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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
Cox Iv, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
Anti Cox Iv, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies <t>anti–COX-IV</t> and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).
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SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies anti–COX-IV and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: SNX10 vesicles contain mitochondrial proteins and LC3B. (A) U2OS cells with stable inducible expression of SNX10-EGFP were pre-treated with doxycycline for 16 h before the addition of DFP (1 µM) for 24 h. The cells were fixed and stained with antibodies against mitochondrial proteins. Scale bars: 10 µm. Insets: 8.57 × 8.57 µm. (B and D) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h and stained with antibodies anti–COX-IV and anti-LC3B in B or anti-LAMP1 in D, prior to acquisition with a Nikon CREST X-Light V3 spinning disk microscope using a 60× oil objective (NA 1.42). Scale bar: 10 µm. Insets: 4.41 × 4.41 µm (B), 5.52 × 5.52 µm (D). (C–E) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for (B and D).

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: Expressing, Staining, Microscopy, Control

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.

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: 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.

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: Expressing, Staining, Transfection, Control, Microscopy, Software, Comparison, cDNA Synthesis, Knockdown, Binding Assay

SNX10 is a negative modulator of COX-IV turnover. (A) U2OS cells were reverse transfected with the indicated siRNA (20 nM) for 72 h, then treated or not with DFP (1 µM) for 24 h and with BafA1 (50 nM) the last 16 h, followed by western blotting for the indicated proteins. (B–D) Quantification of the data in A from n = 5, 3, and 6 independent experiments. Bars show mean values of the protein levels normalized to actin relative to control conditions (siCtrl control) ± SEM. Significance is assessed by two-way ANOVA followed by Tukey’s post hoc test. Data distribution was assumed to be normal. (E) U2OS cells with stable expression of mScarlet-RAB5 were reverse transfected with the indicated siRNA (20 nM) for 72 h, then treated or not with DFP for 24 h. The cells were fixed and stained with anti–COX-IV antibody before image acquisition. Scale bar: 10 µm. Insets: 3.69 × 3.69 µm. (F) Quantification of COX-IV intensity from E represented as z-score from two independent experiments (>250 cells per experiment). The statistical significance between the control and the other conditions was calculated with ordinary one-way ANOVA followed by Tukey’s multiple comparison test. 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. BafA1; bafilomycin A1. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: SNX10 is a negative modulator of COX-IV turnover. (A) U2OS cells were reverse transfected with the indicated siRNA (20 nM) for 72 h, then treated or not with DFP (1 µM) for 24 h and with BafA1 (50 nM) the last 16 h, followed by western blotting for the indicated proteins. (B–D) Quantification of the data in A from n = 5, 3, and 6 independent experiments. Bars show mean values of the protein levels normalized to actin relative to control conditions (siCtrl control) ± SEM. Significance is assessed by two-way ANOVA followed by Tukey’s post hoc test. Data distribution was assumed to be normal. (E) U2OS cells with stable expression of mScarlet-RAB5 were reverse transfected with the indicated siRNA (20 nM) for 72 h, then treated or not with DFP for 24 h. The cells were fixed and stained with anti–COX-IV antibody before image acquisition. Scale bar: 10 µm. Insets: 3.69 × 3.69 µm. (F) Quantification of COX-IV intensity from E represented as z-score from two independent experiments (>250 cells per experiment). The statistical significance between the control and the other conditions was calculated with ordinary one-way ANOVA followed by Tukey’s multiple comparison test. 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. BafA1; bafilomycin A1. Source data are available for this figure: .

