Journal: bioRxiv

Article Title: Ubiquitin Ligase ITCH Regulates Life Cycle of SARS-CoV-2 Virus

doi: 10.1101/2024.12.04.624804

Figure Lengend Snippet: (A) Culture medium from vT2-WT and vT2-ITCH-KO cells expressing HA-tagged ubiquitin (Ub) and Flag-2×Strep tagged E was harvested for Strep AP. A decrease of the extracellular E protein induced by ITCH ablation was visualized by immunoblot (left) and dot blot analysis (right). (B) Lysates from HEK293 cells expressing Flag-tagged E with ITCH or ITCH-CS were subjected to Flag IP, followed by immunoblotting for autophagosome cargo receptors. E specifically precipitated p62 and ITCH promoted their interaction. (C-E) HEK293 cells were transfected with Flag-tagged E with ITCH or ITCH-CS. 24 h later, cells were analyzed by immunofluorescence with Flag, ITCH and p62 or LC3B or LAMP1 antibodies. ITCH enhanced the colocalization between E and p62 (C) or LC3B (D), while no change in the colocalization between E and LAMP1 (E) was noted. Scale bar, 10 μm. (F) Culture media and denatured lysates from control (CTRL) and p62 knock down (#1, #2) HEK293 cells expressing HA-tagged ubiquitin (Ub), Flag-tagged E were subjected to Flag IP (with incorporation of a washing step with urea before elution for culture media samples), followed by immunoblotting or dot blot analysis. p62 depletion resulted in the accumulation of intracellular E (both unmodified and ubiquitinated), while decreasing the level of extracellular E (n=3). (G, H) vT2-WT and vT2-ITCH-KO cells infected with SARS-CoV-2 at 1 MOI for 10 h were subjected to immunofluorescence analysis with E and p62 or LC3B antibodies. ITCH-ablation decreased the colocalization between E and p62 (G) or LC3B (H). Scale bar, 10 μm. (I) A model of the function of ITCH in promoting autophagosome-mediated SARS-CoV-2 virion egress. ITCH-dependent ubiquitin modification enhances E binding with S and M binding with non-ubiquitinated E, resulting in the increase in virion formation and p62-dependent autophagosome targeting for release.

Article Snippet: The following primary antibodies were used: Flag (Sigma, F1804); Flag (Sigma, F3165); Flag (Cell Signaling Technology, 14793S); GAPDH (Invitrogen, MA5-27912); β-tubulin (Cell Signaling Technology, 2128S);s (BioLegend, 688102); Strep (Invitrogen, MA5-17283); CBD (New England BioLabs, E8034S); ubiquitin (Cell Signaling Technology, 58395S); K63-linkage-specific antibody (Enzo Life Sciences, BML-PW0600-0100); K48-linkage-specific antibody (Cell Signaling Technology, 8081S); Spike (Proteintech, 28867-1-AP); M (Proteintech, 28882-1-AP); E (Proteintech, 28904-1-AP); ITCH (Santa Cruz, sc-28367); ITCH (Novus Biologicals, NB100-68142); p62 (Cell Signaling Technology, 88588S and 7695S); GM130 (Proteintech, 11308-1-AP); LAMP1 (Cell Signaling Technology, 9091S); OPTN (Cayman Chemical, 100002); LC3 (Proteintech,14600-1-AP); LC3B (Cell Signaling Technology, 3868S); SARS-CoV-2 Membrane protein (Cell Signaling Technology, 15333S); SARS-CoV-2 Envelope protein (Cell Signaling Technology, 74698S); furin (Proteintech, 18413-1-AP); Cathepsin L (Proteintech, 10938-1-AP); NDP52 (Proteintech, 12229-1-AP); NBR1 (Proteintech, 16004-1-AP); FAM134B (Proteintech, 21537-1-AP); RTN3 (Proteintech, 12055-2-AP) and NIX (Proteintech, 12986-1-AP).

