Journal: bioRxiv

Article Title: Autophagy Induced by Palmitic Acid: a Brake in NAFLD Neutrophils

doi: 10.1101/2021.04.02.438261

Figure Lengend Snippet: (A) The CD11a, CD11b, CD18 and Rap1 protein complexes were modified with polyubiquitin chains (Lys48). CD11a, CD11b, CD18, Rap1 and the polyubiquitinated proteins were immunoprecipitated from neutrophils and then evaluated by immunoblotting. (B-C) Inhibition of the degradation of CD11a, CD11b, CD18 and Rap1 increased neutrophil adhesion. The protein degradation was blocked by BafA1, CQ or MG132 in control and PA-treated neutrophils. Quantitative analysis of LC3B, p62, CD11a, CD11b, CD18 and Rap1 (B, n = 3) was performed, and neutrophil adhesion was detected with a fluorescence microplate reader (C, n = 8). Data represent the mean ± s.e.m. (* P < 0.05 and ** P < 0.01 versus the control group, # P < 0.05 and ## p < 0.01 versus the PA-treated group; Significance calculated using two-way ANOVA). (D-E) Knockdown of ATG5 significantly inhibited the autophagy and increased the adhesion of HL-60 cells. The HL-60 cells were infected with LV-GFP-shATG5 (to block autophagy) and LV-GFP (Vector) (negative controls). Quantitative analysis of LC3B, p62, ATG5, CD11a, CD11b, CD18 and Rap1 (D, n = 3) was performed to evaluate autophagic flux and protein accumulation. The adhesion of HL-60 cells was detected by a fluorescence microplate reader (E, n = 8). Data represent the mean ± s.e.m. (* P < 0.05 and ** P < 0.01 versus the control group, ## p < 0.01 versus the PA-treated group; Significance calculated using two-way ANOVA). (F) Reciprocal co-IP of CD11a, CD11b, CD18 and Rap1 with Hsc70. The CD11a, CD11b, CD18, Rap1 and Hsc70 protein complexes were immunoprecipitated and evaluated by immunoblotting individually. (G) Portion of immunogold electron micrograph showing the localization of Hsc70 (5 nm, white arrows) with MPO, lactoferrin or MMP-9 (10 nm, black arrows) in control and PA-treated neutrophils. Scale bars as indicated. (H-I) Knockdown of Hsc70 blocked the degradation of CD11a, CD11b, CD18 and Rap1 and increased the adhesion of HL-60 cells. The cells were infected with LV-GFP-shHsc70 and LV-GFP (Vector) (negative controls). Quantitative analysis of Hsc70, CD11a, CD11b, CD18 and Rap1 was performed (H, n = 3). Neutrophil adhesion was detected by a fluorescence microplate reader (I, n = 8). Data represent the mean ± s.e.m. (* P < 0.05 and ** P < 0.01 versus the control group, ## p < 0.01 versus the PA-treated group; Significance calculated using two-way ANOVA).

Article Snippet: Anti-ATG5 Ab (NB110-53818), BafilomycinA1 (CAS88899-55-2) were purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Modification, Immunoprecipitation, Western Blot, Inhibition, Control, Fluorescence, Knockdown, Infection, Blocking Assay, Plasmid Preparation, Co-Immunoprecipitation Assay

Journal: bioRxiv

Article Title: Autophagy Induced by Palmitic Acid: a Brake in NAFLD Neutrophils

doi: 10.1101/2021.04.02.438261

Figure Lengend Snippet: (A) Representative transmission electron micrographs of control and PA-treated HL-60 cells (infected or not infected with LV-GFP-shATG5 or empty lentivectors). Scale bars as indicated. (B) the area ratio of autophagic vacuoles to dHL-60 cells were determined (n = 6). Data represent the mean ± s.e.m. (** P < 0.01 versus the control group, ## p < 0.01 versus the PA-treated group; Significance calculated using two-way ANOVA). (C) Immunoblot for LC3B, p62, ATG5, CD11a, CD11b, CD18 and Rap1 in control and PA-treated HL-60 cells (infected or not infected with LV-GFP-shATG5 or empty lentivectors). (D) Representative fluorescence micrographs of control and PA-treated differentiated HL-60 cells (infected or not infected with LV-GFP-shATG5 or empty lentivectors) adhered to HUVECs. Scale bar, 400 μm.

