Figure S2.
SNX10 modulates COX-IV protein levels.(A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP for 24 h. The cells were fixed and stained with antibodies against the indicated mitochondrial proteins for subsequent analysis. Scale bars: 10 μm. Insets: 8.57 × 8.57 µm. (B) Representative images of U2OS cells transfected with 20 nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10 #1 and siSNX10 #2). Cells were stained with an anti-TOMM20 antibody after fixation. Images were acquired using a Nikon CREST X-Light V3 spinning disk microscope utilizing a 60× oil objective. Scale bar: 10 µm. (C) Quantification of the data shown in B, performed using CellProfiler software. The graph displays the area occupied by TOMM20 per cell (n = 3, >100 cells per condition in each replicate). Significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. (D) U20S cells were reverse transfected with the indicated siRNA (20 nM) for 72 h. The cells were lysed in the well, and the RNA was extracted prior to cDNA synthesis. The graph shows the difference in expression levels upon KD of the different proteins (mean values ± SEM). The values were normalized to TBP using the 2−ΔΔCt method and then compared with siCtrl control. Significance was determined from n = 2 independent experiments by one-way ANOVA followed by Dunnetts’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. (E) Quantification of COX-IV protein expression levels in control (siCtrl) and SNX10 (siSXN10#1, siSXN10#2) depleted cells upon treatment of MG132 and/or DFP across three independent experiments. Band densities were normalized to the housekeeping gene actin. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Šídák’s multiple comparisons test to compare each knockdown group to the control group. Data distribution was assumed to be normal but was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. TBP; TATA-box–binding protein and KD; knockdown.

SNX10 modulates COX-IV protein levels. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP for 24 h. The cells were fixed and stained with antibodies against the indicated mitochondrial proteins for subsequent analysis. Scale bars: 10 μm. Insets: 8.57 × 8.57 µm. (B) Representative images of U2OS cells transfected with 20 nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10 #1 and siSNX10 #2). Cells were stained with an anti-TOMM20 antibody after fixation. Images were acquired using a Nikon CREST X-Light V3 spinning disk microscope utilizing a 60× oil objective. Scale bar: 10 µm. (C) Quantification of the data shown in B, performed using CellProfiler software. The graph displays the area occupied by TOMM20 per cell (n = 3, >100 cells per condition in each replicate). Significance was assessed by ordinary one-way ANOVA followed by Tukey’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. (D) U20S cells were reverse transfected with the indicated siRNA (20 nM) for 72 h. The cells were lysed in the well, and the RNA was extracted prior to cDNA synthesis. The graph shows the difference in expression levels upon KD of the different proteins (mean values ± SEM). The values were normalized to TBP using the 2−ΔΔCt method and then compared with siCtrl control. Significance was determined from n = 2 independent experiments by one-way ANOVA followed by Dunnetts’s multiple comparison test. Data distribution was assumed to be normal but was not formally tested. (E) Quantification of COX-IV protein expression levels in control (siCtrl) and SNX10 (siSXN10#1, siSXN10#2) depleted cells upon treatment of MG132 and/or DFP across three independent experiments. Band densities were normalized to the housekeeping gene actin. Data are presented as mean ± SEM. Statistical analysis was performed using one-way ANOVA followed by Šídák’s multiple comparisons test to compare each knockdown group to the control group. Data distribution was assumed to be normal but was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. TBP; TATA-box–binding protein and KD; knockdown.

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