universal type i ifn  (PBL Assay)

Journal: PLOS Pathogens

Article Title: Tripartite motif-containing 34 (TRIM34) protein interacts with the nucleocytoplasmic transport machinery and negatively modulates antiviral responses

doi: 10.1371/journal.ppat.1014142

Figure Lengend Snippet: Human 293T (A, C) and human A549 (B, D) cells were treated with recombinant universal type I IFN or infected with IAV during 24h. TRIM34 gene expression was evaluated by RT-qPCR (A, B) and by Western blot, using an anti-TRIM34 specific antibody, and an anti-GAPDH specific antibody, used as a loading control (C, D) , and compared to the levels in mock-treated or mock-infected cells. Data in A and B represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. **p < 0.01 , ***p < 0.001 , ****p < 0.0001; one-way ANOVA followed by Dunnett’s post-hoc test. Protein bands in C and D were quantified using the ImageJ software and normalized to the levels of GAPDH expression (numbers below the blots). TRIM34 positively affects IAV replication in vitro. (E) Human 293T cells were transfected with the pCAGGS plasmid expressing TRIM34-FLAG protein or a pCAGGS empty plasmid, as control. At 24h post-transfection (hpt), cells were mock-infected or infected with IAV (MOI 1). (F) Human 293T cells were transfected twice with a non-targeted (NT) control siRNA or TRIM34 siRNA at two consecutive days, 24 h apart. On day 3, cells were mock-infected or infected with IAV (MOI 1). (E, F) At 0-, 24-, and 48-hours post-infection (hpi), cell culture supernatants were collected and titrated by immunofocus assay in MDCK cells. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05; Student’s t-test with Holm-Šídák correction .

Article Snippet: Furthermore, human 293T, A549 or BEAS-2B cells were treated with different concentrations of recombinant human type I IFN (11200–2, PBL Assay Science) during 24h.

Techniques: Recombinant, Infection, Gene Expression, Quantitative RT-PCR, Western Blot, Control, Software, Expressing, In Vitro, Transfection, Plasmid Preparation, Cell Culture

Journal: PLOS Pathogens

Article Title: Tripartite motif-containing 34 (TRIM34) protein interacts with the nucleocytoplasmic transport machinery and negatively modulates antiviral responses

doi: 10.1371/journal.ppat.1014142

Figure Lengend Snippet: (A, B) Human 293T cells were transfected with the plasmid pCAGGs-TRIM34-FLAG or the empty control plasmid. (A) At 24h post-transfection, cells were non-treated or treated with type I IFN. At 24h post-IFN-treatment, CXCL10, IFNB1, IFNL1 and IFIT2 expression was measured by RT-qPCR and mRNA levels were expressed as fold-change (increases) in comparison to mock-treated cells transfected with the empty plasmid, used as control. (B) At 24h post-IFN-treatment, cells were infected with rVSV-GFP (MOI 0.1) and viral titers were measured by lysis plaque assay at 24hpi. Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 for comparisons between empty and TRIM34 overexpressed cells using Student’s t-test with Holm-Šídák correction . Human 293T cells (C, F) , human lung adenocarcinoma-derived cells A549 cells (D, G), or the non-tumorigenic human bronchial epithelial BEAS-2B (E, H) were transfected twice with NT control siRNA or TRIM34 siRNA, 24h apart, for two consecutive days. On day 3, cells were treated with IFN for an additional 24h. (C, D, E) TRIM34, and (F, G, H) CXCL10, IFNB1, IFNL1 and IFIT2 expression was measured by RT-qPCR and mRNA levels were expressed as fold-change (increases) in comparison to mock-treated cells, transfected with the NT siRNA, used as control. (A-H) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05 for comparisons between control and TRIM34 knocked-down cells using Student’s t-test with Holm-Šídák correction .

Article Snippet: Furthermore, human 293T, A549 or BEAS-2B cells were treated with different concentrations of recombinant human type I IFN (11200–2, PBL Assay Science) during 24h.

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Comparison, Infection, Lysis, Plaque Assay, Derivative Assay

Journal: Inflammation

Article Title: Pharmacological Characterization of GLPG3667, a Tyrosine Kinase 2-Selective Inhibitor, for the Treatment of Inflammatory and Autoimmune Diseases

doi: 10.1007/s10753-026-02519-1

Figure Lengend Snippet: Type I IFN-induced responses evaluated by RNA sequencing in peripheral blood cells of healthy donors. ( A ) PC analysis was performed using the 500 most variable genes. The percentage of variance retained by the two first components is represented. The donor effect was removed (using the function ‘removeBatchEffect’) from the R package limma, before plotting, and the gene expression levels were normalized using the variance-stabilizing transformation. Samples are colored by the time after exposure to IFN-α. Time is represented in hours. ( B ) Heatmap of the top DEGs (log 2 fold change > 1 and adjusted p value < 0.01 compared with baseline) grouped using hierarchical clustering. The gene expression values were scaled using a Z-score normalization (for each condition, the gene expression value was subtracted from the gene average value and divided by its SD [x – mean/SD]). ( C ) Total number of DEGs (log 2 fold change > 1 and adjusted p value < 0.05) induced by IFN-α at each time point. ( D ) Volcano plot of DEGs affected by IFN-α + placebo at 9 h vs. placebo at baseline. Each gene was plotted as log 2 fold change (x-axis) and −log 10 (adjusted p value [FDR]) (y-axis). The horizontal dashed line indicates the cutoff of −log 10 (adjusted p value) of > 1.3 (adjusted p value < 0.05), and the vertical dashed lines indicate log 2 fold change > 1. ( E ) Gene set enrichment analysis following differential gene expression analysis of peripheral blood cells exposed to placebo + IFN-α at 9 h vs. placebo at 0 h. Hallmark gene sets upregulated (red) and downregulated (blue) are shown. Only significantly modified pathways (adjusted p value < 0.05) are shown as bar plots with the normalized enrichment score on the x-axis. Abbreviations: DEG, differentially expressed gene; FDR, false discovery rate; IFN, interferon; IL, interleukin; JAK, Janus kinase; PC, principal component; SD, standard deviation; STAT, signal transducer and activator of transcription; TNF-α, tumor necrosis factor-α

Article Snippet: After the incubation period, release of IFN-γ-induced protein 10 (IP-10) was triggered with 3000 U/mL IFN-α (Universal Type I IFN-Alpha, 11100-1, PBL Assay Science, PBL) for 24 h at 37 °C.

Techniques: RNA Sequencing, Gene Expression, Transformation Assay, Modification, Standard Deviation