Article Snippet: Anti-ATG5 Ab (NB110-53818), BafilomycinA1 (CAS88899-55-2) were purchased from Novus Biologicals (Centennial, CO, USA).

Techniques: Transmission Assay, Control, Infection, Western Blot, Fluorescence

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

doi: 10.1038/labinvest.2009.8

Figure Lengend Snippet: Apoptosis, autophagy and inflammation pathway genes evaluated in the present study

Article Snippet: Atg5 , autophagy-related 5 (yeast) , GO:0000045: autophagosome formation , NM_053069 , Mm00504340_m1.

Techniques: Transformation Assay, Binding Assay, Transduction, Full Display Name, Activation Assay

Journal: Laboratory investigation; a journal of technical methods and pathology

Article Title: Sepsis Induces Extensive Autophagic Vacuolization in Hepatocytes –a clinical and laboratory based study

doi: 10.1038/labinvest.2009.8

Figure Lengend Snippet: Apoptosis, autophagy and inflammation pathway gene expression on cecal ligation and puncture in total liver cells

Article Snippet: Atg5 , autophagy-related 5 (yeast) , GO:0000045: autophagosome formation , NM_053069 , Mm00504340_m1.

Techniques: Gene Expression, Ligation

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 3 TRIM45 regulates autophagy proteins associated with NLRP3. a qRT-PCR analysis of p62, Atg5, and beclin1 mRNA levels in BV2 cells; n = 3 per group. b The autophagy proteins p62, beclin1, Atg5 and LC3 in BV2 cells were measured by western blotting. c Changes in ROS in BV2 cells in the different groups; n = 3 per group. d JC-1 staining to detect mitochondrial membrane potential and cellular morphology was examined by a phase-contrast microscope. (scale bar = 75 μm); n ≥ 3 per group. Data in a, c, d were analysed by one-way ANOVA followed by Dunnett’s post hoc test. Data are presented as the mean ± S.E.M. from three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Quantitative RT-PCR, Western Blot, Staining, Membrane, Microscopy

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 4 TRIM45 regulates Atg5 based on its E3 ubiquitin ligase activity. a Western blot analysis of Atg5 expression in BV2 cells in response to different interventions. b BV2 cells with TRIM45 knockdown or control cells were treated with cycloheximide (CHX) for 0, 2 and 4 h. c BV2 cells were immunoprecipitated with IgG and anti-Atg5 antibodies, and the expression of TRIM45 and Atg5 was detected by western blotting. d Protein confidence analysis of Atg5 and TRIM45 was performed by high-throughput interactive group (TRIM45 (green), Atg5 (blue)). e BV2 cells were transfected with HA-TRIM45 for 48 h. BV2 cells were immunoprecipitated with an anti-Atg5 antibody and then subjected to western blot analysis with an anti-NLRP3 antibody. f Co-IP analysis of Atg5 ubiquitination in BV2 cells. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. g HEK293T cells were transfected with TRIM45 and HA-tagged ubiquitin plasmids with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies. h, i HEK293T cells were transfected with plasmids TRIM45 and HA-tagged k63 or k48 ubiquitin with or without LPS + ATP treatment. The cell extracts were collected for IP with anti-Atg5 beads, followed by IP analysis with the indicated antibodies

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Ubiquitin Proteomics, Activity Assay, Western Blot, Expressing, Knockdown, Control, Immunoprecipitation, High Throughput Screening Assay, Transfection, Co-Immunoprecipitation Assay

Journal: Journal of neuroinflammation

Article Title: TRIM45 aggravates microglia pyroptosis via Atg5/NLRP3 axis in septic encephalopathy.

doi: 10.1186/s12974-023-02959-8

Figure Lengend Snippet: Fig. 5 TRIM45 regulates the NLRP3 pathway in an Atg5-dependent manner. a, b Western blot analysis and quantification of NLRP3, Gsdmd-N, Atg5 and IL-1β levels in BV2 cells transfected with siTRIM45 or HA-TRIM45 and stimulated by LPS + ATP; n = 3 per group. c BV2 cells were infected with siTRIM45 and Flag-Atg5; n = 4 per group. d BV2 cells were infected with HA-TRIM45 and siAtg5; n = 4 per group. e, f Statistical analysis of Figures c and d. g, h Flow cytometry was used to analyse BV2 pyroptosis; n = 3 per group. Data in a, b, h were analysed by one-way ANOVA followed by Dunnett’s post hoc test. Data in c, d were analysed by unpaired t test. Data are presented as the mean ± S.E.M. from at least three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001