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

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

SNX10 modulates piecemeal mitophagy of OXPHOS components. (A) U2OS cells stably expressing the reporter pSu9-Halo-mGFP were reverse transfected with siCtrl or siSNX10 for 72 h. Cells were treated with TMR (100 nM) for 20 min, washed three times with PBS, and then treated with DFP (1 µM), DMOG (1 µM), or left untreated (control) for 24 h before lysis. (B and C) The relative Free Halo Tag expression was quantified using the formula (Free Halo/[Free Halo + Full Length]) normalized to actin. Data in B were log 2 transformed. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison tests to compare treatment groups to the control. Data represent mean ± SEM from three independent experiments. Data distribution was assumed to be normal but was not formally tested. (D) U2OS cells with inducible expression of SNX10-EGFP were treated with or without DFP (1 µM) for 24 h and stained with antibodies anti–COX-IV and anti-p62, followed by acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. Pixel intensity plot line graphs from control and DFP insets were generated with GraphPad Prism using two different y axis to enhance visualization. Scale bar: 10 µm. Insets: 4.08 × 4.08 µm. (E) U2OS cells subjected to reverse transfection with sip62 (20 nM) for 72 h were stained with an anti–COX-IV and anti-p62 antibody, prior to acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. 100×/1.45 NA oil immersion objective. Scale bar: 10 µm. (F) Quantification of COX-IV intensity from E represented as z-score from one experiment with individual data points corresponding to a single field of view (>30 cells per siRNA). Significance was determined by an unpaired two-tailed t test. Data distribution was assumed to be normal, but this was not formally tested. (G) Representative images of U2OS cells subjected to reverse transfection with siSNX10 (20 nM) for 72 h, followed by treatment with either IN1 or MRT for 24 h before fixation. After fixation, cells were stained with a COX-IV antibody, and images were captured using an ImageXpress Micro Confocal (Molecular Devices) at 20× magnification. (H and I) Quantification of COX-IV intensity from G represented as z-score from one independent experiment, with individual data points corresponding to a single field of view (>200 cells were analyzed for each condition). Significance was determined by one-way ANOVA followed by Šídák’s multiple comparisons 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. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: SNX10 modulates piecemeal mitophagy of OXPHOS components. (A) U2OS cells stably expressing the reporter pSu9-Halo-mGFP were reverse transfected with siCtrl or siSNX10 for 72 h. Cells were treated with TMR (100 nM) for 20 min, washed three times with PBS, and then treated with DFP (1 µM), DMOG (1 µM), or left untreated (control) for 24 h before lysis. (B and C) The relative Free Halo Tag expression was quantified using the formula (Free Halo/[Free Halo + Full Length]) normalized to actin. Data in B were log 2 transformed. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison tests to compare treatment groups to the control. Data represent mean ± SEM from three independent experiments. Data distribution was assumed to be normal but was not formally tested. (D) U2OS cells with inducible expression of SNX10-EGFP were treated with or without DFP (1 µM) for 24 h and stained with antibodies anti–COX-IV and anti-p62, followed by acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. Pixel intensity plot line graphs from control and DFP insets were generated with GraphPad Prism using two different y axis to enhance visualization. Scale bar: 10 µm. Insets: 4.08 × 4.08 µm. (E) U2OS cells subjected to reverse transfection with sip62 (20 nM) for 72 h were stained with an anti–COX-IV and anti-p62 antibody, prior to acquisition with a Nikon Ti2-E microscope with a Yokogawa CSU-W1 SoRa spinning disk 100×/1.45 NA oil immersion objective. 100×/1.45 NA oil immersion objective. Scale bar: 10 µm. (F) Quantification of COX-IV intensity from E represented as z-score from one experiment with individual data points corresponding to a single field of view (>30 cells per siRNA). Significance was determined by an unpaired two-tailed t test. Data distribution was assumed to be normal, but this was not formally tested. (G) Representative images of U2OS cells subjected to reverse transfection with siSNX10 (20 nM) for 72 h, followed by treatment with either IN1 or MRT for 24 h before fixation. After fixation, cells were stained with a COX-IV antibody, and images were captured using an ImageXpress Micro Confocal (Molecular Devices) at 20× magnification. (H and I) Quantification of COX-IV intensity from G represented as z-score from one independent experiment, with individual data points corresponding to a single field of view (>200 cells were analyzed for each condition). Significance was determined by one-way ANOVA followed by Šídák’s multiple comparisons 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. Source data are available for this figure: .

