Figure 6. Mitochondrial zinc handling deficits in SLC30A9-knockdown HeLa cells.

(A) qPCR analysis of HeLa cells transfected with SLC30A9 esiRNA and harvested 24 h post-transfection. (B) Mitochondrial zinc content in control and SLC30A9-transfected HeLa cells was evaluated using Rhod-2,am. The cells were exposed to zinc for 24 h. Average fluorescence in each region of interest containing clearly identifiable mitochondria was recorded and normalized to the values in the cells that were transfected with scrambled shRNA. (C) The delayed loss of mitochondrial zinc in SLC30A9-deficient cells exposed to nominally zinc-tree medium after 24 h long zinc load. The cells were treated as above; at t = 0 the zinc-containing medium was replaced with nominally zinc free medium and images were taken every 60 s. The fluorescence intensity was normalized to the values recorded at t = 0, which was taken as 100%. In Panels A and B, data represent three independent trials involving 7–10 cells. Data are shown ± SEM. Asterisks represent statistical significance at P < 0.05, for Student’s test in Panel A. In Panel B, the curves were fitted using a third order polynomial curve (smooth gray lines) and an extra sum-of-squares test was performed to answer whether one curve can adequately fit both datasets. That hypothesis was rejected at P < 0.0001 level. Here and below, statistical analysis and data plotting were performed using Prism 9.