Article Snippet: Small interfering RNAs against Atg5 (sc-41446, Santa Cruz) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, Transfection, Infection, Flow Cytometry

Journal: Autophagy

Article Title: TRIM44 links the UPS to SQSTM1/p62-dependent aggrephagy and removing misfolded proteins

doi: 10.1080/15548627.2021.1956105

Figure Lengend Snippet: TRIM44 promtoes aggregates deaggregation and clearance via autophagy. (a) Cells were treated with MG132 and immunostained with antibodies to ubiquitin (red) and LC3B-II (green). Arrows indicate ubiquitin-positive aggregates that colocalize with LC3B-positive autophagosomes. Scale bars: 10 μm. (b) Confocal images of TRIM44[OE-CON] and TRIM44[OE] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA (10 mM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (c) Confocal images of TRIM44[KD-CON] and TRIM44[KD] U266 cells after treatment with MG132 (0.5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM). Arrows indicate cells with remaining aggregates. Scale bars: 10 µm. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or PP242 (10 nM) and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (0.5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. (d, e) Confocal images of WT or ATG5 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (d). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (e). (f, g) Confocal images of WT or BECN1 KO TRIM44[OE-CON] and TRIM44[OE] U266 cells were treated with MG132 (0.5 µM) for 16 h. Aggregates (marked by arrows) were identified by staining with the antibody against ubiquitin (f). Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of BECN1 and TRIM44 were assayed by western blot (g). (h, i) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) transfected with NC or ATG5 siRNA after treatment with MG132 (0.5 µM) for 16 h. The status of aggregates after MG132 treatment was quantified in the histogram. Scale bars: 10 µm. The status of aggregates after MG132 treatment was quantified in the histogram. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm. The protein level of ATG5 and TRIM44 were assayed by western blots (h). (j) Confocal images of RPMI-TRIM44[Tet-on] cells treated with or without DOX (1 µg/mL) together with MG132 (5 µM) for 16 h followed by a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA. Arrows indicate cells with remaining aggregates. The relative level of remaining aggregates is determinded by quantifying the percentage of cells with remaining aggregates after a 24-h chase period in normal culture media with DMSO (vehicle), or 3-MA and normalized to the percentage of cells with aggregates formed by the 16 h MG132 (5 µM) treatment in corresponding cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001. Scale bars: 10 μm.

Article Snippet: Anti-TRIM44 polyclonal antibody (Proteintech Group, 11,511-1-AP); anti-Ub antibody (Biolegend, 646,301); anti-mCherry antibody (ThermoFisher, PA5-34,974); anti-VIM antibody (ThermoFisher, MA5-11,883); anti-NFE2L2/NRF2 antibody (ThermoFisher, PA5-27,882); anti-20S proteasome antibody (MilliporeSigma, ST1049); anti-TUBG/γ-Tubulin antibody (MilliporeSigma, T5326); anti-ATG5 antibody (Novus Biologicals, NBP2-24,389); Anti-GFP antibody (Santa Cruz Biotechnology, sc-9996); anti-ACTB/β-Actin antibody (Santa Cruz Biotechnology, sc-47,778), anti-HA antibody (Santa Cruz Biotechnology, sc-805); anti-BECN1 antibody (Cell Signaling Technology, 4122); anti-HDAC6 antibody (Cell Signaling Technology, 7558); anti-LC3B antibody (Cell Signaling Technology, 3868); anti-SQSTM1/p62 antibody (Cell Signaling Technology, 88,588); anti-cleaved PARP antibody (Cell Signaling Technology, 5625); Alexa Fluor 594 donkey anti rabbit IgG(H + L) (Invitrogen, A21207); Alexa Fluor 546 goat anti mouse IgG(H + L) (Invitrogen, A11003).

Techniques: Ubiquitin Proteomics, Transfection, Staining, Western Blot