human carcinoma epithelial lung a549  (InvivoGen)

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: Human A549 cells were infected with MR766 MC or chimeric MR766 MC virus with NS1 CWA protein (MR766 MC chimera) at an m.o.i. of 1. In ( A ), virus progeny production (PFU.mL -1 ) was examined using a conventional plaque-forming assay. In ( B ), intracellular viral RNA production was determined by RT-qPCR at 48 h p.i. In ( C ), the percentage of ZIKV-infected cells based on FACS analysis using anti-E mAb 4G2. In ( D ), LDH activity was measured at 48h p.i and expressed as a percentage relative to mock-infected cells (control). Asterisks indicate that the differences between experimental samples are statistically significant, using an unpaired t test (*** p < 0.001, ** p < 0.01, * p < 0.05).

Article Snippet: Human embryonic kidney HEK-293T (CRL-1573, ATCC, VA, USA), human carcinoma epithelial lung A549 (InvivoGen, Toulouse, France), and monkey kidney normal VeroE6 (CCL-81, ATCC, VA, USA) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) growth medium (Thermo Fisher Scientific, Les Ulis, France) supplemented with heat-inactivated fetal bovine serum (FBS) (Dutscher, Strasbourg, France) and antibiotics (Dutscher, Strasbourg, France) at 37 °C.

Techniques: Infection, Virus, Quantitative RT-PCR, Activity Assay, Control

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: A549 cells were infected with MR766 MC or chimeric MR766 MC virus with NS1 CWA protein (MR766 MC chimera) or mock-infected (no virus) for 24 h or 48 h at an m.o.i. of 1. In ( A ), visualization of intracellular rNS1 protein. Cells infected for 24h were stained with anti-E mAb 4G2 or anti-NS1 mAb 4G4 as primary antibody (green) for confocal immunofluorescence analysis. Nuclei were stained with DAPI (blue). The same magnification was used throughout. Scale bar, 25 μM. In ( B ), cell supernatant samples from three independent infections (exp. 1 to exp. 3) were analyzed in duplicates (A, B) by dot-blotting using mAb 4G4. The mean of signal intensity of each duplicate was determined using Image J software to estimate the relative amounts of secreted soluble NS1 protein. Results are the mean (± SEM) of three independent assays. Asterisks indicate that the differences between experimental samples are statistically significant, using an unpaired t test (** p < 0.01).

Article Snippet: Human embryonic kidney HEK-293T (CRL-1573, ATCC, VA, USA), human carcinoma epithelial lung A549 (InvivoGen, Toulouse, France), and monkey kidney normal VeroE6 (CCL-81, ATCC, VA, USA) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) growth medium (Thermo Fisher Scientific, Les Ulis, France) supplemented with heat-inactivated fetal bovine serum (FBS) (Dutscher, Strasbourg, France) and antibiotics (Dutscher, Strasbourg, France) at 37 °C.

Techniques: Infection, Virus, Staining, Immunofluorescence, Software

Journal: PLOS Neglected Tropical Diseases

Article Title: The NS1 protein of contemporary West African Zika virus potentiates viral replication and reduces innate immune activation

doi: 10.1371/journal.pntd.0012146

Figure Lengend Snippet: A549 cells were infected with MR766 MC or the chimeric MR766 MC with NS1 ZIKV-15555 (MR766 MC chimera) at an m.o.i. of 1. In ( A ), the relative abundance of IFN-β and ISG mRNA was determined at 48 h p.i. by RT-qPCR. Housekeeping gene RPLPO36B4 mRNA served as an internal reference. The results are the mean (± S.D.) of three replicates. Asterisks indicate that the differences between MR766 MC and MR766 MC chimera for each cellular factor are statistically significant, using an unpaired t test (**** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05). In ( B ), Immunoblot assay was performed on cell lysates using anti-ISG15 or anti-IFIT1 antibodies as indicated. Anti-E mAb 4G2 was used to detect ZIKV E protein. β-actin was detected as protein-loading control for lysate samples.

Article Snippet: Human embryonic kidney HEK-293T (CRL-1573, ATCC, VA, USA), human carcinoma epithelial lung A549 (InvivoGen, Toulouse, France), and monkey kidney normal VeroE6 (CCL-81, ATCC, VA, USA) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) growth medium (Thermo Fisher Scientific, Les Ulis, France) supplemented with heat-inactivated fetal bovine serum (FBS) (Dutscher, Strasbourg, France) and antibiotics (Dutscher, Strasbourg, France) at 37 °C.

Techniques: Infection, Quantitative RT-PCR, Western Blot, Control