Fig. 2

HK1⁻HK2⁺ liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition. a HK1⁻HK2⁺ liver cancer cells are sensitive to HK2 knockdown-induced growth inhibition. Top panel, HK2 knockdown by DOX-inducible shHK2^DOX after 72 h DOX (25 ng/ml) induction. Bottom panel, after a 7-day exposure to DOX to induce shRNA expression, relative cell numbers were measured using the MTT assay. Data are expressed as “growth inhibition” [100 × (1 − MTT assay values of DOX-treated cells/MTT assay values of untreated cells)]. b Effect of DOX-induced HK2 knockdown on colony formation by HK1⁻HK2⁺ and HK1⁺HK2⁺ liver cancer cells. Triplicate wells of each cell line are shown, stained at 15–20 days after plating. c, d DOX-induced HK2 silencing has limited or no effect on c cell proliferation or d colony formation in HK1⁺HK2⁺ cancer cells, regardless of tissue of origin. e DOX-induced shRNA HK2 knockdown inhibited colony formation by H460HK1^KO/shHK2^DOX isogenic cells but not by isogenic H460/shHK2^DOX cells. f HK2 knockdown inhibits glucose consumption (upper panels) and lactate production (lower panels) in Hep3B/shHK2^DOX, Huh7/shHK2^DOX, and SUM159/shHK2^DOX cells. Triplicate wells of cells were exposed to DOX or vehicle for 72 h. Media were then refreshed and, after 24 h, glucose consumption and lactate production were measured. g HK2 knockdown decreases ¹⁸F-FDG/PET signal in a mouse subcutaneous Hep3B/shHK2^DOX xenograft model. Tumors on the left flank: Hep3B/shCtrl^DOX. Tumors on the right flank: Hep3B/shHK2^DOX. ¹⁸F-FDG PET/CT scans were performed for each mouse before and after mice were switched to a DOX diet for 4 days. h Following the PET scans in panel g, HK2 knockdown was confirmed in tumor extracts by Western blotting. i Cytostasis induced in HK1⁻HK2⁺ liver cancer cells by HK2 knockdown is reversible. Hep3B/shHK2^DOX cells and Hep3B/shCtrl^DOX cells were treated with DOX for 2 days then re-plated in equal numbers in 96-well plates on day 0 in the continued presence of DOX. Cell proliferation during days 0–5 in the presence of DOX was measured (left panel). Cells from the two conditions (+DOX, −DOX) were pooled and cultured in DOX-free medium for 7 days (days 5–12) to allow the degradation of shRNAs in the cells and re-expression of HK2. The cells were then re-plated in 96-well plates and grown in the absence of DOX to generate the growth curves for days 12–16 (right panel). Relative cell proliferation was measured by the MTT assay and presented as fold change compared to the value on day 0. Each data point in panels a, c, and f represents mean ± SD of triplicate samples