FIGURE 2.
HK2 promotes CSC stemness and tumor growth. (A‐B) Immunoblotting analyses of H1048 (A) and H69 (B) cells with or without HK2 depletion were performed with the indicated antibodies. (C) The sphere formation ability of H1048 cells or H1048 cells with or without HK2 depletion in their first and the second passage was examined. Representative images were displayed (top), and the diameters and number of spheres were determined (bottom). Data shown are the mean ± SD (n = 3). ***, P < 0.001. Scale bars: 100 μm. (D‐E) Immunoblotting analyses of H1048 (D) and H69 (E) cells with or without HK2 overexpression were performed with the indicated antibodies. (F) The sphere formation ability of H1048 cells with or without HK2 overexpression in their first and the second passage was examined. Representative images were displayed (top), and the diameters and number of spheres were determined (bottom). Data shown are the mean ± SD (n = 3). **, P < 0.01, ***, P < 0.001. Scale bars: 100 μm. (G) Immunodeficient mice (n = 10) were subcutaneously inoculated with an equal number (2 × 106) of H1048 cells with or without HK2 shRNA expression. Tumor sizes (top) and volumes (bottom) were measured and calculated. Data represent the means ± SD of ten mice per group. ***, P < 0.001. (H) IHC staining of mouse tumor tissues derived from H1048 cells with or without HK2 shRNA expression was performed with the indicated antibodies. Representative images were displayed (left), and the IHC scores were calculated (right). Data shown are the mean ± SD (n = 5). ***, P < 0.001. Scale bars: 100 μm. (I) Different numbers of H1048 cells with or without HK2 overexpression were subcutaneously injected into NOD/SCID mice (n = 10). Images of the tumors are shown on the left. Scale bar (right): 1 cm. Stem cell frequencies were estimated as the ratio 1/x with the upper and lower 95% confidence intervals (middle). ELDA analysis was performed (right). Abbreviations: HK2, hexokinase 2; CSC, cancer stem cell; SD, standard deviation; IHC, immunohistochemistry; ELDA, extreme limiting dilution analysis