Figure 3.
SNX10 localizes nearby mitochondria. (A) SNX10-EGFP WT or the Y32S mutant, stably expressed in U2OS cells, underwent GFP pulldown for the subsequent analysis of their interactome using mass spectrometry assays. A total of 53 proteins were identified as significant SNX10-EGFP interactors compared with the EGFP control, as analyzed by R lima using a cut-off value of log2FC >1 and adjusted P value <0.05. The Venn diagram illustrates the distinct and shared interactors between the SNX10 WT and Y32S mutant, where the identified significant SNX10-EGFP–interacting proteins are listed below. (B) The interacting proteins of SNX10 WT were enriched for GO term analysis. Their cellular component (upper pie chart) and biological processes (lower pie chart) enrichment is expressed in percentage toward the significant hits. Graphs were plotted using Plotly (Python package). (C) U2OS with stable inducible expression of SNX10-EGFP were treated with doxycycline for 24 h before incubation with MitoTracker Red for 30 min, followed by live imaging with an acquisition speed of 1 frame every 500 ms. Scale bar: 10 µm. (D) U2OS cells stably expressing mScarlet-RAB5 and with inducible expression of SNX10-EGFP were stained with MitoTracker Deep Red FM in the presence or absence of DFP (1 µM). Scale bar: 10 µm. (E) U2OS SNX10-EGFP cells were treated or not with DFP (1 µM) for 24 h in the absence or presence of the ULK1 inhibitor MRT68921 (1 µM) for 1 h. MitoTracker Deep Red FM (100 nM) was added for 1 h, followed by immunofluorescence staining with antibody against LC3B. Scale bars: 10 µm. Insets 5.77 × 5.77 µm. (F and G) Quantification of the percentage of the co-occurrence of LC3 on SNX10 structures (F) and of LC3 on MitroTracker-SNX10–positive structures (G). Data are mean ± SEM with individual data points corresponding to a single field of view (n = 4 [F] and n = 3 [G], corresponding experiment shown in the same color, >150 cells per experiment). The statistical significance was calculated with ordinary one-way ANOVA, followed by Tukey’s multiple comparison test. Data distribution was assumed to be normal, but this was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. GO; Gene Ontology.

SNX10 localizes nearby mitochondria. (A) SNX10-EGFP WT or the Y32S mutant, stably expressed in U2OS cells, underwent GFP pulldown for the subsequent analysis of their interactome using mass spectrometry assays. A total of 53 proteins were identified as significant SNX10-EGFP interactors compared with the EGFP control, as analyzed by R lima using a cut-off value of log2FC >1 and adjusted P value <0.05. The Venn diagram illustrates the distinct and shared interactors between the SNX10 WT and Y32S mutant, where the identified significant SNX10-EGFP–interacting proteins are listed below. (B) The interacting proteins of SNX10 WT were enriched for GO term analysis. Their cellular component (upper pie chart) and biological processes (lower pie chart) enrichment is expressed in percentage toward the significant hits. Graphs were plotted using Plotly (Python package). (C) U2OS with stable inducible expression of SNX10-EGFP were treated with doxycycline for 24 h before incubation with MitoTracker Red for 30 min, followed by live imaging with an acquisition speed of 1 frame every 500 ms. Scale bar: 10 µm. (D) U2OS cells stably expressing mScarlet-RAB5 and with inducible expression of SNX10-EGFP were stained with MitoTracker Deep Red FM in the presence or absence of DFP (1 µM). Scale bar: 10 µm. (E) U2OS SNX10-EGFP cells were treated or not with DFP (1 µM) for 24 h in the absence or presence of the ULK1 inhibitor MRT68921 (1 µM) for 1 h. MitoTracker Deep Red FM (100 nM) was added for 1 h, followed by immunofluorescence staining with antibody against LC3B. Scale bars: 10 µm. Insets 5.77 × 5.77 µm. (F and G) Quantification of the percentage of the co-occurrence of LC3 on SNX10 structures (F) and of LC3 on MitroTracker-SNX10–positive structures (G). Data are mean ± SEM with individual data points corresponding to a single field of view (n = 4 [F] and n = 3 [G], corresponding experiment shown in the same color, >150 cells per experiment). The statistical significance was calculated with ordinary one-way ANOVA, followed by Tukey’s multiple comparison test. Data distribution was assumed to be normal, but this was not formally tested. * = P < 0.05, ** = P < 0.01, *** = P < 0.001, and **** = P < 0.0001; nonsignificant differences are not depicted. GO; Gene Ontology.

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