Journal: Journal of Virology

Article Title: Insights into the Gene Expression Profiles of Active and Restricted Red/Green-HIV + Human Astrocytes: Implications for Shock or Lock Therapies in the Brain

doi: 10.1128/JVI.01563-19

Figure Lengend Snippet: Upregulation of interferon signaling pathways. (A) Pathway image from IPA core analysis illustrating gene expression associated with interferon signaling. (B) The molecular activity prediction (MAP) tool indicates the potential outcomes of activating the interferon signaling. The color intensity indicates the degree of upregulation (red). Predicted activation (orange), inhibition (blue), and unknown association (yellow) are also shown. Gray indicates molecules that were present in the data set but did not pass the cutoff values of a false discovery rate (FDR) of ≤0.05 and a log2 fold change (FC) of either ≥1 or ≤−1. (C) Individual populations (red and yellow) were sorted, and STAT1 gene expression was measured by RT-PCR at 2 weeks postinfection. Data are mean fold changes ± standard error of mean (SEM) compared to D116A-infected cells. Data are cumulative for three individual astrocyte donors. Statistical analyses were performed using one-way ANOVA with Tukey’s post hoc test for multiple comparisons (*, P < 0.05).

Article Snippet: Commercially available gene expression assays for human ICAM1 (no. Hs00164932_m1), CCL2 (no. Hs00234140_m1), and STAT1 (no. Hs01013996_m1) were used with the TaqMan fast advanced master mix according to the manufacturer’s directions (Thermo Fisher).

Techniques: Protein-Protein interactions, Gene Expression, Activity Assay, Activation Assay, Inhibition, Reverse Transcription Polymerase Chain Reaction, Infection

Journal: Journal of Virology

Article Title: Insights into the Gene Expression Profiles of Active and Restricted Red/Green-HIV + Human Astrocytes: Implications for Shock or Lock Therapies in the Brain

doi: 10.1128/JVI.01563-19

Figure Lengend Snippet: Genes altered in the neuroinflammation signaling pathway

Article Snippet: Commercially available gene expression assays for human ICAM1 (no. Hs00164932_m1), CCL2 (no. Hs00234140_m1), and STAT1 (no. Hs01013996_m1) were used with the TaqMan fast advanced master mix according to the manufacturer’s directions (Thermo Fisher).

Techniques: Expressing, Clinical Proteomics, Membrane, Immunopeptidomics

Journal: Gut Pathogens

Article Title: Cytokines derived from innate lymphoid cells assist Helicobacter hepaticus to aggravate hepatocellular tumorigenesis in viral transgenic mice

doi: 10.1186/s13099-019-0302-0

Figure Lengend Snippet: ILC-derived IFN-γ triggered epithelial–mesenchymal transition via STAT1 signaling in Helicobacter hepaticus -associated HCC. a Hepatic E-cadherin relative expression at different ages in wild type B6 and HBs-Tg. b Hepatic E-cadherin relative expression in H. hepaticus adopted HBs-Tg mice with IFN-γ antibody. c Hepatic signaling pathways detected by western blotting at 13 months old. d Detection of E-cadherin and EMT-related genes in primary hepatocytes with IFN-γ and inhibitor. Data are expressed as median ± percentile. Mann–Whitney U was used. P < 0.05 represents statistical difference. *P < 0.05, **P < 0.01

Article Snippet: STAT1 activity inhibition was done by incubation with 20 μM Fludarabine (Selleckchem, USA) for 24 h, followed by IFN-γ treatment for another 24 h.

Techniques: Derivative Assay, Expressing, Protein-Protein interactions, Western Blot, MANN-WHITNEY

Journal: International Journal of Molecular Sciences

Article Title: Methionine Restriction Attenuates Scar Formation in Fibroblasts Derived from Patients with Post-Burn Hypertrophic Scar

doi: 10.3390/ijms26125876

Figure Lengend Snippet: Expression of signaling pathways in MetR-treated hypertrophic scar fibroblasts. Significantly decreased levels of phosphorated ( A , B ) NF-κB, ( A , C ) STAT1, and ( A , D ) STAT3 proteins were observed in HTSFs treated with MetR medium compared to those seen in control cells. Significantly decreased levels of phosphorylated ( E , F ) SMAD2 and ( E , G ) SMAD3 proteins were observed in HTSFs treated with MetR medium compared to those seen in control cells. Significantly increased levels of phosphorylated (p) ( H , I ) JNK1, ( H , J ) p38, and ( H , K ) ERK1/2 proteins were observed in HTSFs treated with MetR medium compared to those seen in control cells. HTSFs treated with normal high-glucose Dulbecco’s modified Eagle’s medium were used as the controls. ** p < 0.01 vs. control. Data represent the mean ± SD; n = 3.