Techniques: Expressing, Western Blot, Dot Blot, Transfection, Immunofluorescence, Control, Knockdown, Infection, Modification, Binding Assay

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) Enzyme-linked immunosorbent assay (ELISA assay) showed that the platelet autophagy biomarkers in cirrhotic patients were significantly reduced compared to the healthy controls. (B–C) The levels of autophagy biomarkers negatively correlated with the Child-Pugh score and the severity of TP. (D) Transmission electron microscopy showed that TP patients had significantly reduced platelet autophagosomes compared to healthy controls. (E) The serum endogenous H 2 S levels in TP patients were significantly reduced compared to healthy controls. (F) Spearman correlation analysis indicated that serum H 2 S levels positively correlated with platelet autophagy markers LC3 and ATG7 and negatively correlated with SQSTM1 in TP patients. * indicates p <0.05. Scale bar: 1 µm.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Enzyme-linked Immunosorbent Assay, Transmission Assay, Electron Microscopy

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) NaHS increased the relative LC3-II expression and attenuated the SQSTM1 expression dose-dependently. (B) Compared with the Baf A1 treatment alone, the LC3-II expression was significantly increased after NaHS+Baf A1 treatment indicated that the platelet autophagosome synthesis was increased by H 2 S. * indicates p <0.05. “+” indicates using Bafilomycin (Baf) A1 treatment, “−” indicates using culture medium treatment. NC, negative control.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Expressing, Negative Control

Journal: Journal of Clinical and Translational Hepatology

Article Title: Hydrogen Sulfide Promotes Platelet Autophagy via PDGFR-α/PI3K/Akt Signaling in Cirrhotic Thrombocytopenia

doi: 10.14218/JCTH.2024.00101

Figure Lengend Snippet: (A) Establishment of mice model of cirrhotic thrombocytopenia. (B) NaHS injection increased the platelet LC3-II expression and reduced the SQSTM1 expression in a dose-dependent manner. After injection with hydroxocobalamin (HC, H 2 S scavenger), the platelet LC3-II expression was significantly reduced, while the SQSTM1 expression was significantly increased. (C) After injection of different concentrations of NaHS or HC, there was no significant change in the platelet count of the mice at an indicated time. (D) After the injection of different concentrations of NaHS, the platelet aggregation rate significantly decreased on Day 15. * indicates p <0.05. i.p, intraperitoneal injection; biw, twice a week; NS, 0.9% normal saline; MPA, microscopic polyangiitis.

Article Snippet: The platelet lysate was centrifuged at 12,000 g for 5 m. The supernatant was collected for ELISA assays to measure the levels of ATG7 (Human ATG7 ELISA Kit, Proteintech KE00276), BECN1 (Human Beclin 1 ELISA Kit, ABclonal RK00973), LC3 (Total LC3B ELISA Kit, Cell Signaling Technology #35172), and SQSTM1 (Human SQSTM1/Sequestosome-1 ELISA Kit, ABclonal RK04613).

Techniques: Injection, Expressing, Saline

Journal: bioRxiv

Article Title: Development of the ULK1-Recruiting Chimeras (ULKRECs) to enable proximity-induced and ULK1-dependent degradation of mitochondria

doi: 10.1101/2024.04.15.589474

Figure Lengend Snippet: ULKRECs rely on ULK1 for the redirection of autophagy components to mitochondria and subsequent mitophagic degradation (A) Western blot of mouse embryonic fibroblasts (MEF) WT and MEF ULK1/2 -/- treated with 1 μM ULKRECs, 5 μM CCCP or both and probed for ULK1, Mfn2 and Actin – quantification of the relative intensity of Mfn2 shown in (B), normalised to Actin. Representative immunofluorescence of MEF WT (C) and MEF ULK1/2 -/- (D) treated with 5 μM CCCP, 1 μM ULKRECs or both and immunostained for Alexa 488 ATP5a and Alexa 647 LC3B. Quantification of the colocalised LC3 to ATP5a spot area shown in WT vs ULK1/2 -/- treated MEF cells shown in (E). Data shown are mean ± SD, n = 3, *p<0.05, **p<0.01, ***p<0.001, Two-way ANOVA with Tukey’s multiple comparisons test.

Article Snippet: Plates were incubated overnight at 4°C with 1:400 LC3B (D11) XP Rabbit mAb Alexa Fluor 647 Conjugate (Cell Signalling Technologies, #65299) and 1:400 ATP5a (Cell Signalling Technologies, #18023) lightning-linked to Alexa Fluor 488.

