a549  (ATCC)

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: Madin-Darby Canine Kidney (MDCK) epithelial (American Type Culture collection, ATCC CCL-34), human embryonic kidney 293T (ATCC CRL-11268), human lung epithelial carcinoma A549 (ATCC CCL-185), and African green monkey kidney epithelial Vero E6 (ATCC CRL-1586) cells were kindly provided by Prof. Luis Enjuanes (National Center for Biotechnology, CNB-CSIC, Spain).

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) Schematic representation of recombinant PR8 viruses expressing Venus, mCherry or TRIM34. A modified IAV-PR8 NS segment encoding NS1, either Venus (top), mCherry (middle) or TRIM34-FLAG (bottom), and NEP are indicated. Orange boxes at the beginning and end of each viral segment represent the viral 3′ and 5′ noncoding regions (NCR). Blue and white boxes indicate the viral NS1 and NEP genes, respectively. The thosea asigna virus (TAV) 2A autoproteolytic cleavage site used for the expression of NS1 and Venus, mCherry or TRIM34 and the porcine teschovirus (PTV) 2A autoproteolytic cleavage site used for the expression of Venus, mCherry or TRIM34 and NEP are indicated in gray. (B, C, D, E) Human A549 or MDCK cells were mock-infected or infected (MOI 0,1 and 1) with the recombinant viruses (rIAV-Venus, rIAV-mCherry or rIAV-TRIM34). (B) At 24hpi, MDCK cells were fixed, permeabilized and visualized for Venus and mCherry expression. Cells were stained with anti-FLAG (TRIM34) and anti-NS1 (infected-cells control) antibodies. DAPI was used for nuclear staining. (C - D) MDCK cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOIs 0.1 (C) or 1 (D) and viral titers were determined at 48 and 72hpi in MDCK cells. (E) A549 cells were infected with the viruses rIAV-Venus, rIAV-mCherry and rIAV-TRIM34 at MOI 1, and viral titers were determined at 48hpi in MDCK cells. Data 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 for comparisons between rIAV-TRIM34 with rIAV-Venus and rIAV-mCherry using one-way ANOVA followed by Dunnett’s post-hoc test.

Article Snippet: Madin-Darby Canine Kidney (MDCK) epithelial (American Type Culture collection, ATCC CCL-34), human embryonic kidney 293T (ATCC CRL-11268), human lung epithelial carcinoma A549 (ATCC CCL-185), and African green monkey kidney epithelial Vero E6 (ATCC CRL-1586) cells were kindly provided by Prof. Luis Enjuanes (National Center for Biotechnology, CNB-CSIC, Spain).

Techniques: Recombinant, Expressing, Modification, Virus, Infection, Staining, Control

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: Madin-Darby Canine Kidney (MDCK) epithelial (American Type Culture collection, ATCC CCL-34), human embryonic kidney 293T (ATCC CRL-11268), human lung epithelial carcinoma A549 (ATCC CCL-185), and African green monkey kidney epithelial Vero E6 (ATCC CRL-1586) cells were kindly provided by Prof. Luis Enjuanes (National Center for Biotechnology, CNB-CSIC, 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: Madin-Darby Canine Kidney (MDCK) epithelial (American Type Culture collection, ATCC CCL-34), human embryonic kidney 293T (ATCC CRL-11268), human lung epithelial carcinoma A549 (ATCC CCL-185), and African green monkey kidney epithelial Vero E6 (ATCC CRL-1586) cells were kindly provided by Prof. Luis Enjuanes (National Center for Biotechnology, CNB-CSIC, Spain).

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

Journal: bioRxiv

Article Title: Transgene Expression Kinetics and Replication Potential of Recombinant Adenovirus Serotype 4 in a Mouse Model and its Use as a Herpes Simplex Virus Vaccine

