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.