Techniques: Western Blot, Immunofluorescence

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: BITC increased the expression levels of LC3B and p62 proteins AGS cells. (A) AGS cells were treated with 10 µM BITC for 8, 12, or 24 h. Whole cell lysate was prepared, and LC3B and p62 levels were determined by western blotting. β-actin was used as a loading control. (B) AGS cells were treated with 5, 10, or 15 µM BITC for 24 h. Whole cell lysate was prepared, and LC3B and p62 levels were determined by western blotting. β-actin was used as a loading control. (C) AGS cells were treated with 5, 10, or 15 μM BITC for 12 h and mRNA level was assessed by q-PCR. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01, and *** p <0.001 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Control

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: BITC-induced LC3B conjugation in AGS cells was not dependent on class III PI3K. (A) For the single treatment, AGS cells were treated with 2.5, 5, 10, or 15 μM BITC for 24 h. For the combined treatment, the cells were pre-treated with 0.5 µM wortmannin for 2 h followed by 10 µM BITC for 24 h. Then, the whole cell lysate was prepared, and the class III PI3K protein level was measured by western blotting. β-actin was used as a loading control. (B) The cytotoxicity of wortmannin was detected by MTT assay after treating the cells with different concentrations of wortmannin for 24 and 48 h. (C) AGS cells were pre-treated with 0.25 or 0.5 µM wortmannin for 2 h followed by 10 or 15 µM BITC for 24 h. After cell lysis, the change in LC3B II level was detected by western blotting. β-actin was used as a loading control. Data were calculated from three independent experiments and expressed as the mean ± SEM. ** p <0.01 and *** p <0.001 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Conjugation Assay, Western Blot, Control, MTT Assay, Lysis

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: Additive effect of BITC and bafilomycin A1 on LC3B accumulation in AGS cells. (A) AGS cells were treated with BITC with or without 100 nM bafilomycin A1 for 24 h. Whole cell lysate was prepared, and the LC3B II level was measured by western blotting. β-actin was used as a loading control. (B) Increased LC3B protein expression was further confirmed by immunostaining with anti-LC3B antibody and was detected via Alexa Flour 488-conjugated secondary antibody (green) under a fluorescence microscope (scale-bar=50 μm). DAPI (blue) was used for nuclear staining. (C) The cytotoxicity of bafilomycin A1 was investigated by MTT assay after treating the cells with different concentrations of Bafilomycin A1 for 24 and 48 h. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01, *** p <0.001 versus the control, and # p <0.05 versus BITC treatment alone.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Western Blot, Control, Expressing, Immunostaining, Fluorescence, Microscopy, Staining, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: No relationship between BITC-induced LC3 B accumulation and cytotoxicity. (A) AGS cells were transfected with 50 nM LC3B siRNA or control siRNA for 12 h and incubated with 10 µM BITC for 8 h. Whole cell lysate was prepared, and siRNA efficacy was confirmed by detecting the LC3B protein expression via western blotting. β-actin was used as a loading control. (B) AGS cells were transfected with 50 nM LC3B siRNA or control siRNA for 12 h, followed by 10 µM BITC treatment for 24 h. Cell viability changes were evaluated by MTT assay. Data were calculated from three independent experiments and expressed as the mean ± SEM. * p <0.05, ** p <0.01 versus the control.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Transfection, Control, Incubation, Expressing, Western Blot, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Benzyl Isothiocyanate-Induced Cytotoxicity via the Inhibition of Autophagy and Lysosomal Function in AGS Cells

doi: 10.4062/biomolther.2022.019

Figure Lengend Snippet: The proposed mechanism of BITC-induced cytotoxicity through autophagy inhibition and lysosomal dysfunction in AGS cells. In human gastric adenocarcinoma AGS cells, BITC induced cell death by caspase-dependent apoptosis and inhibition of cytoprotective autophagy. It inhibited autophagy degradation through lysosomal dysfunction, which decreased both the quality and quantity of cathepsin proteins. Moreover, BITC may inhibit the autophagy initiation and elongation steps, since it decreased the expression levels of Beclin1, Atg5-Atg12 complex, and class III PI3K proteins. However, because of increased mRNA transcription and decreased lysosomal degradation, BITC also caused the accumulation of LC3B and p62. Nevertheless, there was no direct relationship between BITC-induced LC3B II accumulation and cytotoxicity.

Article Snippet: Anti-rabbit IgG (H+L), F(ab’)2 fragment (Alexa Fluor ® 488 conjugate), normal goat serum, LC3B siRNA II, and control siRNA were purchased from Cell Signaling Technology.

Techniques: Inhibition, Expressing