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: Stable Transfection, Expressing, Transfection, Control, Lysis, Transformation Assay, Comparison, Staining, Microscopy, Generated, Two Tailed Test

SNX10 regulates mitochondrial homeostasis and cell death in vivo. (A) Schematic diagram of human SNX10, zebrafish Snx10a, and Snx10b proteins. The percentage identity of the orthologues amongst each other and to the human counterpart is indicated. Also, the percentage identity of the zebrafish PX domains in comparison with the PX domain of human SNX10 is shown. (B) Temporal expression pattern of snx10a and snx10b . The graph shows the fold change in transcript levels relative to β-actin in whole zebrafish embryos from 1 to 5 dpf. Error bars indicate mean ± SEM. Data are collected from three individual experiments using 30 larvae for each experiment. (C) Dorsal and lateral view of the spatial expression pattern of snx10a and snx10b at 3 dpf as demonstrated by WM-ISH using an internal probe. Scale bars: 200 μm. Images are representative from three experiments. (D) Representative immunoblots of Snx10 and β-tubulin on whole embryo lysates from control (scrambled guide), single snx10a KO, and snx10ab DKO animals. β-tubulin served as a loading control. (E) Representative immunoblots of Snx10, Samm50, Cox-IV, and actin on whole embryo lysates from control (scrambled guide) and snx10ab DKO treated with 100 µM DMOG or DMSO control for 24 h at 2 dpf. (F) Quantification of the Cox-IV signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. (G) Quantification of the Samm50 signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. Data distribution was assumed to be normal but was not formally tested. (H) Representative images of TUNEL assay on control (scrambled sgRNA) and snx10ab DKO larvae treated with 100 µM DMOG or DMSO control for 24 h at 3 dpf. Orientation lateral. Scale bar: 500 μm. (I) Quantification of the mean fluorescent intensity from demarcated brain regions of images in H. A total of 45 control larvae (scrambled sgRNA) and 41 snx10ab DKO larvae were used for quantification, respectively. Values were normalized to control DMSO values. Control larvae were treated with DMOG as a comparison to snx10ab DKO larvae. n = 2 independent experiments. Plots demonstrate data distribution and median value (red line). Significance was determined by two-way ANOVA followed by Tukey’s post hoc test to compare all groups. Data distribution was assumed to be normal but was not formally tested. (J) Quantification of ROS levels obtained via FACS analysis of control (scrambled sgRNA), snx10ab DKO, and positive control larvae at 3 dpf incubated with MitoSOX. The values were presented as relative values after normalizing to control. Error bars indicate mean ± SEM. Quantification was from at least two independent experiments. Data distribution was assumed to be normal but was not formally tested. (K) Representative whole mount images shown as maximum intensity projection from z-stack of TUNEL assay performed on control (scrambled sgRNA) and snx10ab DKO larvae treated with or without 100 µM NAC at 3 dpf. Orientation lateral. Scale bar: 500 µm. (L) Quantification of the number of white puncta (dots) from the demarcated whole brain region shown in K. A total of >20 control larvae (scrambled gRNA) and >20 snx10ab DKO larvae treated or not with 100 µM NAC were used for quantification. Values were normalized to control values. Data are collected from three individual experiments. Plots show data distribution and median value (red line). Significance was determined by one-way Brown–Forsythe and Welch’s ANOVA tests to compare all groups. 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. NAC; N-acetyl cysteine. Source data are available for this figure: .

