Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 1. VER-50589 exhibits antiviral effects against EV71 in vitro. (A) Chemical structure of VER-50589. (B–E) RD and HeLa cells were cultured with five- fold dilutions of VER-50589 or ribavirin. After 48 h, cytotoxicity was assessed using an MTT assay. (F–H) RD cells were infected with EV71 (MOI = 0.1) for 2 h and incubated with five-fold dilutions of VER-50589 or ribavirin for 20 h. VP1 protein levels were measured using western blotting (F). EV71 mRNA levels were evaluated using qPCR (G, H). (l) Immu nofluorescence assay detected EV71 mRNA in EV71- infected RD cells treated with VER-50589. (J) EV71- infected RD cells were treated with different con centrations of VER-50589 for 48 h, and the cell freeze-thaw liquid was harvested for the plaque assay. (K)The number of infectious clones of the samples was calculated. (L–M) EV71-infected RD cells were treated with VER-50589 (4 μM) or ribavirin (4 μM) under the same experimental conditions. VP1 protein and viral mRNA levels were measured using western blotting and qPCR, respectively. ***P < 0.001; **P < 0.01; *P < 0.05.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: In Vitro, Cell Culture, MTT Assay, Infection, Incubation, Western Blot, Plaque Assay, Clone Assay

Journal: Antiviral research

Article Title: Antiviral activity of the HSP90 inhibitor VER-50589 against enterovirus 71.

doi: 10.1016/j.antiviral.2023.105553

Figure Lengend Snippet: Fig. 4. VER-50589 inhibits HSP90 and mediates AKT and RAF phosphorylation. (A–B) RD cells were transfected with HSP90-specific siRNA and cultured for 24 h. The p-AKT, AKT, RAF, p-RAF, and HSP90 levels were detected using western blotting (A). HSP90 mRNA levels were detected using qPCR (B). (C–D) RD cells were transfected with HSP90-specific siRNA for 24 h and infected with EV71 (MOI = 8) for 24 h. The VP1, p-AKT, AKT, p-RAF, RAF, and HSP90 levels were detected using western blotting (C). The mRNA levels of EV71 were detected using qPCR (D). (E) Cherry-HSP90 plasmid-carrying RD cells were infected with EV71 (MOI = 8) for 24 h. The expression levels of HSP90, p-AKT, AKT, p-RAF, RAF, and VP1 were detected using western blotting. (F) RD cells were infected with the EV71 virus with different MOI (0.01, 0.1, 1, and 10) and subjected to western blotting. (G) RD cells were infected with EV71 (MOI = 0.1) and harvested at 8 h, 12 h, 18 h, and 22 h. Protein levels were detected using western blotting. (H) RD cells were treated with five-fold dilution of VER-50589 and cultured for 20 h. Different protein levels were detected using western blotting. (I) RD cells were incubated with EV71 (MOI = 0.1) for 2 h, and the medium was replaced with one containing a five-fold series dilution of VER-50589 for another 20 h incubation. Proteins were extracted for western blotting analysis.

Article Snippet: The following antibodies were used according to manufacturer’s recommendations: anti-phospho-AKT (CST, Cat: 4060S), anti-total AKT (CST, Cat: 4691S), anti-HSP90 (Abcom, Cat: ab208035), anti-phospho-ERK1/2 (Santa Cruz, Cat: sc-81492), anti-total ERK1/2 (Santa Cruz, Cat: sc-514302), anti-phospho-RAF (CST, Cat: 2969S), antitotal RAF (CST, Cat: 148145S), anti-total VP1 antibody (CST, cat: 9167), goat anti-rabbit IgG (Cat: SA00001-2, Proteintech, China), goat antimouse IgG (Cat: SA00001-1, Proteintech, China), β-actin (Cat: 60008- 1, Proteintech, China), and GAPDH (Cat: 10494-1, Proteintech, China).

Techniques: Phospho-proteomics, Transfection, Cell Culture, Western Blot, Infection, Plasmid Preparation, Expressing, Virus, Incubation

Journal: Science Advances

Article Title: A non-immunological role for γ-interferon–inducible lysosomal thiol reductase (GILT) in osteoclastic bone resorption

doi: 10.1126/sciadv.abd3684

Figure Lengend Snippet: ( A ) mRNA expression of the osteoclast markers cathepsin K and tartrate-resistant acid phosphatase (TRAP), as well as GILT in M-CSF–stimulated WT bone marrow cells treated with or without RANKL as measured by quantitative polymerase chain reaction (qPCR) ( n = 8 to 9). Expression was normalized to 18 S ribosomal RNA and made relative to M-CSF control samples. ( B ) Representative Western blot image depicting GILT protein levels following lysis of WT BMMØs (−RANKL) or osteoclasts (+RANKL). Mature GILT expression was normalized to total protein levels ( n = 5). ( C ) Detection of GILT (green) and cathepsin K (red) by immunofluorescence microscopy in BMMØs (M-CSF) or osteoclasts (M-CSF + RANKL). Scale bars, 50 μm. Colocalization of the GILT and cathepsin K signals was assessed by calculating the Pearson’s correlation coefficient in BMMØs (0.743 ± 0.04) and osteoclasts (0.703 ± 0.07) ( n = 3 to 4 images). ( D ) STAT1 phosphorylation status within WT osteoclast precursors left untreated (−) or treated for 15 min (+) with RANKL (200 ng/ml) or IFN-γ (100 U/ml). Levels of phosphorylation were normalized to total STAT1 levels and made relative to untreated controls ( n = 3). ( E ) GILT protein expression by WT or STAT1 −/− osteoclast precursors following 48-hour treatment with M-CSF + RANKL (15 ng/ml + 100 ng/ml) or M-CSF only. Mature GILT levels were normalized to total protein levels and made relative to M-CSF–only controls ( n = 3). (A to C) Cells were differentiated for 6 days with M-CSF (15 ng/ml) and RANKL (100 ng/ml), or M-CSF (15 ng/ml) only, throughout the entirety of each experiment. (D and E) Precursor cells were expanded in M-CSF (15 ng/ml) for 48 hours before described treatments. (A to E) Error bars are presented as means ± SEM. * P < 0.05, ** P < 0.01 by paired (A to D) or unpaired (E) Student’s t test.

