Fig. 3: Praja2 restrains AMPK signaling and supports the glycolytic pathway.

a U87MG cells were transiently transfected with siRNAs (siRNAc or siPraja2). Twenty-four hours later, cells were left untreated (time point 0) or deprived of glucose for the indicated times. Lysates were immunoblotted with anti-pThr172-AMPKα, anti-AMPKα1, and anti-praja2 antibodies. b Quantitative analysis of the experiments shown in panel a. A mean value ± SEM of three independent experiments is reported. P value: *** = 0.00070; * = 0.028. c Same as in a, with the exception that glucose-supplemented cells were treated with 1 mM AICAR for the indicated times. d Quantitative analysis of the experiments shown in panel c. A mean value ± SEM of three independent experiments is reported. P value: ** = 0.0042;* = 0.014. e U87MG cells were transiently transfected with siRNAs (siRNAc, siPraja2 or siPraja2, and siKSR2). Twenty-four hours later, cells were left untreated (time point 0) or cells were treated with 1 mM AICAR for the indicated times. Lysates were immunoblotted with indicated antibodies. f Quantitative analysis of the experiments shown in panel e. A mean value ± SEM of three independent experiments is reported. P value: * = 0.014; * = 0.034. g Oxygen consumption rate (OCR) in U87MG cells transiently transfected with the indicated siRNAs. Reported data are the mean values ± SEM of four independent experiments. OCR was measured in real-time, under basal conditions, or in the presence of the indicated mitochondrial inhibitors: oligomycin, FCCP, antimycin A plus rotenone. h Indices of mitochondrial respiratory function, as calculated from the OCR profile of U87MG cells transiently transfected with the indicated siRNAs: basal OCR, maximal respiration, spare respiratory capacity, ATP production. Reported data were the mean values ± SEM of four measurements deriving from four independent experiments. P value: * = 0.011; * = 0.025; ** = 0.0069; ** = 0.0093; ** = 0.0061; ** = 0.0.0058 ; ** = 0.0026. i Oxygen consumption rate (OCR) in U87MG cells transiently transfected with reported siRNAs. When indicated, cells were pretreated with the AMPK inhibitor SBI-0206965 (5 μM for 4 h). Reported data were the mean values ± SEM of four independent experiments. j Indices of mitochondrial respiratory function, as calculated from the OCR profile of praja2-silenced and control U87MG cells: basal OCR, maximal respiration, spare respiratory capacity, ATP production. When indicated, cells were pretreated with the AMPK inhibitor SBI-0206965 (5 μM for 4 h). Reported data were the mean values ± SEM of four independent experiments. P value *** = 0.00014; * = 0.039; *** = 0.0.00013; ** = 0.0035; *** = 0.000090; * = 0.012.