Journal: Journal of Neuroinflammation

Article Title: The histone deacetylase inhibitor suberoylanilide hydroxamic acid attenuates human astrocyte neurotoxicity induced by interferon-γ

doi: 10.1186/1742-2094-9-113

Figure Lengend Snippet: Effects of SAHA on IFN-γ-induced phosphorylation of STAT1 and STAT3 in human astrocytes. Human astrocytes were incubated with or without SAHA at the concentrations indicated for 1 h. The cells were subsequently stimulated with IFN-γ for 30 minutes. Astrocytes in the control group were incubated with medium only. Cell lysates were separated by 8% SDS-PAGE and immunoblotted for phospho-Tyr 701 -STAT1 (pSTAT1) and total STAT1 (A) or phospho-Tyr 705 -STAT3 (pSTAT3) and total STAT3 (C) . The density ratios of phosphorylated to total protein are shown as mean ± S.E.M. of three independent experiments (B, D) . *Significantly different from IFN-γ stimulation only.

Article Snippet: The protein was transferred to a PVDF membrane at 70 V for 2 h. The membrane was blocked with 5% skim milk plus 3% bovine serum albumin (BSA) in PBS at room temperature (RT) for 1 h. Subsequently, the membrane was incubated with specific rabbit antibodies against phospho-Tyr 701 -STAT1 (1:2,000), total STAT1 (1:1,000), phospho-Tyr 705 -STAT3 (1:2,000) or total STAT3 (1:1,000) at 4°C overnight and then treated with horseradish peroxidase-conjugated anti-rabbit IgG antibody (1:2,000) at RT for 1 h. All antibodies used for immunoblotting were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Phospho-proteomics, Incubation, Control, SDS Page

Journal: BioMed Research International

Article Title: Helicobacter pylori Outer Membrane Protein 18 (Hp1125) Is Involved in Persistent Colonization by Evading Interferon- γ Signaling

doi: 10.1155/2015/571280

Figure Lengend Snippet: Δomp18 H. pylori induces more nitric oxide (NO) production in macrophages and mice stomachs. The murine macrophage RAW264.7 was used in this experiment and was infected by H. pylori 26695 or Δomp18 H. pylori 26695 with or without IFN- γ (50 pg/mL). (a) Western blot analysis of phosphorylated signal transducer and regulator of transcription 1 (STAT1) protein expression in murine macrophage RAW264.7 cells. Actin was a loading control. (b) Analysis of NO secretion from murine macrophage RAW264.7 cells by ELISA. (c) Analysis of NO secretion in gastric tissues from C57BL/6 mice infected by H. pylori SS1 and Δomp18 H. pylori SS1 at week 8 by ELISA. n = 10/group, 6–8 weeks old. ∗∗∗ P < 0.01. Data are means ± SEM from 3 independent experiments.

Article Snippet: They were then probed with specific antibodies against NapA (obtained from our lab) [ ], STAT1 (Cell Signaling Technology, #5375), and CagA (Abcam, ab90490), followed by anti-mouse or rabbit horseradish peroxidase-conjugated IgG.

Techniques: Infection, Western Blot, Expressing, Control, Enzyme-linked Immunosorbent Assay

Journal: The Journal of Immunology Author Choice

Article Title: Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells

doi: 10.4049/jimmunol.1901472

Figure Lengend Snippet: Functional characterization of A549-ISG15–knockout cell lines. ( A ) CRISPR/Cas9 genome editing was used to knockout ISG15 expression in A549 cells, followed by single-cell cloning [following previously reported procedures ]. Four independent clones were treated with 1000 IU/ml IFN-α for 24 and 48 h or left untreated, and protein expression was tested by immunoblot analysis of ISG15, MxA, IFIT1, and β-actin. Control cells were naive A549 cells. Representative image from two independent experiments. ( B ) A549 and A549-ISG15 −/− (B8) cells were treated with 1000 IU/ml IFN-α for 30 min, then extensively washed and media without IFN replaced. Cells were harvested at 0 and 30 min and 24 h after IFN-α removal and phopho-STAT1, total STAT1, MxA, ISG15, and β-actin were detected following immunoblot analysis. ( C ) A549 and A549-ISG15 −/− (clone B8) were treated with 1000 IU/ml IFN-α for 24 h. Expression of ISGs was tested using reverse transcription quantitative PCR (RT-qPCR) with primers specific for HERC5 , USP18 , IFIT1 , and MxA . Relative expression was determined following SYBR Green quantitative PCR (qPCR) using ΔΔCt method. β-Actin expression was used to normalize between samples. Error bars represent the SD of the mean from three independent RNA samples. ( D ) A549 and A549-ISG15 −/− (clone B8) were treated with 1000 IU/ml for 24 h. Cells were washed, and fresh media (without IFN-α) were replaced. Cells were processed for immunoblot analysis using Abs specific for MxA and β-actin at 24 h post–IFN-α and every 24 h thereafter for 72 h. Controls were cells without IFN-α. Representative image from two independent experiments. ( E ) A549 and A5549-ISG15 −/− (clone B8) were treated with 1000 IU/ml for 24 and 48 h (or left untreated). Whole-cell lysates were analyzed by immunoblotting with Abs specific for USP18, ISG15, and β-actin. Image is representative of more than three independent experiments.