Article Snippet: Rabbit α-mouse P-S727 STAT1 (#9177), P-Y701 STAT1 (#9167), and total STAT1 (#9172) antibodies (all Cell Signaling Technologies) were used to evaluate STAT1 signaling and were probed using a goat α-rabbit IgG-HRP secondary antibody (#7074, Cell Signaling Technology).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Lysis, Immunofluorescence, Microscopy

Journal: Infection and Immunity

Article Title: Legionella pneumophila inhibits type I interferon signaling to avoid cell-intrinsic host cell defense

doi: 10.1128/iai.00365-23

Figure Lengend Snippet: L. pneumophila inhibits cell surface expression of tetherin but does not affect the phosphorylation of STAT1 and STAT2. THP-1 cells were infected with GFP-expressing wild-type L. pneumophila and dotA deficient mutant (Lp∆) at an MOI of 10 and treated or not with 10 ng/mL IFN-β. (A) The flow cytometry analysis performed on the GFP+/infected cells using a fluorescent antibody targeting the cell surface ISG protein tetherin at 20 h.p.i.. (B) The flow cytometry analysis performed on the GFP+/infected cells at an MOI of 10 using a fluorescent antibody targeting phosphorylated STAT1 30 minutes post-infection. (C) The flow cytometry analysis performed on the GFP+/infected cells at an MOI of 10 using a fluorescent antibody targeting phosphorylated STAT2 30 minutes post-infection. The histograms are from one experiment representative of three independent experiments. The bar graphs show the mean fluorescent intensity fold changes compared with the uninfected untreated cells for each flow cytometry analysis. Individual points represent independent experiments. An asterisk indicates multiple unpaired t-tests with Welch correction relative to the uninfected IFN-β treated cells. Significance is indicated as follows: **P ≤ 0.01, ***P ≤ 0.001, and ****P ≤ 0.0001.

Article Snippet: Next, 50 µL of the stain cocktail [1× Brillant Stain Buffer (BD Bioscience) and either 5 µL per sample of Alexa Flour 647 Anti-Total Stat1 (N-Terminus) (BD Biosciences) and 5 µL per sample of PE anti-STAT1 Phospho (Tyr701) (BioLegend) or 2 µL per sample of Stat2 (D9J7L) Rabbit mAb (PE) (Cell Signaling Technology) and 2 µL per sample of P-Stat2 (P-690) Rabbit mAb (Alexa 647) (Cell Signaling Technology)] was added to each sample.

Techniques: Expressing, Infection, Mutagenesis, Flow Cytometry

Journal: Journal of Virology

Article Title: Interferon Regulatory Factor 1 Restricts Gammaherpesvirus Replication in Primary Immune Cells

doi: 10.1128/JVI.00638-14

Figure Lengend Snippet: IRF-1 functions downstream of the type I IFN receptor to attenuate MHV68 replication in primary macrophages. Bone marrow-derived macrophages from control (BL6) and IRF-1−/− mice were mock infected or infected with MHV68 at an MOI of 10 PFU/cell. (A) Antiviral activity in the culture supernatants was analyzed at the indicated times postinfection using an EMCV bioassay and expressed as the number of units of type I IFN based on the standard curve generated with recombinant IFN-β in the same assay. (B, C) Total RNA was harvested at the indicated times postinfection, and the levels of IFN-β and IFN-α mRNA were measured by quantitative RT-PCR; data from 2 to 3 independent experiments were pooled, each data point was derived from 2 to 3 replicates within each experiment, and error bars represent SEMs. (D) Protein levels of ISG15, total Stat1, MHV68 ssDBP, and β-actin were measured at the indicated times postinfection. (E) Macrophages of the indicated genotypes were infected with MHV68 at an MOI of 1 PFU/cell. The virus yield in triplicate cultures was measured at the indicated times postinfection. Data are representative of those from two independent experiments, and error bars represent SEMs.

Article Snippet: The antibodies used were anti-β-actin (1:20,000; Novus Biological, Littleton, CO), anti-total Stat1 (1:3,000; Santa Cruz Biotechnology, Santa Cruz, CA), anti-IRF-1 (1:1,000; Cell Signaling Technology, Danvers, MA), anti-ssDBP (1:1,000 [ 30 ]), anti-ISG15 (1:4,000; a gift of Debbie Lenschow), and a secondary goat antimouse or antirabbit horseradish peroxidase-conjugated secondary antibody (1:25,000; Jackson ImmunoResearch, West Grove, PA).

Techniques: Derivative Assay, Control, Infection, Activity Assay, Bioassay, Generated, Recombinant, Quantitative RT-PCR, Virus