Fig. 2.

HIF1α is required for the in vitro growth and invasion and the in vivo tumor growth of GBM cells. A, B Knockout of HIF1A retards cell growth. HIF1α protein levels for sgRNA control (WT) or two HIF1A KO clones (KO-1, KO-5) are shown by Western blot confirming successful KO (A), and crystal violet staining (B) depicts difference in cell growth among HIF1A KO or WT U251 cells; 5 × 10³ cells were seeded in a 6-well plate and cultured for 7 days. C, D HIF1A knockout inhibits GBM cell invasion. WT or HIF1A-KO U251 cells were seeded in the upper chambers of a 24-well trans-well plate (1 × 10⁵ cells/well) 4–5 days before staining the invasive cells in the lower chambers (C). Number of invasive cells were counted and are shown as average number of cells/vision (D). E HIF1A knockout inhibits tumor sphere formation. WT and HIF1A-KO U251 were seeded in a low-touch 6-well plate (1 × 10⁵cells/well). After 7 days of culture, tumor spheres formed in serum-free NeuroCult culture medium were photographed (E, upper panel) and total number of tumor spheres/well were counted (E, lower panel). F HIF1A KO U251 cells are prone to apoptosis and sensitive to CoCl₂-induced apoptosis compared with WT U251 cells. Western blot depicts levels of apoptotic proteins cleaved-caspase 3 (cCasp3) and PARP or cleaved PARP, in WT U251 cells or HIF1A KO clones KO1 or KO5, with or without CoCl2 treatment. G, H HIF1A knockout eliminates tumor growth in vivo. NSG mice received orthotopic transplantation of WT or HIF1A KO U251 cells (5 × 10⁴ cells/mouse) and bioluminescence imaging was performed. (G). Survival curves for mice that received WT or HIF1A-KO cells were estimated within the period of 100-day observation (H). I, J Knockdown of HIF1A by mixed HIF1A-sh silencers in primary GBM cells retarded GBM growth in NSG recipients compared with scrambled-sh (Sr-sh) control cells. Survival curves for mice that received high-titer lentivirus-transduced Glio-1 or Glio-2 cells at 5 × 10⁴ cells/mouse were estimated within 100–120 days after intracranial implantation. Error bars represent standard deviation (SD) over three independent experiments, each performed in triplicate. Data shown are representative of three independent experiments