a549 lung carcinoma cells  (InvivoGen)

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: 3P SARS2 VLPs are superior to 4P SARS2 VLPs. ( A ) 4P and 3P SARS2 Luc-PS9 VLPs were produced by co-transfection of either four or three plasmids into 293T producer cells. These plasmids encode for the viral structural proteins, along with a luciferase reporter coupled to PS9 packaging signal (Luc-PS9). ( B ) Luciferase assay showed ∼7-fold higher luminescence intensity in 293T-hACE2 cells upon using 3P versus 4P VLPs. ( C ) VLP concentrate (10 μl for M and E, 2 μl for S2 and N) was loaded in each lane. Western blot analysis suggests incorporation of all SARS2 structural components in VLPs, with 3P SARS2 Luc-PS9 VLPs displaying more intense protein bands compared with 4P VLPs. ( D ) Higher Luc-PS9 transcript levels were observed using RT-PCR in the case of 3P SARS2 Luc-PS9 VLPs. ( E ) Cryo-TEM images of 3P SARS2 Luc-PS9 VLPs showed spherical ∼100-nm-sized VLPs with double-layered membrane structures. ( F ) 3P SARS2 EGFP-PS9 VLPs produced with EGFP reporter efficiently infected 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Fluorescence images were acquired 24 h post-infection. ( G ) Flow cytometry VLP entry time-course showed peak fluorescence for 3P SARS2 EGFP-PS9 VLPs at 24 h followed by decrease at larger times. This was observed both for 293T-hACE2 and A549-hACE2-TMPRSS2 cells. Abbreviations: aa, amino acid; nt, nucleotide. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: A549 lung carcinoma-overexpressing human ACE2 and TMPRSS2 (“A549-hACE2-TMPRSS2”) cells (Catalog #: a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA).

Techniques: Produced, Cotransfection, Luciferase, Western Blot, Reverse Transcription Polymerase Chain Reaction, Membrane, Infection, Fluorescence, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Tuning VLP viral tropism by altering viral glycoprotein. ( A ) Different types of VLPs were produced using the 3P system by varying the viral glycoprotein (VSV-G, SARS2 spike, SARS spike, or MERS spike) and reporter genes (luciferase or EGFP). ( B ) One microgram of the viral glycoprotein was used to create various 3P Luc-PS9 VLPs and these were used to infect five cell types: WT 293T (293T), 293T-hACE2, A549-hACE2-TMPRSS2, Calu-3, and 293T-DPP4. SARS2 and SARS spike VLPs displayed similar tropism and entered only hACE2-expressing cells (293T-hACE2, A549-hACE2-TMPRSS2, and Calu-3), with SARS exhibiting higher luminescence intensity compared with SARS2. MERS VLPs infected Calu-3 cell at low level and efficiently entered 293T-DPP4 cells. VSV-G VLPs entered all cell types. ( C ) 3P EGFP-PS9 VLPs were produced with 4 μg VSV-G, 1 μg SARS2 spike, or 1 μg MERS spike plasmid. VSV-G VLPs entered all cell types. SARS2 VLPs only entered ACE2 cells. MERS VLPs only entered DPP4 cells. Methods provide detailed steps for VLP production. Data are mean ± STD. * P < .05, ** P < .01, *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: A549 lung carcinoma-overexpressing human ACE2 and TMPRSS2 (“A549-hACE2-TMPRSS2”) cells (Catalog #: a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA).

Techniques: Produced, Luciferase, Expressing, Infection, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Tuning the tropism and infectivity of SARS-CoV-2 virus-like particles for mRNA delivery

doi: 10.1093/nar/gkaf133

Figure Lengend Snippet: Streamlining VLP technology using a 2P system. ( A ) Four constructs were developed with two independent promoters driving expression of reporter gene and SARS2 structural proteins. The promoters were separated by insulator and terminator sequences to minimize promoter interference: synthetic polyA (spa); a G-rich sequence from β-actin (Tactb); chicken hypersensitive site 4 (cHS4); and a synthetic MAR sequence 8 (sMAR8) at the end of the E protein. (B, C) Each of these constructs was transfected into 293T cells along with spike plasmid to produce four different 2P SARS2 Luc-PS9 VLPs. 2P.2 VLPs displayed highest luminescence intensity ( B ). Its signal was comparable to 4P VLP but lower than 3P VLP ( C ). ( D ) Western blots of SARS2 structural proteins showed different patterns of protein expression for different 2P plasmids. 2P.2 SARS2 Luc-PS9 VLPs displayed more intense protein bands compared with 2P.3 and 2P.4, but this was lower than 2P.1. ( E ) 2P.2.EGFP VLPs were created by replacing the luciferase reporter with EGFP. VLP entry of 2P.2.EGFP SARS2 VLPs into A549-hACE2-TMPRSS2 and 293T-hACE2 cells was measured using flow cytometry. Data are mean ± STD. *** P < .001, **** P < .0001, NS: not significant.

Article Snippet: A549 lung carcinoma-overexpressing human ACE2 and TMPRSS2 (“A549-hACE2-TMPRSS2”) cells (Catalog #: a549-hace2tpsa) were purchased from InvivoGen (San Diego, CA).

Techniques: Construct, Expressing, Sequencing, Transfection, Plasmid Preparation, Western Blot, Luciferase, Flow Cytometry

Journal: Journal of traditional and complementary medicine

Article Title: In vivo evaluation of Andrographis paniculata and Boesenbergia rotunda extract activity against SARS-CoV-2 Delta variant in Golden Syrian hamsters: Potential herbal alternative for COVID-19 treatment.

doi: 10.1016/j.jtcme.2024.05.004

Figure Lengend Snippet: Fig. 6. Antiviral activity of andrographolide upon infection of ACE2TMPRSS2 over expressing A549 cells with different SARS-CoV-2 VOCs. A monolayer of ACE2-TMPRSS2 overexpressing A549 cells infected with different SARS-CoV-2 VOCs (Alpha, Delta, and Omicron) at 25TCID50 and treated with different dosages of andrographolide ranging from 1.56 to 50 μM for 2 days. The infected cells were fixed and stained for viral nucleoproteins. (A) The representative of fluorescent images of SARS-CoV-2 infected cells. Scale bar, 2 mm. The fluorescent intensity of SARS-CoV-2 nucleoprotein-positive cells were measured. (B) Dose-response curve by fluorescent intensity were analyzed. (C) Inhibition of SARS-CoV-2 infection (%) over andrographolide were plotted and IC50 were calculated. (n = 2 biolog ical replicates).

Article Snippet: A human ACE2-and TMPRSS2-expressing A549 lung carcinoma cell line (A549-hACE2-TMPRSS2) (a549-hace2tpsa) was obtained from InvivoGen.

Techniques: Activity Assay, Infection, Expressing, Staining, Inhibition

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