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: Non-tumorigenic human bronchial epithelial cell line BEAS-2B (ATCC CRL-9690), was obtained from Carlos López-Larrea (Health Research Institute of the Principality of Asturias (ISPA), Spain).

Techniques: Transfection, Plasmid Preparation, Control, Expressing, Quantitative RT-PCR, Comparison, Infection, Lysis, Plaque Assay, Derivative 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: (A) Human 293T cells were transfected with the pCAGGS plasmids encoding TRIM34-FLAG or the empty plasmid, as control. At 24h post-transfection, cells were mock-infected or infected with IAV or IAV-ΔNS1. At 24hpi, the levels of CXCL10, IFNB1, IFNL1, and IFIT2 mRNAs were evaluated by RT-qPCR, and expressed as fold-change (increases) in comparison to mock-infected cells, transfected with the empty plasmid, used as control. (B) Human 293T cells were transfected twice with the NT control siRNA or TRIM34 siRNA every 24h for two consecutive days. On day 3, cells were infected with IAV or IAV-ΔNS1 (MOI 1) for an additional 24h. The levels of CXCL10, IFNB1, IFNL1 and IFIT2 were measured by RT-qPCR and mRNA levels were expressed as fold- change (increases) in comparison to mock-infected cells transfected with the NT siRNA, used as controls. Human A549 (C) or BEAS-2B (D) cells were transfected twice with the NT control siRNA or TRIM34 siRNA every 24h for two consecutive days. On day 3, cells were infected with IAV for an additional 24h. (B, C, D) The levels of CXCL10, IFNB1, IFNL1 and IFIT2 were measured by RT-qPCR and mRNA levels were expressed as fold change (increases) in comparison to mock-infected cells transfected with the NT siRNA, used as controls. (A-D) 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, or NT siRNA and TRIM34 knocked-down cells, using Student’s t-test with Holm-Šídák correction. (E, F) A549 cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOI 1, and the levels of (E) IAV NP-vRNA, and (F) CXCL10, CCL2, IFNL1 and IFIT2 mRNAs were determined at 24hpi. (E, F) Data represents means and SDs of results from triplicate wells. Three different experiments were performed, with similar results. *p < 0.05, **p < 0.01, ***p < 0.001 for comparisons between rIAV-TRIM34 with rIAV-Venus and rIAV-mCherry using one-way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: Non-tumorigenic human bronchial epithelial cell line BEAS-2B (ATCC CRL-9690), was obtained from Carlos López-Larrea (Health Research Institute of the Principality of Asturias (ISPA), Spain).

Techniques: Transfection, Plasmid Preparation, Control, Infection, Quantitative RT-PCR, Comparison

Journal: The Journal of Gene Medicine

Article Title: Development of CpG‐Depleted CFTR Plasmid‐Based Nanoparticles for Nonviral Gene Therapy in Lung Cystic Fibrosis Disease

doi: 10.1002/jgm.70087

Figure Lengend Snippet: CFTR protein expression from different CFTR plasmids. (a) CFTR gene expression was assessed in untreated 16HBE14o‐, untreated CFBE41o‐ (UNT), and CFBE41o‐ cells transfected with HPAEs/CFTR plasmids (pWT‐ CFTR 1, pWT‐ CFTR 2, pOP‐ CFTR 1, pOP‐ CFTR 2) 48 h after transfection via western blot. (b) The semiquantification of western blot was normalized with the loading control protein α‐tubulin. (c) CFTR gene expression from untreated 16HBE14o‐, untreated CFBE41o‐ (UNT), and CFBE41o‐ cells transfected with HPAEs/CFTR plasmids 48 h after transfection was assessed by immunofluorescence, using a 20× fluorescence microscopy objective; the dimension of the scale bar is 50 μm. The yellow arrows show the CFTR protein expression in treated CFBE41o‐ cells with different CFTR plasmids. Western blot and immunofluorescence images are representative of three independent experiments ( n = 3). All data were collected from at least three independent experiments ( n ≥ 3). All bar graph data are depicted as means ± SDs. ** p < 0.01, *** p < 0.001; ns: not significant, as compared with pWT‐ CFTR 1.

Article Snippet: The 16HBE14o‐ human bronchial epithelial cell line, purchased from Merck (Cat num.

Techniques: Expressing, Gene Expression, Transfection, Western Blot, Control, Immunofluorescence, Fluorescence, Microscopy

Journal: The Journal of Gene Medicine

Article Title: Development of CpG‐Depleted CFTR Plasmid‐Based Nanoparticles for Nonviral Gene Therapy in Lung Cystic Fibrosis Disease

doi: 10.1002/jgm.70087

Figure Lengend Snippet: Time courses of CFTR protein expression of different CFTR plasmids (pOP‐ CFTR 2 and pOP‐ CFTR 2‐CMV). (a) CFTR protein expression of different CFTR plasmids with the loading control protein α‐tubulin was assessed by western blot at 24 h, 48 h, and 7 days after transfection. (b) Quantification of CFTR mRNA levels via RT‐qPCR, normalized to GAPDH and then compared with 16HBE14o‐ cells, following treatment with HPAE complexes containing different CFTR plasmids at 24 h, 48 h, and 7 days after transfection. (c) CFTR protein was semiquantified, normalized with the loading control protein α‐tubulin, and then compared with 16HBE14o‐ cells, after treatment with HPAEs complexing to different CFTR plasmids by western blot 24 h after transfection, 48 h after transfection, and 7 days after transfection. Western blot images are representative of three independent experiments ( n = 3). All data were collected from at least three independent experiments ( n ≥ 3). All bar graph data are depicted as means ± SDs. **** p < 0.001; ns: not significant, as compared with pOP‐CFTR2‐CMV.

Article Snippet: The 16HBE14o‐ human bronchial epithelial cell line, purchased from Merck (Cat num.

Techniques: Expressing, Control, Western Blot, Transfection, Quantitative RT-PCR