Fig. 2.

Partial loss of mtDNA in iNHAs through shRNA-mediated silencing of TFAM promoted tumorigenesis. (A) Western blotting of TFAM and 2 key subunits of OXPHOS complexes, COXII and NDUFB2, in TFAM knockdown iNHAs and shRNA controls, with actin probed as a loading control. (B) Partial depletion of mtDNA greatly enhanced the production of L-lactate (~2-fold) in iNHAs relative to corresponding controls. (C) Stable knockdown of TFAM resulted in a ~30–40% decrease in relative mtDNA copy number in iNHAs compared with scrambled controls. (D) TFAM knockdown cells proliferated at a lower rate compared with controls. (E) NSG mice carrying TFAM-deficient iNHA orthotopic xenografts displayed enhanced tumorigenicity with a significantly worse survival time compared with the control group (n = 5/group). (F) Representative H&E staining of TFAM-deficient xenograft tumors showed some pathological characteristics of DIPG/pediatric high-grade astrocytoma compared with the control. Scale bar: 0.1 mm. (G) Quantitative PCR assays demonstrating a significantly lower mtDNA number in TFAM knockdown xenografts than iNHA non-targeting control (NTC) cells. *P < 0.05; **P < 0.01; ***P < 0.001.