doi: 10.64898/2026.05.15.725395

Figure Lengend Snippet: Construction and characterization of Ad4 expressing luciferase or HSV-2 gD. (A) The E3 region of the adenovirus serotype 4 (Ad4) genome is replaced by left and right arms (LA, RA) to allow recombination with a cassette with LA, a CMV promoter, a luciferase or HSV-2 gD gene, a bovine growth hormone (BGH) poly A site, and an RA. The recombinant Ad4 genome is approximately 37 kb in length and retains genes in the E1 region responsible for viral replication. The genome map abbreviations are as follows; left inverted terminal repeat (LITR), E1-early transcriptional gene region 1 (consisting of E1A and E1B), E2-early transcriptional gene region 2 (consisting of E2A and E2B), E3-early transcriptional gene region 3, E4-early transcriptional gene region 4, and right inverted terminal repeat (RITR). (B) In vitro Ad4-Luc infection of A549 cells showing cytopathic effects (MOI 0.1, 3 days after infection, 5x magnification). (C) Uninfected A549 cells for comparison (5x magnification). (D) Relative luminescence of A549 cells infected with Ad4-Luc, Ad4-EGFP, or uninfected. Bars show median values with interquartile range (IQR), with individual data points. ## p=0.009 (Ad4-Luc versus Ad4-EGFP); ** p=0.002 (Ad4-Luc versus uninfected); ns: not significant. (E) Western blot of purified soluble HSV-2 gD protein (Sol gD2), cell lysates of A549 cells infected with Ad4-gD2 (Ad4-gD2), a replicate lysate (Ad4-gD2 rep), and uninfected A549 cells after staining with anti-HSV-2 gD antibody.

Article Snippet: The previously described SpeI site was utilized to linearize pRAd4-EGFP or pRAd4-Luc and this was transfected using PEIpro® (Polyplus Cat# 101000033) into the A549 human carcinoma lung cell line (ATCC cat. CCL-185).

Techniques: Expressing, Luciferase, Recombinant, In Vitro, Infection, Comparison, Western Blot, Purification, Staining

Journal: iScience

Article Title: Drug screening to identify compounds to eliminate Burkholderia pseudomallei through Hcp protein

doi: 10.1016/j.isci.2026.115367

Figure Lengend Snippet: Netilmicin sulfate alone and in combination exerted protective effects on an infected A549 cell model (A) Cytotoxic effects of the drugs on A549 cells. (B) Protective effects of different concentrations of NETS on A549 cells. The early interventions on the abscissa reflect the incubation of A549 cells with the drug for 2 h before B. pseudomallei HNBP001 infection. Late intervention was defined as the introduction of the drug to A549 cells 2 h after infection with B. pseudomallei HNBP001 . The ordinate is the survival rate of the infected A549 cells. NC: the blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Bp+30 μM NETS: The infected A549 cells were treated with 30 μM NETS; the other conditions were the same, but different concentrations of NETS were used. (C) Protective effects of several drugs on infected A549 cells; the ordinate is the survival rate of infected A549 cells. NC, blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Other: The infected A549 cells were treated with drugs (including SXT, GM, NETS, CAZ, and NETS+CAZ). (D) Number of intracellular bacteria in infected A549 cells after drug treatment; the ordinate represents the number of bacteria in infected A549 cells. NC is the blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Drug group: 2 × MIC drug was used to treat infected A549 cells. All drugs, including SXT, GM, NETS, CAZ, and NETS+CAZ. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no significant difference. Data are represented as mean ± SEM.).

Article Snippet: The A549 human lung carcinoma epithelial cell line was purchased from Procell (Wuhan, China; Cat# CL-0016).

Techniques: Infection, Incubation, Negative Control, Bacteria

Journal: iScience

Article Title: Drug screening to identify compounds to eliminate Burkholderia pseudomallei through Hcp protein

doi: 10.1016/j.isci.2026.115367

Figure Lengend Snippet: Netilmicin sulfate alone and in combination exerted protective effects on an infected A549 cell model (A) Cytotoxic effects of the drugs on A549 cells. (B) Protective effects of different concentrations of NETS on A549 cells. The early interventions on the abscissa reflect the incubation of A549 cells with the drug for 2 h before B. pseudomallei HNBP001 infection. Late intervention was defined as the introduction of the drug to A549 cells 2 h after infection with B. pseudomallei HNBP001 . The ordinate is the survival rate of the infected A549 cells. NC: the blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Bp+30 μM NETS: The infected A549 cells were treated with 30 μM NETS; the other conditions were the same, but different concentrations of NETS were used. (C) Protective effects of several drugs on infected A549 cells; the ordinate is the survival rate of infected A549 cells. NC, blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Other: The infected A549 cells were treated with drugs (including SXT, GM, NETS, CAZ, and NETS+CAZ). (D) Number of intracellular bacteria in infected A549 cells after drug treatment; the ordinate represents the number of bacteria in infected A549 cells. NC is the blank negative control group of fresh 10% DMEM. Bp: infected A549 cell group. Drug group: 2 × MIC drug was used to treat infected A549 cells. All drugs, including SXT, GM, NETS, CAZ, and NETS+CAZ. (∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001, and ∗∗∗∗ p < 0.0001; ns, no significant difference. Data are represented as mean ± SEM.).

Article Snippet: A549 human lung carcinoma cell line , Procell , Cat#CL-0016.

Techniques: Infection, Incubation, Negative Control, Bacteria