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: Viruses

Article Title: Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3

doi: 10.3390/v14071499

Figure Lengend Snippet: hPL-induced innate immunity response type I interferon and antiviral-related genes on GBM cells. ( A ) GBM cells were cultured under FBS, 2.5% and 5% hPL for 6 h, 24 h, and 48 h. The cell protein lysates were employed to analyze the innate immunity pathways by immunoblotting using anti-phospho-STAT1, anti-total STAT2, anti-phospho-STAT2, anti-total STAT2, anti-phospho-IRF3, anti-total IRF3, anti-phospho-NFκB, anti-total NFκB p65, and anti-GAPDH. ( B ) Quantification of protein expressions was normalized to GAPDH. Data are mean ± SD of three independent experiments. Student’s t -test, *, p < 0.05; **, p <0.01 compared with GBM cells supplemented with FBS. ( C ) GBM cells were cultured under FBS, 2.5% and 5% hPL mediums for 24 h and 48 h. The mRNA level of antiviral-related genes IFN-α , IFN-β , IRF3 , and MxA were confirmed by qPCR. The level of transcripts was normalized to GAPDH. Data are mean ± SD of three independent experimental samples. Student’s t -test, *, p < 0.05; **, p < 0.01; ***, p < 0.001 compared with FBS.

Article Snippet: The antibodies were anti-PeV VP0 (LTK BioLaboratories, Taoyuan, Taiwan) [ , ], anti-phospho STAT2 (Cell Signaling, 88410, Danvers, MA, USA), anti-total STAT2 (Cell Signaling, #72604), anti-phospho STAT1 (Cell Signaling, #9167), anti-total STAT1 (Cell Signaling, #14994), anti-phospho IRF3 (Abcam, #ab76493, Cambridge, England), anti-total IRF3 (Cell Signaling, #11904), anti-phospho NFκB p65 (Abcam, #ab86299), anti-total NFκB p65 (Cell Signaling, #8242) and anti-GAPDH (Proteintech, 60004-1-Ig, Rosemont, IL, USA).

Techniques: Cell Culture, Western Blot

Journal: Viruses

Article Title: Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3

doi: 10.3390/v14071499

Figure Lengend Snippet: hPL-supplemented growth medium-induced expression of IFN-signaling pathway under PeV-A3 infection of GBM cells. ( A ) GBM cells were infected with PeV-A3 at MOI = 1 for 6 h, 24 h, and 48 h under FBS- or hPL-supplemented growth mediums. Cellular protein lysates were extracted to analyze IFN-signaling pathway by immunoblotting using anti-PeV VP0, anti-phospho-STAT1, anti-total STAT1, anti-phospho-STAT2, anti-total STAT2, anti-phospho-IRF3, anti-total IRF3, anti-phospho-NFκB, anti-total NFκB p65, and anti-GAPDH. ( B ) Quantification of different protein expressions was normalized to GAPDH. Data are mean ± SD of three independent samples. Student’s t -test, *, p < 0.05 compared with FBS.

Article Snippet: The antibodies were anti-PeV VP0 (LTK BioLaboratories, Taoyuan, Taiwan) [ , ], anti-phospho STAT2 (Cell Signaling, 88410, Danvers, MA, USA), anti-total STAT2 (Cell Signaling, #72604), anti-phospho STAT1 (Cell Signaling, #9167), anti-total STAT1 (Cell Signaling, #14994), anti-phospho IRF3 (Abcam, #ab76493, Cambridge, England), anti-total IRF3 (Cell Signaling, #11904), anti-phospho NFκB p65 (Abcam, #ab86299), anti-total NFκB p65 (Cell Signaling, #8242) and anti-GAPDH (Proteintech, 60004-1-Ig, Rosemont, IL, USA).

Techniques: Expressing, Infection, Western Blot

Journal: Viruses

Article Title: Human Platelet Lysate Induces Antiviral Responses against Parechovirus A3

