u2os cells expressing snx10 egfp wild type  (Proteintech)

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) SNX10 graphical view. The PX domain is represented in green, and the arrows indicate the position of the natural variants (SNPs) linked to ARO (R16L, Y32S, R51P, R51Q). B) Confocal imaging of U2OS cell lines stably expressing doxycycline inducible SNX10-EGFP WT or the indicated ARO-linked mutants. Nuclei were stained with Hoechst. Scale bar = 20µm. C) Representative immunoblot showing the expression levels of SNX10-EGFP and the indicated ARO mutants. The membrane was blotted using and antibody anti GFP and using Actin as loading control. D and E) Representative immunofluorescence images of U2OS cells stably expressing SNX10-EGFP WT or the Y32S mutant (green) immuno-stained with anti-EEA1 (D) or anti-LAMP1 antibodies (E) (magenta) after treating cells with 5µM VPS34-IN1 for 2h. Nuclei were stained with Hoechst. Scale bar = 10µm F) U2OS cells with stable inducible expression of SNX10-EGFP were infected with lentiviral particles to express mScarlet-RAB5/RAB7/RAB9 vectors. Nuclei were stained with Hoechst. Scale bars, 10 μm. G) U2OS SNX10-EGFP cells fixed for CLEM analysis. The area analyzed and showed in (ii) is indicated with a square in the confocal image (i). (iii) shows the transmission EM and (iv) the z-slide from the tomogram. The white arrows indicate clathrin coated vesicles. (v) green: endosomes; red: lipid droplets; yellow: vesicles; and pink: clathrin coated vesicles Scale bars, 10 μm (i and ii), 1 μm (iii and iv).

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Imaging, Stable Transfection, Expressing, Staining, Western Blot, Membrane, Control, Immunofluorescence, Mutagenesis, Infection, Transmission Assay

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) Graphical description of the plasma membrane EGFR staining. Live cells are put on ice, and (1) incubated for 20 min with the primary anti-EGFR antibody, then washed and (2) incubated with a secondary antibody for 20 min, followed by (3) incubation with EGF for 15 or 50 min at 37°C before fixation and imaging. B) U2OS SNX10-EGFP cells were incubated with anti EGFR antibody as described in A), then stimulated with EGF and fixed. Cells were stain with an anti-EFG antibody after fixation. Scale bar: 10µm. C) After 72 h of siRNA transfection, U2OS SNX10-EGFP cells were serum starved for 2 h and then incubated with 50 ng/ml EGF + 10 µg/ml Cycloheximide (CHX) for the indicated times. The cells were lysed followed by western blotting for the indicated proteins. D) Quantification of EGFR and pEGFRTyr1068 protein levels normalized to GAPDH in n= 3 independent experiments. Significance was determined by two-way ANOVA followed by Šídák’s multiple comparisons test. E) Cells were reverse transfected with 20nM siRNA: siCtrl (control) and two different siSNX10 oligoes (siSNX10#1 and siSNX10#2) prior to fixation and staining for endogenous EEA1. Images were taken with Zeiss Axio Observer widefield microscope (Zen Blue 2.3, Zeiss) and a 20x objective was used. Scale bar: 10 µm. F) quantification of the data shown in E) was performed using CellProfiler software. The values were obtained from analyzing >1000 cells per condition and they were normalized to control siRNA (siCtrl). The graphs display the mean values ±SEM from n = 3 independent experiments. The significance was assessed by ordinary one-way ANOVA followed by Bonferroni’s post hoc test. (* = p < 0.05). G) Representative electron microscopy images of U2OS cells (control and siSNX10) showing immunogold-labelled EGFR containing endosomes. One experiment was performed. The number of endosomes without EGFR-gold and with EGFR-gold of which diameter was measured were 8 (siCtrl), 45 (siSNX10), and 23 (siCtrl), 10 (siSNX10) respectively. H) The graph displays the mean values ± SEM from the endosomal diameters. Significance was determined by unpaired t-test with Welch’s correction. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs.