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: SNX10 regulates mitochondrial homeostasis and cell death in vivo. (A) Schematic diagram of human SNX10, zebrafish Snx10a, and Snx10b proteins. The percentage identity of the orthologues amongst each other and to the human counterpart is indicated. Also, the percentage identity of the zebrafish PX domains in comparison with the PX domain of human SNX10 is shown. (B) Temporal expression pattern of snx10a and snx10b . The graph shows the fold change in transcript levels relative to β-actin in whole zebrafish embryos from 1 to 5 dpf. Error bars indicate mean ± SEM. Data are collected from three individual experiments using 30 larvae for each experiment. (C) Dorsal and lateral view of the spatial expression pattern of snx10a and snx10b at 3 dpf as demonstrated by WM-ISH using an internal probe. Scale bars: 200 μm. Images are representative from three experiments. (D) Representative immunoblots of Snx10 and β-tubulin on whole embryo lysates from control (scrambled guide), single snx10a KO, and snx10ab DKO animals. β-tubulin served as a loading control. (E) Representative immunoblots of Snx10, Samm50, Cox-IV, and actin on whole embryo lysates from control (scrambled guide) and snx10ab DKO treated with 100 µM DMOG or DMSO control for 24 h at 2 dpf. (F) Quantification of the Cox-IV signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. (G) Quantification of the Samm50 signal intensity from blots in E normalized to control DMSO signal intensity from n = 4 experiments. Error bars indicate mean ± SEM, unpaired Student’s t test was performed to assess significance. Data distribution was assumed to be normal but was not formally tested. (H) Representative images of TUNEL assay on control (scrambled sgRNA) and snx10ab DKO larvae treated with 100 µM DMOG or DMSO control for 24 h at 3 dpf. Orientation lateral. Scale bar: 500 μm. (I) Quantification of the mean fluorescent intensity from demarcated brain regions of images in H. A total of 45 control larvae (scrambled sgRNA) and 41 snx10ab DKO larvae were used for quantification, respectively. Values were normalized to control DMSO values. Control larvae were treated with DMOG as a comparison to snx10ab DKO larvae. n = 2 independent experiments. Plots demonstrate data distribution and median value (red line). Significance was determined by two-way ANOVA followed by Tukey’s post hoc test to compare all groups. Data distribution was assumed to be normal but was not formally tested. (J) Quantification of ROS levels obtained via FACS analysis of control (scrambled sgRNA), snx10ab DKO, and positive control larvae at 3 dpf incubated with MitoSOX. The values were presented as relative values after normalizing to control. Error bars indicate mean ± SEM. Quantification was from at least two independent experiments. Data distribution was assumed to be normal but was not formally tested. (K) Representative whole mount images shown as maximum intensity projection from z-stack of TUNEL assay performed on control (scrambled sgRNA) and snx10ab DKO larvae treated with or without 100 µM NAC at 3 dpf. Orientation lateral. Scale bar: 500 µm. (L) Quantification of the number of white puncta (dots) from the demarcated whole brain region shown in K. A total of >20 control larvae (scrambled gRNA) and >20 snx10ab DKO larvae treated or not with 100 µM NAC were used for quantification. Values were normalized to control values. Data are collected from three individual experiments. Plots show data distribution and median value (red line). Significance was determined by one-way Brown–Forsythe and Welch’s ANOVA tests to compare all groups. 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. NAC; N-acetyl cysteine. Source data are available for this figure: .

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: In Vivo, Comparison, Expressing, Western Blot, Control, TUNEL Assay, Positive Control, Incubation

