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: PLoS Pathogens

Article Title: IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

doi: 10.1371/journal.ppat.1002422

Figure Lengend Snippet: (A) Autophagy-associated proteins contribute differently to the autophagy network. Autophagy-associated proteins are plotted according to their context connectivity and context centrality. Autophagy context connectivity (x axis) is the ratio of interaction in autophagy network over the interaction in the whole human protein interaction network. Autophagy context centrality (y axis) is the ratio of betweenness (log normalized values) in autophagy network over the betweenness in the human protein interaction network. A protein with high degree and high betweenness ratios is respectively defined as highly devoted and highly central in the autophagy context. Proteins in red represent autophagy-associated proteins targeted by at least one RNA virus protein. The four proteins that are not connected to the autophagy network are not represented. (B) IRGM interacts with ATG10, ATG5, SH3GLB1 and MAP1LC3C. IRGM was tested by yeast two-hybrid against 35 different autophagy-associated proteins. Positive interactions with ATG10, ATG5, SH3GLB1 and MAP1LC3C were found. A reduced yeast two-hybrid matrix containing positive interactions and the appropriate empty vector controls is shown. One experiment representative of three is shown. (C) IRGM co-localizes with autophagy-associated proteins. GFP-IRGM was expressed in HeLa cells together with FLAG-ATG10, FLAG-ATG5, FLAG-SH3GLB1 or FLAG-MAP1LC3C. Fixed cells were then stained with an anti-FLAG antibody and GFP-IRGM and FLAG-tagged proteins co-localisation was visualized on merged images by confocal microscopy. Scale bars, 5 µM. (D) IRGM interacts with autophagy-associated proteins. HEK293T cells were co-transfected with GST and GST-tagged expression vectors encoding the indicated proteins and FLAG-IRGM. Interaction was assayed by co-affinity purification (AP) using glutathione-sepharose beads. FLAG-IRGM was detected using anti-FLAG antibody after (AP-GST, WB: FLAG) and before (total cell lysate-TCL, WB: FLAG) co-AP. GST alone and GST-tagged proteins were detected by using anti-GST antibody (AP-GST, WB: GST). One experiment representative of two is shown. (E) Endogenous IRGM co-localizes with endogenous SH3GLB1 and ATG5 in MeV infected cells. HeLa cells were infected with MeV Edmonston (MOI = 1) for 24 hrs. Cells were fixed in acetone and both IRGM and/or SH3GLB1 or ATG5 were detected using specific antibodies. IRGM/SH3GLB1 or IRGM/ATG5 protein co-localisation was visualized on merged images obtained by confocal microscopy. Scale bars, 5 µM. (F) Endogenous ATG5 interacts with FLAG-IRGM. HeLa cells were transfected or not with FLAG-IRGM encoding vector and infected with MeV (MOI = 1) 24 hrs post-transfection. Cells were lysed 24 hrs post-infection. Flag-tagged IRGM was immunoprecipitated and endogenous ATG5 binding was detected by western blot (top panel). Overexpression and immunoprecipitation of FLAG tagged IRGM was confirmed by a western blot using (bottom panel).

Article Snippet: Anti- Glutathione-S-Transferase (GST) peroxidase (A7340), anti-FLAG M2 peroxidase (A8592), anti-MAP1LC3B (L7543), anti-Actin (A2066), anti-Myc (C3956), anti-ATG5 (A0856) and anti-eGFP (G6795) were from Sigma (St Louis, Mo, USA).

Techniques: Plasmid Preparation, Staining, Confocal Microscopy, Transfection, Expressing, Affinity Purification, Infection, Immunoprecipitation, Binding Assay, Western Blot, Over Expression

Journal: PLoS Pathogens

Article Title: IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

doi: 10.1371/journal.ppat.1002422

Figure Lengend Snippet: (A–C) MeV-induced autophagy is dependent on IRGM. GFP-LC3-HeLa (A–B) or HeLa cells (C) were treated with si-control, si-ATG5 or si-IRGM and either left uninfected or infected with MeV Edmonston (MOI = 3) for 24 hrs. Autophagy was monitored either by evaluating the number of GFP-LC3+ vesicles per cell profile by confocal microscopy (A, B) or by detection of LC3-I and LC3-II by western-blot (C). Representative profiles for each condition (A) and the corresponding graph representing the number of GFP-LC3+ vesicles per cell profile (B) are shown, error bars, mean ± SD of three independent experiments. (C) One experiment representative of three is shown. (D–F) HCV-induced autophagy is dependent on IRGM. GFP-LC3-Huh7.5 (D–E) or Huh7.5 (F) cells were treated with the indicated si-RNA and either left uninfected or infected with HCV JFH-1 (MOI = 1) for 24 hrs. Autophagy was monitored as above. (E) Error bars, mean ± SD of three independent experiments. (F) One experiment representative of two is shown. (G–I) Influenza A-induced autophagy is not impaired by IRGM absence. GFP-LC3-A549 (G–H) or A549 (I) cells were treated with the indicated si-RNA and either left uninfected or infected with influenza A/H1N1/New Caledonia (MOI = 1) for 24 hrs. Autophagy was monitored as above. (H) Error bars, mean ± SD of two independent experiments. (I) One experiment representative of two is shown. (J) HIV-1-induced autophagy is dependent on IRGM. Monocyte-derived macrophages (MDM) were treated with the indicated si-RNA and were either left uninfected or infected with HIV-1 for 24 hrs. LC3-I and LC3-II detection was carried out by western-blot as in C, F, I. One experiment representative of two is shown with the quantification number representing the intensity of LC3-II/GAPDH bands normalized to the uninfected condition. Student's t test; * p<0.05; ** p<0.01; # p>0.05.

