ADPGK regulates energy metabolism. (a) Lactate concentrations (n = 4 independent experiments) and (b) glucose uptake (in 1 hour; n = 3 independent experiments) in control and KO1 cells. (c) Changes of enzymatic activities of Hexokinase (HK), Phosphofructokinase (PFK) and Lactate dehydrogenase (LDH) in ADPGK KO clones compared to control cells. N = 3 independent experiments. (d) Changes of respiratory chain complex III and ATP synthase activities in ADPGK KO clones compared to control cells. N = 3 independent experiments. (e) Mitochondrial membrane potential measured in control and ADPGK KO cells after 24 h stimulation with PMA/Iono using JC-1 dye. N = 3 independent experiments. (f) Sum of thymidine degradation intermediates (thymin, thymidine, dihydrothymine) in lysates of control and ADPGK KO cells treated with PMA/Iono for 0 h or 1 h. N = 3 independent experiments. (g) Acridine Orange staining of control and KO1 cells upon 1 h and 24 h PMA/Iono stimulation. N = 3 independent experiments. (h) Representative immunoblots for mTOR phosphorylation after different PMA/Iono activation periods of control and KO1 cells. N = 3 independent experiments. (i) Immunoblots of pS6K and pRibS6 after different PMA/Iono activation periods in control and KO1 cells. In all experiments stimulation was induced as described in the text using 10 ng/mL PMA and 10 µM Ionomycin. *p < 0.05, **p < 0.01. All images of blots represent cropped blots of appropriate protein size. For full length blots see Supplemental Fig. 3.
|