s nx10 is highly expressed in the brain of zebrafish larvae and its depletion elevates oxidative stress. (A) Dorsal view of spatial expression pattern of snx10a and snx10b at 2, 4, and 5 dpf as demonstrated by WM-ISH using an internal antisense (AS) probe. Scale bar = 200 μm. Images are representative of three experiments. Control 2 dpf larvae hybridized to a sense probe (S). (B) Illustration of sgRNA-binding regions on snx10a and snx10b gene, respectively. (C) Temporal expression levels of bnip3 and bnip3l transcripts in DMSO- and DMOG-treated WT zebrafish larvae at 3 dpf. The graph shows the fold change in transcript levels relative to β-actin and normalized to DMSO 2 −ΔΔCt levels. Error bars indicate mean ± SEM. Data are collected from three individual experiments. Significance was determined by two-way ANOVA test to compare all groups with the two variables. (D) Temporal expression levels of cox-iv and samm50 transcripts in control and snx10ab _DKO zebrafish larvae treated with or without 100 µm DMOG at 3 dpf. The graph shows the fold change in transcript levels relative to β-actin and normalized to DMSO 2 −ΔΔCt levels. Error bars indicate mean ± SEM. Data are collected from three individual experiments. Significance was determined by one-way ANOVA test to compare all groups with the individual variable. 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. (E) Representative dot plots showing the region selected for FACS analysis from control and snx10ab DKO zebrafish larvae at 3 dpf using MitoSOX reagent. H 2 O 2 was added to the water for 1 h as a positive control. (F) Representative FACS plot showing oxidative stress in control and snx10ab DKO zebrafish larvae at 3 dpf using the MitoSOX reagent. H 2 O 2 added in water served as positive control.

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: s nx10 is highly expressed in the brain of zebrafish larvae and its depletion elevates oxidative stress. (A) Dorsal view of spatial expression pattern of snx10a and snx10b at 2, 4, and 5 dpf as demonstrated by WM-ISH using an internal antisense (AS) probe. Scale bar = 200 μm. Images are representative of three experiments. Control 2 dpf larvae hybridized to a sense probe (S). (B) Illustration of sgRNA-binding regions on snx10a and snx10b gene, respectively. (C) Temporal expression levels of bnip3 and bnip3l transcripts in DMSO- and DMOG-treated WT zebrafish larvae at 3 dpf. The graph shows the fold change in transcript levels relative to β-actin and normalized to DMSO 2 −ΔΔCt levels. Error bars indicate mean ± SEM. Data are collected from three individual experiments. Significance was determined by two-way ANOVA test to compare all groups with the two variables. (D) Temporal expression levels of cox-iv and samm50 transcripts in control and snx10ab _DKO zebrafish larvae treated with or without 100 µm DMOG at 3 dpf. The graph shows the fold change in transcript levels relative to β-actin and normalized to DMSO 2 −ΔΔCt levels. Error bars indicate mean ± SEM. Data are collected from three individual experiments. Significance was determined by one-way ANOVA test to compare all groups with the individual variable. 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. (E) Representative dot plots showing the region selected for FACS analysis from control and snx10ab DKO zebrafish larvae at 3 dpf using MitoSOX reagent. H 2 O 2 was added to the water for 1 h as a positive control. (F) Representative FACS plot showing oxidative stress in control and snx10ab DKO zebrafish larvae at 3 dpf using the MitoSOX reagent. H 2 O 2 added in water served as positive control.

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: Expressing, Control, Binding Assay, Positive Control

Model showing the role of SNX10 as a modulator of endocytic transport and piecemeal mitophagy. In vitro: Under normal conditions (control), SNX10 localizes to early endosomes (RAB5 and EEA1 positive) and late endosomes (CD63 positive) together with endocytic cargo (as EGFR). SNX10-positive structures are also observed near mitochondria. Upon hypoxia-mimicking conditions (induced by DFP or DMOG) SNX10 vesicles co-localize with CD63, LC3B, and p62 and incorporate selected mitochondrial components (including COX-IV), indicating a role for SNX10 in selective mitochondrial degradation. Upon SNX10 knockdown (SNX10 KD), early endosomes appear smaller and more numerous, with a corresponding reduced degradation of EGFR. In contrast, the turnover of mitochondrial COX-IV and ATP synthase is increased, along with reduced oxygen consumption rate and ATP production, reflecting impaired mitochondrial function. The arrow thicknesses indicate the extent of the different pathways under different conditions. In vivo: In zebrafish larvae, Snx10ab DKO leads to decreased levels of mitochondrial proteins (Cox-IV and Samm50), increased levels of ROS, and elevated cell death in the brain region, as shown by TUNEL staining. The snx10ab DKO–mediated cell death can be rescued by treatment with the antioxidant NAC, suggesting that Snx10 modulates piecemeal mitophagy to limit oxidative stress and maintain mitochondrial homeostasis. NAC; N-acetyl cysteine.

