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 ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal lavage fluid from uninoculated or inoculated wild-type, IL-23p19 −/− , IL-17RA −/− and IL-22 −/− mice with high PMNs were evaluated for (A) S100A8 and (B) S100A9 concentrations by ELISA. The results are cumulative data of 1 to 3 repeat experiment(s) testing lavage samples collected on day 7 post-inoculation. LF, lavage fluid. SEM, standard error of the mean.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: The Acute Neutrophil Response Mediated by S100 Alarmins during Vaginal Candida Infections Is Independent of the Th17-Pathway

doi: 10.1371/journal.pone.0046311

Figure Lengend Snippet: Vaginal tissue sections from uninoculated or inoculated mice with high PMNs were stained with anti-S100A8 or S100A9 antibodies. Images are shown at ×400 magnification. Arrows represent epithelium positively stained for S100A8 or S100A9. Images show representative results of 15 uninoculated and 30 inoculated animals on day 7 post-inoculation.

Article Snippet: Briefly, tissues were treated with peroxidase, goat serum, avidin and biotin blocking buffers and then incubated with monoclonal rat anti-mouse S100A8 or S100A9 antibody (10 μg/ml; R&D Systems), monoclonal mouse anti-human AE1/AE3 antibody (epithelial cytokeratin markers, 5 μg/ml; MP Biomedicals, Solon, OH), or isotype controls (rat IgG2a, rat IgG2b and mouse IgG1) overnight at 4°C.

Techniques: Staining