Article Snippet: Anti- Glutathione-S-Transferase (GST) peroxidase (A7340), anti-FLAG M2 peroxidase (A8592), anti-MAP1LC3B (L7543), anti-Actin (A2066), anti-Myc (C3956), anti-ATG5 (A0856) and anti-eGFP (G6795) were from Sigma (St Louis, Mo, USA).

Techniques: Infection, Confocal Microscopy, Western Blot, Derivative Assay

Journal: PLoS Pathogens

Article Title: IRGM Is a Common Target of RNA Viruses that Subvert the Autophagy Network

doi: 10.1371/journal.ppat.1002422

Figure Lengend Snippet: (A–B) Overexpression of MeV-C, HCV-NS3 and HIV-NEF modulates autophagosome formation. (A–B) GFP-LC3 HeLa cells were transfected with a GST encoding vector (control) or a vector encoding MeV-C, HCV-NS3 and HIV-NEF proteins. Twenty four hours post transfection the number of autophagic vesicles was determined by confocal microscopy. Representative profiles for each condition (A) and the corresponding graph representing the number of GFP-LC3+ vesicles per cell profile (B) are shown, error bars, mean ± SD of three independent experiments. (C–F) MeV-C, HCV-NS3 and HIV-NEF modulate autophagosome formation partly via IRGM. (C) GFP-LC3 HeLa cells were treated with si-control, si-ATG5 or si-IRGM 24 hrs prior transfection with vector encoding for MeV-C, HCV-NS3 or HIV-NEF. After an additional 24 hrs, cells were fixed and the number of autophagosome was determined by confocal microscopy. Representative profiles for each condition (C) and the corresponding graph representing the number of GFP-LC3+ vesicles per cell profile (D–F) are shown, error bars, mean ± SD of three independent experiments. Student's t test; * p<0.05; ** p<0.01.

Article Snippet: Anti- Glutathione-S-Transferase (GST) peroxidase (A7340), anti-FLAG M2 peroxidase (A8592), anti-MAP1LC3B (L7543), anti-Actin (A2066), anti-Myc (C3956), anti-ATG5 (A0856) and anti-eGFP (G6795) were from Sigma (St Louis, Mo, USA).

Techniques: Over Expression, Transfection, Plasmid Preparation, Confocal Microscopy

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

Journal: Journal of Cell Death

Article Title: Autophagy Induction Protects Against 7-Oxysterol-induced Cell Death via Lysosomal Pathway and Oxidative Stress

doi: 10.4137/JCD.S37841

Figure Lengend Snippet: Exposure to a mixture of 7-oxysterols (2mix) causes lipid accumulation and autophagy dysfunction. THP-1 monocytes or THP-1 differentiated macrophages were treated with or without 2mix for 12 hours to 48 hours. In some experiments, cells were pretreated with the autophagy inhibitor 3MA or the autophagy inducer rapamycin for 1 hour and then exposed to 2mix or treated with the lysosomatropic agent CQ for 24 hours or CQ for 1 hour and 2mix for 24 hours. ( A ) Oil red O-stained THP-1 monocytes counterstained with hematoxylin (24 hours), bars = 100 µm. ( B and C ) Accumulation of autophagy vacuoles in THP-1 differentiated macrophages assayed by TEM and quantified by image analysis. ( B ) Photographs of low power view of control and 2mix-treated cells (two photographs on the left-hand side), bars = 2 µm or a higher magnified photograph (photograph on the top right-hand side) demonstrates autophagic vacuoles with double-membrane structure containing undigested cytoplasmic materials, bar = 1 µm. ( C ) Quantification of numbers of autophagic vacuoles, * P < 0.05, ** P < 0.01 versus control and 3MA + 2mix, respectively. ( D ) Atg5-immunostained THP-1 monocytes analyzed by flow cytometry. n = 6–8, * P < 0.05, ** P < 0.01 and *** P < 0.001. The inset shows a representative histogram of flow cytometry results (control: gray field histogram; 2mix: black-lined empty histogram). ( E ) LC3β-immunostained THP-1 monocytes analyzed by flow cytometry; n = 6–20, * P < 0.05. ( F ) Western blot analysis of LC3 I/II in the whole cell lysate of THP-1 monocytes. Values mentioned below the Western blot image are the ratios of LC3-II to LC3-I. ( G ) p62/ SQSTM1 -immunostained THP-1 monocytes analyzed by flow cytometry; n = 3–4. a P < 0.05 vs CQ and P < 0.01 vs 3MA + 2mix and CQ + 2mix. b P < 0.05 vs CQ and P < 0.01 vs CQ + 2mix and P < 0.001 vs 3MA + 2mix. * P < 0.05.

Article Snippet: Different groups of cells were collected, fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin in PBS, and incubated overnight with the following rabbit primary antibodies at 4°C: anti-LC3β antibody (Santa Cruz Biotechnology Inc.) or anti-Atg5 (Thermo Scientific) or anti-p62/ SQSTM1 (Medical and Biological Laboratories Co., Ltd).

Techniques: Staining, Flow Cytometry, Western Blot