Fig. 3

Hsp90ab1 siRNA knockdown in HSMM increases substrate metabolism and mitochondrial respiration. a Seahorse extracellular acidification rate (ECAR) curve from a glycolysis stress test of HSMM transfected with either negative control (NC) siRNA or siRNA to Hsp90ab1 (siHsp90ab1) measured over time. The ECAR curve shows the active glycolytic activity and maximal capacity of glycolysis obtained by sequential injections of glucose (25 mM) and oligomycin (2 μM), respectively. Injection of 2-DG (50 mM) reversed the glucose-induced ECAR increase. Data represent mean + SEM of n = 3 independent samples. *p < 0.05 and **p < 0.01 vs. NC, Student’s t-test. b Area under the curve (AUC) of ECAR/DNA glycolysis stress test after glucose and oligomycin injections after adjustment from baseline AUC (before glucose injection) plotted as fold change relative to results of HSMM transiently transfected with NC siRNA. HSMM transfected with siHsp90ab1 show increased glucose- and oligomycin-induced glycolysis. c Oxygen consumption rate (OCR) curve from a Seahorse fatty acid oxidation (FAO) assay measured over time. Palmitate (400μM), CCCP (1μM), and etomoxir (50μM) were sequentially injected into the assay plate containing HSMM transiently transfected with NC or siHsp90ab1. Data represent n = 6 independent samples. d Uncoupled mitochondrial respiration measured by AUC of OCR/protein after CCCP injection adjusted by subtracting basal OCR AUCs (prior to palmitate injection) plotted as fold change relative to results of HSMM transiently transfected with NC siRNA. HSMM transfected with siHsp90ab1 show increased uncoupling relative to NC. Data represent mean + SEM of n = 6 independent samples. *p < 0.05 vs NC, Student’s t-test. e OCR curve for bioenergetics profiling of HSMM. Oligomycin (2μM), CCCP (1μM), and rotenone (2μM) were sequentially injected and OCR was measured over time. f Basal and uncoupled mitochondrial respiration. AUC of OCR/DNA was calculated by subtracting AUC after rotenone injections (which represents non-mitochondrial respiration) from AUC prior to oligomycin injection (basal mitochondrial respiration, left side of graph) or AUC after CCCP injection that was adjusted after subtracting AUC prior to oligomycin (uncoupled mitochondrial respiration, right side of graph) and plotted as fold change relative to results of HSMM transiently transfected with NC siRNA. HSMM transfected with siHsp90ab1 show no difference in basal mitochondrial respiration compared to NC. In contrast, siHSP90ab1 knockdown increased uncoupled mitochondrial respiration. Data represent mean + SEM of n = 3 independent samples. *p < 0.05 vs. NC, Student’s t-test