Article Snippet: STAT1 , Rabbit , 1:1000 , Cusabio Technology (CSB- PA825331 ).

Techniques: Expressing, Protein-Protein interactions, Control, Modification

Journal: Scientific Reports

Article Title: miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells

doi: 10.1038/srep12987

Figure Lengend Snippet: Cells were transfected with miR-744 mimic or NC mimic (200 nM) for 24 h, then stimulated with type I IFN for 0 min or 15 min. Proteins were harvested and analyzed with western blotting to detect the phosphorylation of TYK2, STAT1, JAK1, and STAT2 ( A ) in the JAK–STAT pathway and p38, ERK, and STAT3 in the non-classical pathway ( B ) (The full-length blots/gels of TYK2, STAT1, JAK1, STAT2, p38, ERK and STAT3 are presented in , , , , , , , respectively). RMCs were pretreated with SB203580 (p38 inhibitor), PD98059 (ERK inhibitor), or PDTC (NF-κB inhibitor) for 0.5 h before stimulation with type I IFN for 6 h. RNA was then harvested and the expression of IFN-inducible inflammatory genes was detected ( C,D ). *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001; NS, not significant.

Article Snippet: The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively).

Techniques: Transfection, Western Blot, Phospho-proteomics, Expressing

Journal: Scientific Reports

Article Title: miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells

doi: 10.1038/srep12987

Figure Lengend Snippet: ( A ) Schematic diagram of potential miR-744 binding sites in the 3′-UTR of PTP1B , predicted by RNAhybrid, and two mutant binding sites. Mutant 1 abolished the binding to miR-744 without changing the nucleotide composition of the sequence, while mutant 2 affected both the nucleotide composition of the sequence and the binding of the 3′-UTR to miR-744. ( B ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or pSicheck2 vector (50 ng per well). Luciferase activity was measured 24 h after transfection. ( C ) RMCs were simultaneously transfected with NC or miR-744 (200 nM) and the PTP1B 3′-UTR-containing vector (50 ng per well) or PTP1B 3′-UTR mutant vector (50 ng per well). Luciferase activity was measured 24 h after transfection, quantified, and expressed as relative luciferase activity. ( D ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics (left) or for 48 h for the inhibitors (right). The levels of PTP1B mRNA were detected after stimulation with type I IFN for 6 h. ( E ) RMCs were transfected with miR-744 mimic or inhibitor and the corresponding control mimic or inhibitor for either 24 h for the mimics or for 48 h for the inhibitors. PTP1B was detected in the whole-cell lysates with western blotting. The ratios of PTP1B to β-tubulin in the control-transfected cells was arbitrarily set at 1(The full-length blots/gels of PTP1B are presented in , , respectively). ( F ) Induction of CCL5 after PTP1B was silenced with siRNA (200 nM) in RMCs. ( G ) Efficiency of siRNA measured with real-time PCR. ( H ) Western blotting analysis of the phosphorylation of STAT1 and STAT3 in PTP1B-silenced RMCs. Cells were treated with type I IFN (1000 U/mL) for the indicated times (The full-length blots/gels of STAT1 and STAT3 are presented in , , respectively). At least three independent experiments were performed. *p ≤ 0.05; **p ≤ 0.01; ***p ≤ 0.001. NS, not significant.

Article Snippet: The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively).