Article Snippet: Confluent monolayers in six-well dishes were lysed with 250 μl 2× Laemmli sample buffer (4% w/v SDS, 20% v/v glycerol, 0.004% w/v bromphenol blue, and 0.125 M Tris–HCl (pH 6.8) with 10% v/v 2-ME) for 10 min, incubated at 95°C for 10 min, sonicated at 4°C with three cycles of 30 s on/30 s off in a Bioruptor Pico (Diagenode) and clarified by centrifugation at 12,000 × g , 4°C for 10 min. SDS-PAGE in Tris-glycine-SDS running buffer and immunoblotting followed standard techniques using the following Abs: mouse monoclonal anti-ISG15 F-9 (catalog no. sc166755; Santa Cruz Biotechnology), rabbit polyclonal anti-MxA (catalog no. 13750-1-AP; Proteintech), goat polyclonal anti-IFIT1 N-16 (catalog no. sc82946; Santa Cruz Biotechnology), mouse monoclonal anti–β-ACTIN, UBA7 (anti-UBE1L B-7; catalog no. sc-390097; Santa Cruz Biotechnology), rabbit monoclonal anti–phosphorylated STAT1 (anti–phospho-STAT1 [Tyr 701 ] 58D6; catalog no. 9167; Cell Signaling Technology), mouse monoclonal anti-PIV5 NP 125 , mouse monoclonal anti-HPIV2 and anti-PIV5 phosphoprotein (P) 161 [Ab cross-reacts with P of both viruses ( )], and mouse monoclonal anti-HPIV3 NP ( ).

Techniques: Functional Assay, Knock-Out, CRISPR, Expressing, Cloning, Clone Assay, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, SYBR Green Assay

Journal: The Journal of Immunology Author Choice

Article Title: Direct Antiviral Activity of IFN-Stimulated Genes Is Responsible for Resistance to Paramyxoviruses in ISG15-Deficient Cells

doi: 10.4049/jimmunol.1901472

Figure Lengend Snippet: IFN-α pretreatment of ISG15-deficient cells leads to virus resistance that is independent of ISGylation. ( A ) Control (naive A549) and four independent clones of A549-ISG15 −/− cells generated by CRISPR/Cas9 genome editing were treated with 1000 IU/ml IFN-α for 16 h or left untreated and then infected with PIV5-W3 (MOI = 10). Cells were harvested at 24 and 48 h p.i. and processed for immunoblot analysis using Abs specific for PIV5 NP, ISG15, STAT1, IFIT1, MxA, and β-actin. This experiment was independently performed twice. ( B ) UBA7-knockout cells were generated using CRISPR/Cas9 genome editing; Cas9-expressing A549 cells were first generated (following transduction with lentiCas9-Blast) and then transduced with lentiGuide-Puro expressing a sgRNA that targeted exon 3 of the UBA7 gene. Knockout cells were single-cell cloned, and three were selected for further analysis. These cells were treated with IFN-α or left untreated, infected, and processed as in (A) using Abs specific for PIV5 NP, ISG15, UBA7, and β-actin. This experiment was independently performed twice.