Journal: The Journal of Cell Biology

Article Title: SNX10 functions as a modulator of piecemeal mitophagy and mitochondrial bioenergetics

doi: 10.1083/jcb.202404009

Figure Lengend Snippet: Model showing the role of SNX10 as a modulator of endocytic transport and piecemeal mitophagy. In vitro: Under normal conditions (control), SNX10 localizes to early endosomes (RAB5 and EEA1 positive) and late endosomes (CD63 positive) together with endocytic cargo (as EGFR). SNX10-positive structures are also observed near mitochondria. Upon hypoxia-mimicking conditions (induced by DFP or DMOG) SNX10 vesicles co-localize with CD63, LC3B, and p62 and incorporate selected mitochondrial components (including COX-IV), indicating a role for SNX10 in selective mitochondrial degradation. Upon SNX10 knockdown (SNX10 KD), early endosomes appear smaller and more numerous, with a corresponding reduced degradation of EGFR. In contrast, the turnover of mitochondrial COX-IV and ATP synthase is increased, along with reduced oxygen consumption rate and ATP production, reflecting impaired mitochondrial function. The arrow thicknesses indicate the extent of the different pathways under different conditions. In vivo: In zebrafish larvae, Snx10ab DKO leads to decreased levels of mitochondrial proteins (Cox-IV and Samm50), increased levels of ROS, and elevated cell death in the brain region, as shown by TUNEL staining. The snx10ab DKO–mediated cell death can be rescued by treatment with the antioxidant NAC, suggesting that Snx10 modulates piecemeal mitophagy to limit oxidative stress and maintain mitochondrial homeostasis. NAC; N-acetyl cysteine.

Article Snippet: Primary antibodies used: Anti-HaloTag (G9211, 1:1,000, ms; Promega), ATP5J (HPA031069, 1:200, rb; Atlas Antibodies), β-Actin (#3700, 1:5,000, ms; Cell Signaling Technology), CD63 (#H5C6, 1:200, ms; DHB) Citrate Synthase (#14309, 1:1,000, rb; Cell Signaling Technology), Clathrin (#SM5011P, 1:200; Acris), COX-IV (#4850S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), EEA1 (#610457; 1:250, ms; BD Biosciences), GAPDH (Ab9484, 1:5,000, ms; Abcam), GAPDH (#5174, 1:1,000, rb; Cell Signaling), GFP (# 632569, 1:1,000, ms; Takara) EGFR (#20-ES04, 1:1,000 for WB and 1:200 for IF, sh; Fitzgerald), P-EGFR (Tyr1068) (#3777, 1:1,000, rb; Cell Signaling), PDH (#2784S, 1:1,000 for WB and 1:200 for IF, rb; Cell Signaling Technology), LAMP1 (#sc-20011, 1:250, ms; Santa Cruz Biotechnology), LC3B (#PM036, 1:250, rb; MBL), SAMM50 (NBP1–84509, 1:200, rb; Novus Biologicals), SNX10 (HPA015605, 1:1,000, rb; Atlas Antibodies), alpha Tubulin (GTX628802, 1:5,000, ms; GeneTex), TIMM23 (# 611223, 1:1,000 for WB 1:250 for IF, ms; BD Biosciences), and TOMM20 (#17764, 1:200, ms; Santa Cruz).

Techniques: In Vitro, Control, Knockdown, In Vivo, TUNEL Assay, Staining