Investigating POLG-driven modulation of cancer stemness: a pilot study in breast cancer cells (IMAGE)
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Figure 2. POLG1 and POLG2 gene silencing drives a significant decrease in the mitochondrial marker MTCO2 and in mitochondrial membrane potential, while increasing mitochondrial superoxide levels. (A) MTCO2 protein levels were evaluated by immunoblotting in MCF-7 POLG1 silenced cells and control cells. The graph shows densitometric analysis of the bands normalized to vinculin, using Licor Empiria Studio software. Results are shown as the mean ± SEM of three independent experiments. (B) Evaluation of the mitochondrial protein TOMM20 by immunoblotting in MCF-7 POLG1 silenced cells and control cells. The graph shows densitometric quantification of the bands, using Licor Empiria Studio software, normalized to vinculin that was used as loading control. Data are shown as mean ± SEM of three independent biological replicates. (C) Mitochondrial membrane potential was assessed using MitoNIR probe, by flow cytometry. Experiments were repeated three times independently, with three technical replicates for each condition. Error bars represent mean ± SEM. Statistical significance (relative to control): **p< 0.01, evaluated by Student’s t test, two-tailed test. (D) The level of mitochondrial superoxide anions was measured by quantifying the mean fluorescence intensity (MFI) of MitoSOX Red probe (5μM), in MCF-7 POLG1 silenced and control cells, using flow cytometry. Experiments were performed three times independently with three technical replicates for each condition. Error bars represent mean ± SEM. Statistical significance (relative to control): *p<0.05, evaluated by Student’s t test, two-tailed test. (E) MTCO2 protein levels were evaluated by immunoblotting in MCF-7 POLG2 silenced cells and control cells. The graph shows the densitometric analysis of the bands normalized to vinculin, assessed by measuring signal intensity with ImageJ. Results are shown as the mean ± SEM of three independent experiments. Statistical significance (relative to control): **p< 0.01, evaluated by Student’s t test, two-tailed test. (F) MCF-7 transduced cells were cultured for 48h and incubated with MitoTracker Deep Red (MTDR; 50nM) and analyzed by FACS analysis. Experiments were repeated three times independently. Error bars represent mean ± SEM. Statistical significance (relative to control): ns not statistically significant. (G) Mitochondrial membrane potential was assessed using MitoNIR probe, by flow cytometry. Experiments were repeated three times independently, with three technical replicates for each condition. Error bars represent mean ± SEM. Statistical significance (in relation to control): **p< 0.01, evaluated by Student’s t test, two-tailed test. (H) The level of mitochondrial superoxide anions was measured by quantifying the fluorescence intensity of MitoSOX Red probe (5μM), in MCF-7 POLG2 silenced and control cells, using flow cytometry. Experiments were performed three times independently with three technical replicates for each condition. Error bars represent mean ± SEM. Statistical significance (relative to control): *p<0.05, evaluated by Student’s t test, two-tailed test. (I) The total ROS levels were measured by flow cytometry using CellRox dye (5μM). Data are shown as mean ± SEM obtained from three biological repeats with three technical replicates for each condition. Statistical significance (related to control): ns not statistically significant, evaluated by Student’s t test, two-tailed test.
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