Journal: Journal of Translational Medicine

Article Title: Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

doi: 10.1186/s12967-023-04796-y

Figure Lengend Snippet: Erbin deficiency aggravated inflammatory response and organ injuries and deteriorated prognosis of sepsis. A Erbin −/− mice and WT mice were subjected to CLP procedure and euthanasia of mice after CLP 6 h, 24 h, and 48h. The serum TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured. B , C Histopathologic changes in the liver tissue, serum AST, and ALT levels were detected after CLP. D – H Histopathologic changes and the injury score of lung tissue, the wet/dry ratio, the protein concentration, and the PMN/total cells in BALF were measured. I The survival rates of WT and Erbin −/− mice subjected to CLP were observed. J BMDMs were treated with MDP (6 h), and the TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured in the supernatant of Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs. Each group had 10 mice. The data are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 from ANOVA followed by Tukey’s post hoc test

Article Snippet: Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.

Techniques: Protein Concentration

Journal: Journal of Translational Medicine

Article Title: Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

doi: 10.1186/s12967-023-04796-y

Figure Lengend Snippet: Erbin deficiency exacerbates autophagy impairment in sepsis mice and MDP-treated BMDMs. A, C WT and Erbin −/− mice were subjected to CLP 6 h, and the mouse liver and lung tissues were examined by transmission electron microscopy (TEM). B, D Autophagosomes were quantified by analysis of ( A ) and ( C ) results. The arrow indicates autophagosomes. Scale bar: 1 μm. E Western blotting was used to estimate the protein levels of LC3-I/II and p62 at 6 h, 24 h, and 48 h after CLP in mice liver tissue. F Western blotting was used to estimate the protein levels of LC3-I/II and p62 at 6 h, 24 h, and 48 h after CLP in mice lung tissue. G CQ (60 mg/kg) was used as a pretreatment before CLP to inhibit autophagosome-lysosome fusion. The protein expression of LC3-I/II and p62 in liver tissues were detected by western blotting. H CQ (60 mg/kg) was used as a pretreatment before CLP to inhibit autophagosome-lysosome fusion. The protein expression of LC3-I/II and p62 in lung tissues was detected by western blotting. I BMDMs were examined by transmission electron microscopy (TEM). The arrow indicates autophagosome. Scale bar: 1 μm. J Autophagosomes were quantified by analysis of results of ( I ). K The LC3-I/II fluorescence was detected by confocal microscopy, scale bar: 50μm. L The LC3-I/II fluorescence staining was quantified by analysis of results of ( K ). M Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP (6 h), and the western blotting was used to estimate the protein levels of LC3-I/II, p62, Beclin1, ATG5, and ATG7. 200 nM RAPA was used as a positive control for autophagy. N CQ (5 μM) was used as a pretreatment before MDP to inhibit autophagosome-lysosome fusion. The LC3-I/II conversion and p62 accumulation were detected by western blotting in BMDMs. * P < 0.05, ** P < 0.01, *** P < 0.001 from ANOVA followed by Tukey’s post hoc test

Article Snippet: Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.

Techniques: Transmission Assay, Electron Microscopy, Western Blot, Expressing, Fluorescence, Confocal Microscopy, Staining, Positive Control

Journal: Journal of Translational Medicine

Article Title: Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

doi: 10.1186/s12967-023-04796-y

Figure Lengend Snippet: Erbin-mediated autophagy response alleviated sepsis-induced inflammatory response and organ injury. A Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were pretreated with CQ (5 μM) for 12 h and then treated with MDP for 6 h. The TNF-α, IL-6, IL-1β, HMGB1, and IL-10 levels were measured in the supernatant of Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs. B The Erbin −/− and WT littermate mice were pretreated with CQ (60 mg/kg) before CLP 1 h, and mice were euthanized after CLP 6 h. The serum TNF-α, IL-6, IL-1β, HMGB1 and IL-10 levels were measured by ELISA. C , D Histopathologic changes in the liver and lung tissues were detected by H&E staining. Each group had 10 mice. Data shown are representative of 3 independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001 from ANOVA followed by Tukey’s post hoc test

Article Snippet: Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.

Techniques: Enzyme-linked Immunosorbent Assay, Staining

Journal: Journal of Translational Medicine

Article Title: Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

doi: 10.1186/s12967-023-04796-y

Figure Lengend Snippet: Erbin enhanced lysosome biogenesis in BMDMs. A BMDMs were treated with 10 μg/ml MDP (6 h) and then stained with LysoTracker Red for 15 min. The fluorescence intensity of treated cells was measured by confocal microscopy. Scale bar: 25 μm. B Fluorescence puncta were quantified by analysis of results of ( A ). C Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP for 6 h, and western blotting was used to estimate the protein levels of LAMP2, CTSB, ATP6V1A, and ATP6V1B2. * P < 0.05, ** P < 0.01, *** P < 0.001 from ANOVA followed by Tukey’s post hoc test

Article Snippet: Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.

Techniques: Staining, Fluorescence, Confocal Microscopy, Western Blot

Journal: Journal of Translational Medicine

Article Title: Erbin accelerates TFEB-mediated lysosome biogenesis and autophagy and alleviates sepsis-induced inflammatory responses and organ injuries

doi: 10.1186/s12967-023-04796-y

Figure Lengend Snippet: Erbin promotes TFEB nuclear translocation and transcription activity. A Erbin fl/fl and Erbin fl/fl/Lyz2−cre BMDMs were treated with MDP for 6 h, and western blotting was used to estimate the protein levels of total TFEB and pTFEB (S211). B THP-1 macrophages were transfected with vector control or HA-Erbin for 36 h prior to western blot assays. C BMDMs were treated with 10μg/ml MDP for 6 h and then incubated with TFEB antibody. The fluorescence intensity of treated cells was measured by confocal microscopy. Scale bar: 25 μm. D THP-1 macrophages were transfected with vector control or HA-Erbin for 36 h. Then, cells were treated with or without MDP (10 μg/ml) for 6 h. Cytosolic and nuclear extracts were prepared and subjected to western blot analyses. Lamin A and β-actin were used as the internal control for nuclear and cytosolic fractions, respectively. E Experiments were performed similarly to those in D , except indicated siRNA was used. F THP-1 macrophages were transfected with siRNA control or si-Erbin for 36 h and treated with or without MDP (10 μg/ml) for 6 h before qPCR analyses. The data shown are representative of 3 independent experiments

Article Snippet: Erbin conditional knockout mice and Lyz2-Cre were obtained from Cyagen (Guangzhou) Biosciences.

Techniques: Translocation Assay, Activity Assay, Western Blot, Transfection, Plasmid Preparation, Incubation, Fluorescence, Confocal Microscopy