doi: 10.3390/v14071499

Figure Lengend Snippet: Enhanced PeV-A3 VP1 gene expression under blockade of STAT1 on PeV-A3 infection of GBM cells. ( A ) GBM cells were pre-treated with fludarabine 50 µM for 30 min under FBS or hPL medium. After treatment, GBM cells were then infected with PeV-A3 at MOI = 1 for 6 h. Expression of PeV-A3 VP1 genes was measured using real-time qPCR. The level of PeV-A3 VP1 was normalized to GAPDH. Data are mean ± SD of three independent samples. Student’s t -test, *, p < 0.05; **, p < 0.01 compared with PeV-A3-infected GBM cells under FBS or hPL mediums. ( B ) GBM cells were pre-treated with fludarabine 50 µM for 30 min under FBS or hPL mediums. After treatment, GBM cells were infected with PeV-A3 at MOI = 1 for 6 h. Cellular protein lysates were subjected to immunoblotting with anti-phospho-STAT1, anti-total-STAT1, and anti-GAPDH (upper panels). Quantification of protein expression levels of PeV-VP0, total-STAT1, and phospho-STAT1 was normalized to GAPDH. Data are mean ± SD of three independent experiments. Student’s t -test, *, p < 0.05 (lower panels). ( C ) The STAT-1 was knockdown by shRNA transfection; the cells were then infected by PeV-A3 at MOI = 1 for 48 h. The expression level of PeV-A3 VP1 RNA was measured by RT-qPCR. Data are mean ± SD of three independent samples. Student’s t -test, **, p < 0.01 compared with PeV-A3-infected GBM cells under FBS or hPL mediums. ( D ) The protein expression of PeV-A3 VP0, STAT1, phospho-STAT1, and GAPDH were detected by immunoblotting assay.

Article Snippet: The antibodies were anti-PeV VP0 (LTK BioLaboratories, Taoyuan, Taiwan) [ , ], anti-phospho STAT2 (Cell Signaling, 88410, Danvers, MA, USA), anti-total STAT2 (Cell Signaling, #72604), anti-phospho STAT1 (Cell Signaling, #9167), anti-total STAT1 (Cell Signaling, #14994), anti-phospho IRF3 (Abcam, #ab76493, Cambridge, England), anti-total IRF3 (Cell Signaling, #11904), anti-phospho NFκB p65 (Abcam, #ab86299), anti-total NFκB p65 (Cell Signaling, #8242) and anti-GAPDH (Proteintech, 60004-1-Ig, Rosemont, IL, USA).

Techniques: Expressing, Infection, Western Blot, shRNA, Transfection, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Selective epigenetic regulation of IFN-γ signature genes by JAK inhibitor in inflammatory diseases

doi: 10.1101/2024.08.05.606293

Figure Lengend Snippet: (A) Flow cytometry assessment of STAT1 and STAT3 protein levels in THP-1 cells treated with PMA and IFN-γ (or without) at various time points. Kinetics of total STAT and phospho-STAT tyrosine (STAT1, Tyr701; STAT3, Tyr705) measured by flow cytometry in IFN-γ-primed macrophages compared to resting macrophages. (B) Kinetics of total STAT1 (or STAT3) and phospho-STAT1 (or STAT3) measured by flow cytometry in JAK inhibitor-treated macrophages compared to resting and JAK inhibitor-untreated macrophages. Resting and IFN-γ-primed macrophages were differentiated for 24 h and then treated with JAKi (or DMSO) for up to 6 h. (A and B) Mean fluorescence intensity (MFI) represents a fold change compared to unstained samples. (C) RT-qPCR analysis of normalized target mRNA relative to TBP mRNA in THP-1 monocyte-derived macrophages under indicated conditions. IFN-γ-primed macrophages were treated with JAK inhibitor at a concentration of 1 μM for up to 6 h. Data show means ± SD from two independent experiments. (D) K-means clustering of differentially expressed (DE) genes in pairwise comparisons between the four conditions. DE genes identified by EdgeR (FDR adjusted P < 0.05, fold change > 2) were used. TPM values of RNA-seq data were filtered to be greater than 4. Non-significant clusters between replications were removed, resulting in three identified clusters. Clusters are indicated on the left. (E-G) Examples of expression for selected genes from clusters identified in the heatmap. Each dot on the bar plot represents one sample, and error bars denote the standard deviation. Error bars represent means ± SD. (H) Gene ontology (GO) analysis of THP-1 RNA-seq using genes positively correlated with HMDM RNA-seq. Heatmap displays the P-value (-Log10) significance of GO term enrichment for genes in each cluster, with clusters shown at the top. Downregulated by IFN-γ, n = 42; JAKi-sensitive, n = 129; JAKi-insensitive, n = 112. (I) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in RA patients. Clusters are indicated on the left. (J) Identification of genes associated with JAKi-sensitive or JAKi-insensitive in COVID-19 patients. Clusters are indicated on the left. For heatmaps of single cells, mean expression values were used, and hierarchical analysis was performed. (K) GO analysis of overlapping JAKi-sensitive and JAKi-insensitive genes in RA or COVID-19 patients. Clusters are indicated on the left. JAKi-sensitive, n = 51; JAKi-insensitive, n = 24. p < 0.05(*), p < 0.01(**), p < 0.001(***) and p < 0.0001(****) by one-way ANOVA. GO analysis was performed using Metascape ( http://metascape.org/ ).

