Fig. 1

Cellular respiration in CAMKK2 deleted HEK293, HepG2 and EA.hy926 cell clones measured with an XF-24 extracellular flux analyzer. A: Immunoblots showing expression of CAMKK2 in HEK293 and HepG2 cells. WT: wild-type (parental), KO: CAMKK2 knockout (CAMKK2^−/−), M: molecular weight ladder, and ns: nonspecific band. B Scatter plot showing CAMKK2 protein levels in HEK293 (HEK), HepG2 cells. The relative expression was normalized based on HEK293. Data presented as Mean ± SEM. N = 3 replicates from 3 independent experiments. Statistical significance from one-way ANOVA followed by multiple comparisons test. C, F Line graphs showing OCR kinetics at different time points following glucose/drug injections in the parental and CAMKK2 deleted HEK293 (C), and HepG2 (F) cell clones. Glu: glucose, Oligo: Oligomycin, FCCP: Carbonyl cyanide 4-(trifluoromethoxy), and Rtn/AA: rotenone/antimycin. The grey and yellow highlighted areas in D indicate basal respiration following injection of glucose and non-mitochondrial respiration following Rtn/AA injection, respectively. Data presented as Mean ± SEM, N = 10 replicates. Arrows indicate pre-glucose injection OCR rate set at 100 for normalization. This allows comparison between different biological replicates. D, G Bar graphs showing OCR (basal respiration) at 30 min after glucose injection. The basal respiration was calculated by subtracting non-mitochondrial respiration rate from 2nd rate measurement after glucose injection. Data presented as Mean ± SEM, N = 20 replicates from 2 independent experiments. Statistical significance from one-way ANOVA followed by multiple comparisons test. “×” indicates fold change. E, H: ECAR versus OCR plots after 30 min of glucose injection. Data presented as Mean ± SEM, N = 20 replicates from 2 independent experiments. The yellow arrows indicate a shift in the metabolic phenotype under CAMKK2 deleted condition