Article Snippet: Confluent monolayers in six-well dishes were lysed with 250 μl 2× Laemmli sample buffer (4% w/v SDS, 20% v/v glycerol, 0.004% w/v bromphenol blue, and 0.125 M Tris–HCl (pH 6.8) with 10% v/v 2-ME) for 10 min, incubated at 95°C for 10 min, sonicated at 4°C with three cycles of 30 s on/30 s off in a Bioruptor Pico (Diagenode) and clarified by centrifugation at 12,000 × g , 4°C for 10 min. SDS-PAGE in Tris-glycine-SDS running buffer and immunoblotting followed standard techniques using the following Abs: mouse monoclonal anti-ISG15 F-9 (catalog no. sc166755; Santa Cruz Biotechnology), rabbit polyclonal anti-MxA (catalog no. 13750-1-AP; Proteintech), goat polyclonal anti-IFIT1 N-16 (catalog no. sc82946; Santa Cruz Biotechnology), mouse monoclonal anti–β-ACTIN, UBA7 (anti-UBE1L B-7; catalog no. sc-390097; Santa Cruz Biotechnology), rabbit monoclonal anti–phosphorylated STAT1 (anti–phospho-STAT1 [Tyr 701 ] 58D6; catalog no. 9167; Cell Signaling Technology), mouse monoclonal anti-PIV5 NP 125 , mouse monoclonal anti-HPIV2 and anti-PIV5 phosphoprotein (P) 161 [Ab cross-reacts with P of both viruses ( )], and mouse monoclonal anti-HPIV3 NP ( ).

Techniques: Virus, Control, Clone Assay, Generated, CRISPR, Infection, Western Blot, Knock-Out, Expressing, Transduction, Gene Knockout

Journal: Virology Journal

Article Title: RUNX1 inhibits the antiviral immune response against influenza A virus through attenuating type I interferon signaling

doi: 10.1186/s12985-022-01764-8

Figure Lengend Snippet: Primers used for qPCR

Article Snippet: Mouse anti-RUNX1(A-2) (sc-365644) antibodies were purchased from Santa Cruz, Dallas, TX, USA; rabbit anti-IRF3 (11312-AP) and rabbit anti-STAT1 (10144–2-AP) were purchased from Proteintech, Rosemont, IL, USA; rabbit anti-P-IRF3 (Ser396) (4D4G) antibodies and rabbit anti-P-STAT1 (Tyr701) (D4A7) antibodies were purchased from Cell Signaling, Danvers, MA, USA; mouse anti-GAPDH antibody (AF0006) was purchased from Beyotime, Shanghai, China; mouse mAbs to viral proteins NP and M1 of IAV were obtained from Dr. Jiyong Zhou [ ].

Techniques: Sequencing

Journal: Virology Journal

Article Title: RUNX1 inhibits the antiviral immune response against influenza A virus through attenuating type I interferon signaling

doi: 10.1186/s12985-022-01764-8

Figure Lengend Snippet: RUNX1 attenuated IRF3 and STAT1 signaling. a The mRNA level of TRAF3 , RIG-I , MAVS , TBK1 , IRF3, and STAT1 in the shRUNX1 and shControl cells were assessed by qRT-PCR. b The mRNA of these genes in A549 cells that were transfected with empty plasmid pCMV-GFP or pCMV-RUNX1 was assessed by qRT-PCR. c , d The protein and the phosphorylation level of IRF3 and STAT1 of these cells were assessed by Western blot. e , f These cells were infected with PR8 (MOI = 5) and collected at 3, 6, and 9 h.p.i. The protein and the phosphorylation levels of IRF3 and STAT1 in these cells were assessed by Western blot. Data are mean ± SD of three independent experiments. Significance is by unpaired T-test; * p < 0.05; ** p < 0.01

Article Snippet: Mouse anti-RUNX1(A-2) (sc-365644) antibodies were purchased from Santa Cruz, Dallas, TX, USA; rabbit anti-IRF3 (11312-AP) and rabbit anti-STAT1 (10144–2-AP) were purchased from Proteintech, Rosemont, IL, USA; rabbit anti-P-IRF3 (Ser396) (4D4G) antibodies and rabbit anti-P-STAT1 (Tyr701) (D4A7) antibodies were purchased from Cell Signaling, Danvers, MA, USA; mouse anti-GAPDH antibody (AF0006) was purchased from Beyotime, Shanghai, China; mouse mAbs to viral proteins NP and M1 of IAV were obtained from Dr. Jiyong Zhou [ ].

Techniques: Quantitative RT-PCR, Transfection, Plasmid Preparation, Phospho-proteomics, Western Blot, Infection