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 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 log 2 FC >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.

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

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 structures containing mitochondria mature into late endosomes. (A and C) U2OS cells with inducible expression of SNX10-EGFP were treated with DFP (1 µM) or DMOG (1 µM) for 24 h, stained with MitoTracker Deep Red FM (100 nM) for 30 min, followed by immunofluorescence with antibodies anti-LC3B and anti-EEA1 (A) or anti-CD63 (C), 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: 5.52 × 5.52 µm (A), 6.62 × 6.62 µm (C). (B–D) Pixel intensity plots for line in control, DFP, and DMOG insets, respectively for A and C.

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, Immunofluorescence, Microscopy, 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: 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

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

Journal: Neural Regeneration Research

Article Title: Tranylcypromine upregulates Sestrin 2 expression to ameliorate NLRP3-related noise-induced hearing loss

doi: 10.4103/NRR.NRR-D-24-00130

Figure Lengend Snippet: TCP upregulates autophagy in mouse cochleae. (A) Representative images of HCs co-immunostained for nuclei (DAPI; blue), the lysosomal marker LAMP1 (green), and the autophagosome marker LC3B (red) to detect HC autophagy in mice after a noise-induced PTS ( n = 3 cochleae from three mice per group). Scale bar: 20 μm. (B) Autolysosomes (LC3B/LAMP1) quantification in the control, NE + saline, and NE + TCP groups. (C) Immunoblot analysis of LC3B expression in mouse cochleae ( n = 3 cochleae from three mice per group). All experiments were replicated thrice. Data are presented as mean ± standard of the mean. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (one-way analysis of variance with Bonferroni’s multiple comparison test). Ctrl: Control; NE: noise exposure; ns: not significant; OHCs: outer hair cells; PTS: permanent threshold shifts; TCP, tranylcypromine.

Article Snippet: First, the membranes were incubated overnight at 4°C with the following primary antibodies: mouse monoclonal anti-SESN2 (1:200, sc-393195, Santa Cruz Biotechnology, Santa Cruz, CA, USA), rabbit polyclonal anti-NLRP3 (1:400, NBP2-12446, Novus Biologicals, Centennial, CO, USA), rabbit polyclonal anti-LC3B (1:250, ab192890, Abcam, Cambridge, UK), rat monoclonal anti-LAMP1 (1:200, 14-1071-82, Invitrogen, Waltham, MA, USA), and mouse monoclonal 4-hydroxynonenal (4-HNE) (1:250, MA5-27570, Invitrogen).

Techniques: Marker, Control, Saline, Western Blot, Expressing, Comparison

Journal: International Journal of Molecular Medicine

Article Title: Elevated neuregulin-1 expression modulates tumor malignancy and autophagy in esophageal squamous cell carcinoma

doi: 10.3892/ijmm.2025.5503

Figure Lengend Snippet: Effect of NRG1 on autophagy activity of ESCC cells. (A) Transfected CE48T, (B) CE81T and (C) CE146T ESCC cells were stained with autophagosome (DAP, 0.1 µ M) or autolysosome dye (DAL, 0.5 µ M). ESCC cells were treated with CQ for autophagy induction and inhibition, respectively. The autophagosome and autolysosome puncta were observed under a confocal microscope. Scale bar, 10 µ m. (D) Protein levels of NRG1, p62 and LC3B following silencing were (E) quantified. (F) Transfected ESCC cells were treated with CQ (20 µ M) to examine LC3B-II turnover using immunoblotting. (G) LC3B-II was used to determine autophagic flux. * P<0.05, ** P<0.01, *** P<0.001 vs. siCtrl. NRG, neuregulin-1; ESCC, esophageal squamous cell carcinoma; DAP, dye of autophagosome; DAL, dye of autolysosome; EBSS, Earle's Balanced Salt Solution; CQ, chloroquine; si, small interfering; Ctrl, control; ACTB, β-actin.

Article Snippet: The membranes were blocked with 5% BSA (cat. no. A5611; Sigma-Aldrich; Merck KGaA) at room temperature for 3 h and incubated with 1,000-fold diluted primary antibodies against NRG1 (cat. no. ab191139, Abcam), phosphorylated (p-)AKT (cat. no. 4060), AKT (cat. no. 4691), p-cellular rapidly accelerated fibrosarcoma (p-cRAF; cat. no. 9427), cRAF (cat. no. 53745), β-actin (cat. no. 3700; all Cell Signaling Technology, Inc.), LC3B (cat. no. ARG55799) and p62 (cat. no. ARG55040; both Arigo Biolaboratories Corp.) at 4°C overnight.

Techniques: Activity Assay, Transfection, Staining, Inhibition, Microscopy, Western Blot, Control

Journal: International Journal of Molecular Medicine

Article Title: Elevated neuregulin-1 expression modulates tumor malignancy and autophagy in esophageal squamous cell carcinoma

doi: 10.3892/ijmm.2025.5503

Figure Lengend Snippet: Clinical association between gene expression of NRG1 and LC3B in patients with ESCC. Based on an ESCC dataset from The Cancer Genome Atlas, Kaplan-Meier plots were used to analyze the association between NRG1 and LC3B1 expression of patients with ESCC and (A) overall, (B) progression-free interval, (C) disease-specific and (D) disease-free interval survival. Association between NRG1 and LC3B2 gene expression and (E) overall, (F) progression-free interval, (G) disease-specific and (H) disease-free interval survival. NRG1, neuregulin-1; ESCC, esophageal squamous cell carcinoma; L, low expression; H, high expression; Either, NRG1(H)cRAF(L) or NRG1(L)/cRAF(H); yrs, years.

Article Snippet: The membranes were blocked with 5% BSA (cat. no. A5611; Sigma-Aldrich; Merck KGaA) at room temperature for 3 h and incubated with 1,000-fold diluted primary antibodies against NRG1 (cat. no. ab191139, Abcam), phosphorylated (p-)AKT (cat. no. 4060), AKT (cat. no. 4691), p-cellular rapidly accelerated fibrosarcoma (p-cRAF; cat. no. 9427), cRAF (cat. no. 53745), β-actin (cat. no. 3700; all Cell Signaling Technology, Inc.), LC3B (cat. no. ARG55799) and p62 (cat. no. ARG55040; both Arigo Biolaboratories Corp.) at 4°C overnight.

Techniques: Gene Expression, Expressing