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. 2022 Feb 12;51:102264. doi: 10.1016/j.redox.2022.102264

Fig. 1.

Fig. 1

Molecular characterization of HAP1 cell lines. A. Relative quantification of VDAC1, VDAC2 and VDAC3 mRNAs in HAP1-ΔVDAC1 and HAP1-ΔVDAC3. Data are normalized to the β-actin and expressed as means ± SEM (n = 3) and compared to HAP1 parental cells (equals one). B. Western blot illustration and relative quantification of VDAC1, VDAC2 and VDAC3 protein levels in HAP1-ΔVDAC1 and HAP1-ΔVDAC3. Data are normalized to the β-tubulin, expressed as means ± SEM (n = 3) and compared to HAP1 parental cells. C. Relative quantification of PGC-1α, NRF1 and TFAM (mitochondrial biogenesis markers) mRNAs in HAP1-ΔVDAC1 and HAP1-ΔVDAC3. Data are normalized to the β-actin and expressed as means ± SEM (n = 4) and compared to HAP1 parental cells. D. Quantification of mitochondrial DNA (mtDNA) in HAP1 cell lines devoid of VDAC1 or VDAC3. mtDNA amount was measured by Real-Time PCR of the mitochondrial gene COXII and normalized to the nuclear gene APP. Data are expressed as means ± SEM (n = 3) and compared to HAP1 parental cells. E. Quantification of mitochondrial content. Western blot illustration and relative quantification of mitochondrion-specific protein SDHA level in HAP1-ΔVDAC1 and HAP1-ΔVDAC3. Data are normalized to the β-Actin, expressed as means ± SEM (n = 3) and compared to HAP1 parental cells. F. Measurement of mitochondrial mass using MitoTracker Green. Representative fluorescence profiles of cells populations for the three cells lines. Histogram reports the average of fluorescence intensity measured by flow cytometry for the three HAP1 cell lines after proper gating of fluorescence. Gates were established based on negative and positive controls. Data are indicated as means ± SEM (n = 3) and compared to HAP1 parental cells. G. Detection of mitochondrial membrane potential by Mitotracker Red and measured by flow cytometry in each population of the three HAP1 cell lines. Histograms show the average fluorescence intensity measured for each gated cell population analyzed. Data are indicated as means ± SEM (n = 3) and compared to HAP1 parental cells. H. Quantification of mitochondrial membrane potential normalized for mitochondrial mass in HAP1 cell lines devoid of VDAC1 and VDAC3. Data are expressed as means ± SEM (n = 3) and compared to parental cell line. Data were analyzed with One-Way or Two-Way ANOVA; with *p < 0.05 **p < 0.01 and ***p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)