Fig. 5

IFI6 modulates mitochondrial ROS production by regulating mitochondrial Ca²⁺ overload. a-b. Representative images (a) and statistical quantification (b) of mitochondrial ROS (mtROS) production assay results in ESCC cells. The indicated cells were stained with MitoSOX, and fluorescence was quantified under a fluorescence microscope. MitoSOX: red, Hoechst: blue. Scale bar: 20 μm. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01. c-d. Quantification (c) and statistical analysis (d) of the relative Rhod-2 AM fluorescence intensity time course in the indicated ESCC cells. The fluorescence intensity at each time point was recorded with an integrated spectrofluorometer at excitation and emission wavelengths of 550 nm and 580 nm, respectively. The relative fluorescence intensity was calculated as a percentage of the baseline fluorescence intensity, which was recorded during the first 60 s (F₀). The arrow indicates the time at which Tg was added. The data are presented as the means and SDs (n = 3). Statistical significance was determined by two-tailed Student’s t-test. **P < 0.01. e. The indicated ESCC cells were incubated in the presence or absence of BAPTA-AM (2 μM) for 1 h, and mitochondrial ROS levels were then measured by MitoSOX staining followed by flow cytometry. f. The indicated ESCC cells were cultured in complete medium or calcium-deficient medium, and mitochondrial ROS levels were determined by MitoSOX staining followed by flow cytometry. g-h. Quantification (g) and statistical analysis (h) of the relative Rhod-2 AM fluorescence intensity time course in the indicated ESCC cells. The fluorescence intensity at each time point was recorded with an integrated spectrofluorometer at excitation and emission wavelengths of 550 nm and 580 nm, respectively. The relative fluorescence intensity was calculated as a percentage of the baseline fluorescence intensity, which was recorded during the first 60 s (F₀). The arrow indicates the time at which Tg was added. The data are presented as the means and SDs (n = 3). Statistical significance was determined by one-way ANOVA. **P < 0.01. i. The indicated ESCC cells were incubated in the presence or absence of the MCU inhibitor DS16570511 (10 μM), and mitochondrial ROS levels were then measured by MitoSOX staining, followed by flow cytometry