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Clinical Proteomics, Membrane, Staining, Incubation, Imaging, Transfection, Western Blot, Control, Microscopy, Software, Electron Microscopy

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) U2OS cells expressing either WT SNX10-EGFP or the Y32S mutant underwent GFP pulldown for the subsequent analysis of their interactome using mass spectrometry assays. The resulting data were depicted in a Venn diagram, illustrating the distinct and shared interactors between the WT and mutant forms of SNX10. Significant identified proteins representing hits that interacted with SNX10 WT are listed below B) The interacting proteins of SNX10 WT were enriched for GO term analysis. The enrichment of the Cellular Component they belong to (pie chart above) and Biological Processes they are involved in (pie chart below) is expressed in percentage towards 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 hrs before incubation with MitoTracker Red for 30 min, followed by live imaging using a Dragonfly 505 (Andor) microscope, 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 and were stained with MitoTracker Deep Red FM in the presence or absence of DFP. Scale bar 10 µm. E) U2OS SNX10-EGFP were treated with or without DFP (1 µM) for 24 hrs and with or without MRT68921 (1 µM) for 1 h. MitoTracker (100 nM) was added for 1 h, followed by immunofluorescence staining with antibodies against LC3B and COX-IV. Scale bars: 10 µm. F and G) Quantification of the percentage of SNX10 localizing on LC3B (F) and LC3 + MitoTracker positive structures localizing on SNX10 (G), normalized to control sample (-MRT/-DFP) (the graph shows mean ± SEM [error bars] from n=3 independent experiments. The statistical significance was calculated with a two-way ANOVA followed by a Tukey’s post analysis test. * = p < 0.05)

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Expressing, Mutagenesis, Mass Spectrometry, Incubation, Imaging, Microscopy, Stable Transfection, Staining, Immunofluorescence, Control

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 hrs before the addition of DFP for 24 hrs. The cells were fixed and stained with antibodies against mitochondrial proteins for subsequent analysis. Scale bars: 10 µm. B) U2OS cells were reverse transfected with the indicated siRNA (20 nM) for 48 hrs, then treated or not with DFP for 24 hrs with BafA1 treatment or not the last 2 hrs, followed by western blotting for the indicated proteins. C-E) Quantification of the data in B) from n=5, 3 and 6 independent experiments. Bars show mean values of the protein levels normalized to b-Actin relative to Control conditions (siCtrl control). Significance is assessed by two-way ANOVA followed by Tukey’s post hoc test. F) U2OS cells were subjected to reverse transfection with siSNX10 (20nM) for 48 hours, followed by treatment with either IN1 or MRT for 24 hours before fixation. Post-fixation, cells were stained with a COX-IV antibody, and images were captured using an ImageXpress Micro Confocal (Molecular Devices) at 20X magnification. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Expressing, Staining, Transfection, Western Blot, Control

Journal: bioRxiv

Article Title: SNX10 regulates the clearance of mitochondrial proteins and mitochondrial bioenergetics

doi: 10.1101/2024.05.15.594320

Figure Lengend Snippet: A) Citrate Synthase activity was determined by spectrophotometry from lysates of U2OS cells transfected with siRNA for 72 hrs, in the presence or absence of DFP for the last 24 hrs. The graph displays mean values normalized to siCtrl. Significance was determined from n = 3 independent experiments, by two-way ANOVA followed by Tukey’s multiple comparison test. B) Mitochondrial oxygen consumption rate (OCR) was assessed in control and SNX10 knocked down cells using the Seahorse XFe24 Analyzer. OCR was measured following sequential addition of Oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), and Rotenone/Antimycin A (Rot/AntiA). C) The four basal OCR measurements per well were averaged to determine the basal OCR value, and non-mitochondrial respiration was subtracted to ascertain the basal respiration associated with each condition. D) ATP production was calculated by subtracting the proton leak from the maximal respiratory capacity. Error bars represent mean ± SEM from n=5. Statistical significance was determined using one-way ANOVA followed by Dunnett’s multiple comparison test. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001 and n.s = not significant in all graphs

Article Snippet: U2OS cells expressing SNX10-EGFP wild type or the Y32S mutant were subjected to GFP pulldown using the ChromoTek GFP-Trap®, following the vendor’s specifications.

Techniques: Activity Assay, Spectrophotometry, Transfection, Comparison, Control