Techniques: Binding Assay, Mutagenesis, Sequencing, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Control, Western Blot, Real-time Polymerase Chain Reaction, Phospho-proteomics

Journal: Scientific Reports

Article Title: miR-744 enhances type I interferon signaling pathway by targeting PTP1B in primary human renal mesangial cells

doi: 10.1038/srep12987

Figure Lengend Snippet: ( A ) RMCs were stimulated with type I IFN (1000 U/mL) and the RNA was collected at the indicated time points. The expression of miR-744 was detected with qRT–PCR. Each point shows the mean relative expression level of miR-744 for three independent experiments. We used RNU48 as the reference gene. ( B ) Schematic diagram of the mechanisms by which miR-744 feed-forward regulates the IFN-activated classical JAK–STAT and non-classical MAPK and NF-κB signaling pathways. Type I IFN triggers an unknown transcription factor to induce miR-744 expression. miR-744 subsequently represses PTP1B expression, leading to the enhanced activation of TYK2, STAT1, STAT3, ERK, p38, and NF-κB, thus enhancing the expression of IFN-induced inflammatory genes.

Article Snippet: The proteins were extracted, separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, blotted, and probed with the specified primary antibodies directed against phosphorylated STAT1 (p-STAT1), STAT1, p-STAT2, STAT2, JAK1, PTP1B, p-P44/42 MAPK (ERK1/2), P44/42 MAPK (ERK1/2), p38 MAPK, p-p38 MAPK, and p-TYK2 (Cell Signaling Technology, diluted 1:1000); anti-STAT3, anti-p-STAT3, and anti-p-JAK1 (Santa Cruz Biotechnology, diluted 1:200); anti-TYK2 and anti-β-tubulin (Abcam, diluted 1:1000 and 1:5000, respectively).

Techniques: Expressing, Quantitative RT-PCR, Protein-Protein interactions, Activation Assay

Journal: Journal of Virology

Article Title: Multimodal characterization of the responsiveness of eight hepatitis D virus genotype isolates to interferon-alpha treatment

doi: 10.1128/jvi.01280-25

Figure Lengend Snippet: Characterization of the antiviral efficacy of IFN-α2a against HDV 1–8 isolates upon HDV de novo infection. ( A ) Schematic of the experimental setting. ( B ) The IC 50 values of IFN-α2a against HDV 1–8 isolates in HuH7 NTCP , HepaRG NTCP , and HepG2 NTCP cells. ( C–J ) The antiviral efficacy of IFN-α2a against HDV 1–8 isolates was tested in HuH7 NTCP and HepG2 NTCP cells upon HDV de novo infection ( n = 4). ( K ) Schematic model of JAK-STAT signaling pathways. ( L ) HuH7 NTCP and HepG2 NTCP cells were treated with IFN-α2a (1,000 IU/mL) for 30 minutes. The levels of STAT1, STAT2, pSTAT1(Y701), and pSTAT2(Y690) were detected by western blot. The ratios of p-STAT1/STAT1 or p-STAT2/STAT2 were calculated by normalizing IFN-α2a-induced p-STAT1 or p-STAT2 level to both GAPDH and the baseline STAT1 or STAT2 expression. ( M ) HuH7 NTCP and HepG2 NTCP cells were treated with IFN-α2a (1,000 IU/mL), and the levels of interferon-stimulated response element (ISRE)-related luciferase values were detected 8 hours post-treatment ( n = 6). ( N and O ) HuH7 NTCP and HepG2 NTCP cells were treated with IFN-α2a (1,000 IU/mL) for 8 hours. A of representative ISGs was analyzed by RT-qPCR ( n = 4). The data were presented either in bar chart ( N ) or heatmap ( O ). HDAg, hepatitis D antigen; RLU, relative light unit; IF, immunofluorescence; Luc, luciferase; ND, not detected. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Proteins were transferred onto nitrocellulose membranes by semidry transfer and incubated with primary antibodies as indicated below: rabbit anti-HDAg polyclonal antibody, mouse anti-β-actin (Sigma, A1978), mouse anti-GAPDH (Proteintech, 60004-1-lg), rabbit anti-ADAR1 (Cell Signaling, 14175), STAT1 monoclonal antibody (Proteintech, 66545-1-lg), STAT2 polyclonal antibody (Proteintech, 16674-1-AP), p-STAT1: phospho-STAT1 (Tyr701) polyclonal antibody (Proteintech, 28979-1-AP), and phospho-STAT2 (Tyr690) antibody (Affinity, AF3342).

Techniques: Infection, Protein-Protein interactions, Western Blot, Expressing, Luciferase, Quantitative RT-PCR, Immunofluorescence