Fig. 1.

Downregulation of TFAM inhibits mitochondrial respiration and promotes glycolysis in colon cancer cells. a The expression of TFAM was downregulated in stable TFAM knockdown DLD1 and HCT116 cells. Protein lysates from control and knockdown cells were analyzed by western blot. Two different shRNA targeting sequences were used for knocking down TFAM. b, c Control and TFAM knockdown DLD1 (b) and HCT116 (c) cells were subjected to the Mito stress test to obtain OCR measurements using the Seahorse XF96 Extracellular Flux analyzer. Results were quantified and the relative levels of OCR associated with basal and maximal respiration, ATP production and reserved capacity were calculated. Data represent the mean ± SD (n = 3, ^¶p < 0.0001 and ^§p < 0.001). d Cellular ATP levels were measured in control and TFAM knockdown DLD1 and HCT116 cells. Data represent the mean ± SD (n = 4, ^#p < 0.05). e, f Control and TFAM knockdown DLD1 (e) and HCT116 (f) cells were subjected to the glycolysis stress test to obtain ECAR measurements using the Seahorse XF96 Extracellular Flux analyzer. Results were quantified and levels of glycolysis, glycolytic capacity and glycolytic reserve were calculated. Data represent the mean ± SD (n = 3, ^¶p < 0.0001 and ^§p < 0.001). g, h Control and TFAM knockdown DLD1 (g) and HCT116 (h) cells were cultured in the control medium (high-glucose), glucose-free medium or treated with 2-DG for 48 h. The relative cell survival was determined by comparing to the control condition. Data represent the mean ± SD (n = 4, ^¶p < 0.0001, ^§p < 0.001, and *p < 0.01 compared to sh-control within individual culture conditions)