Article Snippet: Add antibody (Alexa Fluor® 647 Mouse anti-Total Stat1, BD biosciences, 58560; PE anti-STAT1 Phospho (Tyr701), BioLegend, 666404; APC Mouse anti-Total Stat3, BD biosciences, 560392; PE/Cyanine5 anti-STAT3 Phospho (Tyr705), BioLegend, 651014) cocktail(s) to appropriate tubes, vortex to mix, and incubate for 30 minutes at room temperature in the dark.

Techniques: Flow Cytometry, Fluorescence, Quantitative RT-PCR, Derivative Assay, Concentration Assay, RNA Sequencing Assay, Expressing, Standard Deviation

Journal: Journal of Experimental Pharmacology

Article Title: Synergistic Effects of Azithromycin and STING Agonist Promote IFN-I Production by Enhancing the Activation of STING-TBK1 Signaling

doi: 10.2147/JEP.S433181

Figure Lengend Snippet: Co-treatment of AZM and DMXAA increased pTBK1 expression in activated macrophages after 3 hr of activation. ( A ) Cells were collected and mRNA levels of TBK1 were investigated by quantitative PCR (N=3-5). ( B and C ) Western blot analysis of AZM-activated macrophages with or without DMXAA showed phosphorylation of TBK1 (Ser172). Data are representative of independent over three experiments. ( D ) Confocal microscope images show DAPI (blue) and pTBK1 (Ser172) (green). ( E ) The quantification of immunofluorescence signals (N = 3). ( F and G ) Western blot analysis of AZM-activated macrophages with or without DMXAA showed phosphorylation of STAT1 (Tyr701). Data are presented as mean ± SEM; *p < 0.05, **p < 0.01, and ***p < 0.001.

Article Snippet: Then, proteins from the gel were transferred into nitrocellulose membranes and the antibodies’ unspecific binding was blocked with a blocking buffer for 1 h and then probed with Total TBK1 antibody (clone: D1B4 cat: 3504S, 1:1000) and Phospho-TBK1/NAK (Ser172) (clone: D52C2 cat: 5483S, 1:1000), Total STAT1 antibody (clone: 42H3 cat: 9175S, 1:1000), Phospho-Stat1 (Tyr701) (clone: D4A7 cat: 7649S, 1:1000) and β-ACTIN (clone: 13E5 cat: 4970S, 1:2500) (Cell Signaling, Danvers, MA, USA) overnight at 4°C.

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

Journal: Journal of Experimental Pharmacology

Article Title: Synergistic Effects of Azithromycin and STING Agonist Promote IFN-I Production by Enhancing the Activation of STING-TBK1 Signaling

doi: 10.2147/JEP.S433181

Figure Lengend Snippet: The proposed effect of Azithromycin and STING agonist (DMXAA) on macrophages. Co-treatment of AZM and DMXAA increased the expression of STING-TBK1-IRF3 signaling, leading to the up-regulation of IFN-I production. IFN-I can induce the activation of IFN- γ, resulting in the induction of STAT1 signaling and increasing ISG and pro-inflammatory cytokines expression. This figure was constructed by biorender.com.

Article Snippet: Then, proteins from the gel were transferred into nitrocellulose membranes and the antibodies’ unspecific binding was blocked with a blocking buffer for 1 h and then probed with Total TBK1 antibody (clone: D1B4 cat: 3504S, 1:1000) and Phospho-TBK1/NAK (Ser172) (clone: D52C2 cat: 5483S, 1:1000), Total STAT1 antibody (clone: 42H3 cat: 9175S, 1:1000), Phospho-Stat1 (Tyr701) (clone: D4A7 cat: 7649S, 1:1000) and β-ACTIN (clone: 13E5 cat: 4970S, 1:2500) (Cell Signaling, Danvers, MA, USA) overnight at 4°C.

Techniques: Expressing, Activation Assay, Construct