Figure 5
CS knockdown causes severe defects in mitochondrial respiration, but increases glucose uptake and upregulates glycolytic metabolism.
(A) Δψm assay of CS knockdown cells. Indicated cells were stained with TMRM and then analyzed using a flow cytometer. (B) ROS assay of CS knockdown cells. Cells were treated with CM-H2DCFDA and then analyzed using a flow cytometer. (C) ATP assay of CS knockdown cells. Total cell extracts prepared from indicated cells were subjected to ATP assay using ATP Bioluminescence Assay Kit CLSII. (D) Western blotting of Glut-1, Glut-3, p-AMPK, p-p38 MAPK and p-ERK1 protein in CS knockdown cells. Total proteins isolated from indicated cells were blotted with antibodies as labeled. (E) Glucose uptake assay of CS knockdown cells. Cells were loaded with 2-NDBG and then analyzed using a flow cytometer. (F) Western blotting of proteins involved in the TCA cycle, electron transport chain from complex I to V and glycolysis in CS knockdown cells. Total proteins isolated from indicated cells were blotted with antibodies as labeled. (G) Lactate dehydrogenase assay of CS knockdown cells. Cells were assayed for lactate dehydrogenase activity using the CytoTox 96® Non-Radioactive Cytotoxicity Assay. (H) pH of conditioned media cultured with CS knockdown cells. Cells were cultured until confluent and then incubated in fresh media. The color and pH of the conditioned media were examined. The plotted data were averaged from three independent experiments and the bars represent mean ± SD. The quantity of β-actin serves as a loading control.
