3.3 Validating MitoRAISE in cell lines
As mentioned before, three different parameters can be tested through a single assay of MitoRAISE: ATP contents, ATP synthesis capacity, and inhibitor sensitivity. We tested the three parameters on A549 cells at different cell counts. We were able to see a cell count dependent increase in ATP signals in both MitoRAISE and commercial ATP kit, in which MitoRAISE was able to sensitively reflect the number of cells through ATP contents in more than 1*10^4 cells (Figure 2A). ATP synthesis capacity was measured through MitoRAISE simultaneously, and its results also showed correlation with the cell count from 0.5*10^4 cells (Figure 2B). After the implementation of MitoRAISE and validation from normal cells, it was reexamined on damaged cells. A549 rho-0 cells, with depleted mitochondrial DNA confirmed through gel electrophoresis and qPCR, showed less total ATP contents, no ATP synthesis capacity, and naturally, no response to inhibitors (Supporting Figure 2A-B, Figure 2A-C). A549 cells with damaged mitochondrial membrane potential using either 50 µM 2-[2-(3-Chlorophenyl)hydrazinylyidene]propanedinitrile (CCCP) or 50 µM carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP) was also tested. When treated with CCCP or FCCP, the mitochondrial membrane potential dissipates as seen through the loss of mitochondrial staining in fluorescence microscopy, and no ATP is predicted to be synthesized (Supporting Figure 2C-D). In accordance with the prediction, no ATP was synthesized and no response to inhibitors was found (Figure 2B-C).
We applied MitoRAISE assay to ten different cell lines originating from various organs. They were screened for their entire mitochondrial function through testing the total ATP contents, ATP synthesis capacity, sensitivity to inhibitory substrates, and mitochondrial DNA copy number (mtDNA CN) (Figure 2D-F, Supporting Figure 3). Several positive correlations were found among the parameters: ATP contents and GM-induced ATP synthesis capacity (ρ=0.62), ATP contents and S-induced ATP synthesis capacity (ρ=0.76), ATP contents and mtDNA copy number (ρ=0.73). Just as we expected, there was a positive correlation between ATP contents, ATP synthesis capacity, inhibitor sensitivity, and these values were in correlation with mitochondrial DNA copy number.