) of xenografts derived from NSTCs expressing shVDAC2 or shNT at day 10 and day 20 after tumor cell implantation. Silencing of VDAC2 markedly promotes tumor formation of xenografts derived from NSTCs. p photons, sr steradian (***p < 0.001). g Kaplan–Meier survival analysis of mice bearing xenografts derived from NSTCs expressing shVDAC2 or shNT. Silencing of VDAC2 reduces the survival of tumor-bearing mice. n = 5/group. h, i Quantification of the level of VDAC2 (h) or GSC marker SOX2 (i) in GBM xenografts derived from NSTCs expressing shVDAC2 or shNT by IHC staining (***p < 0.001)

Fig 4: PFK inhibitor compromises the effect of VDAC2 disruption on glycolytic reprogramming and GSC phenotypic transition.a Analysis of PFK activity in shVDAC2-expressing NSTCs treated with or without PFK inhibitor CTZ. CTZ treatment impairs the effect of VDAC2 silencing on the promotion of PFK activity (**p < 0.01). b Analysis of lactate production in shVDAC2-expressing NSTCs treated with or without PFK inhibitor CTZ (**p < 0.01). c Western blot analyses of the GSC markers CD133, SOX2, and OLIG2 in shVDAC2-expressing NSTCs treated with or without PFK inhibitor CTZ. d In vitro limiting dilution assay of shVDAC2-expressing NSTCs treated with or without PFK inhibitor CTZ. Inhibition of PFKP by CTZ effectively compromises the VDAC2 disruption-induced self-renewal of NSTCs. e Quantification of clone formation efficiency of shVDAC2-expressing NSTCs treated with or without PFK inhibitor CTZ (***p < 0.001)

Fig 5: VDAC2 expression inversely correlates with glioma grades and predicts outcome of glioma patients.a, b Representative IHC images (a) and quantification of IHC scores (b) of VDAC2 in human gliomas with grade II, grade III, and grade IV (GBM) (**p < 0.01, *p < 0.05). c VDAC2 mRNA level in human gliomas with grade II, grade III, and grade IV (GBM) from the TCGA database (***p < 0.001). d Kaplan–Meier analysis of VDAC2 expression and overall survival of glioma patients (n = 60) from Southwest Hospital. e, f Kaplan–Meier analysis of VDAC2 expression and overall survival of GBM patients (n = 515) from TCGA database (e) or those (n = 188) from Gravendeel database (f). X-tile software is used to determine the cutoff point of VDAC2 expression for the survival analysis