Fig. 9

Inhibition of PI3K downregulated both glycolysis and OXPHOS in MscL-G22S-activated SCs. MscL-G22S-expressing SCs were pretreated with 10 mM of LY294002 and then mechanically stretched for 24 h. A The real-time ATP rate assay showed a significant reduction of mitochondrial and glycolytic ATP production in MscL-G22S-activated SCs by inhibition of PI3K. ***p < 0.001, mitochondrial ATP production; ^###p < 0.001, glycolytic ATP production. (B-E). The Seahorse experiments showed that in MscL-G22S-activated SCs, inhibition of PI3K significantly abolished the upregulation of glycolysis rate, glycolytic capacity and glycolytic reserve in ECAR (B-C) and the basal respiration, ATP production, maximal respiration, and spare respiratory capacity in OCR (D-E). F-K The mtDNA copy number, mitochondrial length and area as well as the fluorescence intensity of mitochondria in MscL-G22S-activated SCs were significantly reduced by inhibition of PI3K. Scale bars = 2 μm for electron microscope and scale bars = 5 μm for mitochondria staining. L-M Analysis of mitochondrial membrane potential in MscL-G22S-activated SCs measured by JC-1 staining showed significantly less aggregated JC-1, indicating reduced mitochondrial activity caused by inhibition of PI3K. Scale bars = 50 μm. The data shown were representative of three or six independent experiments. NC, negative control; MscL, MscL-G22S-expressing SCs; NC-stretch, negative control with mechanical stretching; MscL-stretch, MscL-G22S-expressing SCs with mechanical stretching. **p < 0.01, ***p < 0.001