Figure 6. HK1 silencing induces severe defects in mitochondrial respiration but increases glucose uptake and promotes glycolytic activity.

(A) Δψ_m assay of HK1-knocked down cells. Cells as indicated were treated with TMRM and then fluorescence intensities were examined and analysed by a flow cytometer. (B) ROS assay of HK1-silenced cells. Cells as indicated were stained with CM-H2DCFDA and then fluorescence intensities were inspected and analysed by a flow cytometer. (C) ATP assay of HK1-knocked down cells. Total cell extracts prepared from cells as indicated were subjected to an ATP assay using the ATP Bioluminescence Assay Kit CLSII according to the manufacturer’s instructions. (D) Western blotting of Glut-1, Glut-3, p-AMPK and p-p38 MAPK expression in HK1-silenced cells. Total protein extracts isolated from cells as indicated were probed with antibodies specific for proteins as labelled. The β-actin level serves as the loading control for total proteins. (E) Glucose uptake assay in HK1-inhibited cells. Cells as indicated were treated with 2-NDBG and then fluorescence intensities were examined and analysed by a flow cytometer. (F) Western blotting of proteins involved in the TCA cycle and glycolysis in HK1-knocked down cells. Total protein extracts prepared from cells as indicated were blotted with antibodies specific for proteins as labelled. (G) Lactate dehydrogenase assay of HK1-silenced cells. Cells as indicated were subjected to a lactate dehydrogenase activity assay using the Lactate Dehydrogenase Assay Kit according to the manufacturer’s procedures. (H) pH measurement in conditioned medium of HK1-inhibited cells. Cells as indicated were cultured until confluent and then incubated in fresh medium. The color and pH value of the conditioned media were imaged and measured. The plotted data are averaged from three independent experiments and the bars represent mean ± SD.