viruses a549 at cells  (ATCC)

Journal: Autophagy

Article Title: Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy

doi: 10.1080/15548627.2020.1725375

Figure Lengend Snippet: IAV PB1-F2 induces autophagy. (A) HEK 293T cells were transfected with HA-PB1-F2PR8 and HA-PB1-F2HM from the A/PR8/H1N1 virus and H5N1/HM virus, respectively. Cell lyses were harvested and analyzed by western blot. (B) A549 cells were transfected with GFP-LC3 and vector, HA-PB1-F2PR8, or HA-PB1-F2HM, respectively. The LC3 puncta formation and the colocalization of LC3 puncta with PB1-F2 were analyzed. Scale bar: 10 μm. It was the representative of 20 cells. (C) HEK 293T cells were transfected with indicated plasmids for 12 h and then treated with or without chloroquine (CQ, 25 μM). 12 h later, cell lysates were analyzed by western blot. (D) A549 cells were transfected with ptf-LC3 (mCherry-GFP-LC3) and vector, HA-PB1-F2PR8, or HA-M2HM (as a control to induce incomplete autophagy), respectively. Cells were analyzed to detect the autophagosome formation. In the zoomed images, fluorescence signals indicated the expression of mRFP-LC3 and EGFP-LC3 (yellow color: incomplete autophagy, red color: complete autophagy). Scale bar: 10 μm. The graph shows the quantification of mRFP+GFP+ autophagosomes by taking the average number of dots in 20 cells (n = average number of dots in 20 cells). Mean ± SD (error bars) were determined for triplicates of three independent experiments (*p < 0.05; **p < 0.01; ns, nonsignificant)

Article Snippet: Cells and viruses Human lung epithelial cell line A549 cells (ATCC CCL-185TM), human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216TM) and Madin–Darby canine kidney (MDCK) cells (ATCC, ATCCPTA-7909) were cultured in HAM’S/F-12 (HyClone, SH30026.01), RPMI 1640 medium (HyClone, SH30809.01) and DMEM/HIGH GLUCOSE (HyClone, SH30022.01), respectively, and supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN biotech, P30-3302) at 37°C with 5% CO 2 .

Techniques: Transfection, Virus, Western Blot, Plasmid Preparation, Control, Fluorescence, Expressing

Journal: Autophagy

Article Title: Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy

doi: 10.1080/15548627.2020.1725375

Figure Lengend Snippet: PB1-F2 overexpression induces mitophagy. (A) A549 cells were co-transfected with indicated plasmids for 24 h and analyzed for mitophagosome formation. In the zoomed images, yellow color indicated the colocalization of GFP-LC3 and dsRed-mito (Mito). Scale bar: 10 μm. It was the representative of 20 cells. (B and C) HEK 293T cells were transfected with indicated plasmids. Lysates were evaluated by western blot. (D) pmRFP-GFP-Mito-transfected A549 cells were co-transfected with HA-PB1-F2PR8 or HA-PB1-F2PR8 and HA-M2HM for 24 h and then analyzed to detect mitophagosome formation. In the zoomed images, fluorescence signals indicated the expression of mRFP-EGFP-mito targeting the mitochondria (yellow color: incomplete mitophagy; red color: complete mitophagy). Scale bar: 10 μm. The graph shows the quantification of mRFP+GFP+ mitophagosomes by taking the average number of dots in 20 cells (n = average number of dots in 20 cells). Mean ± SD (error bars) were determined for triplicates of 3 independent experiments (*p < 0.05; **p < 0.01)

Article Snippet: Cells and viruses Human lung epithelial cell line A549 cells (ATCC CCL-185TM), human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216TM) and Madin–Darby canine kidney (MDCK) cells (ATCC, ATCCPTA-7909) were cultured in HAM’S/F-12 (HyClone, SH30026.01), RPMI 1640 medium (HyClone, SH30809.01) and DMEM/HIGH GLUCOSE (HyClone, SH30022.01), respectively, and supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN biotech, P30-3302) at 37°C with 5% CO 2 .

Techniques: Over Expression, Transfection, Western Blot, Fluorescence, Expressing

Journal: Autophagy

Article Title: Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy

doi: 10.1080/15548627.2020.1725375

Figure Lengend Snippet: PB1-F2 interacts and colocalizes with TUFM. (A) HEK 293T cells were transfected with vector or Flag-TUFM and HA-PB1-F2PR8, respectively. Cell lysates were subjected to IP. (B) HEK 293T cells were transfected with vector or HA-PB1-F2PR8 and Flag-TUFM, respectively. Cell lysates were subjected to IP. An asterisk next to the blot indicated the protein. (C) Lysates of HA-PB1-F2PR8-transfected HEK 293T cells were prepared and immunoprecipitated with the anti-TUFM antibody or control IgG. (D) A549 cells were transfected with vector or HA-PB1-F2PR8. 24 h later, cells were analyzed for the distribution of PB1-F2 and endogenous TUFM. In the zoomed images, yellow color indicated the colocalization of PB1-F2 and TUFM. Scale bar: 10 μm. It was the representative of 20 cells. (E) HEK 293T cells were transfected with Flag-TUFM with PB1-F2PR8 truncation mutant HA-PB1-F2∆N37. Cell lysates were subjected to IP. (F) HEK 293T cells were transfected with Flag-TUFM and PB1-F2PR8 truncation mutant GFP-PB1-F2∆C50 for 24 h cell lysates were subjected to IP and analyzed by western blot. (G) Flag-tagged WT TUFM or domain truncation mutants (TUFM∆domain I, TUFM∆domain II and TUFM∆domain III) were co-transfected with HA-PB1-F2PR8 into HEK 293T cells. Cell lysates were immunoprecipitated with anti-FLAG and immunoblotted for HA-PB1-F2PR8.

Article Snippet: Cells and viruses Human lung epithelial cell line A549 cells (ATCC CCL-185TM), human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216TM) and Madin–Darby canine kidney (MDCK) cells (ATCC, ATCCPTA-7909) were cultured in HAM’S/F-12 (HyClone, SH30026.01), RPMI 1640 medium (HyClone, SH30809.01) and DMEM/HIGH GLUCOSE (HyClone, SH30022.01), respectively, and supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN biotech, P30-3302) at 37°C with 5% CO 2 .

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Control, Mutagenesis, Western Blot

Journal: Autophagy

Article Title: Influenza A virus protein PB1-F2 impairs innate immunity by inducing mitophagy

doi: 10.1080/15548627.2020.1725375

Figure Lengend Snippet: PB1-F2 mediates TUFM-dependent mitophagy. (A and B) HEK 293T cells were transfected with siTUFM or siNC (negative control) for 12 h, and cells were further transfected with HA-PB1-F2PR8 for another 24 h. Cytoplasm and mitochondrial fractions were purified for western blot analysis (Fractions: Cyto, purified cytosolic; Mito, purified mitochondria. Organelle markers: TOMM20, mitochondria; HSPA5/GRP78, endoplasmic reticulum [ER]; LAMP1, lysosome; GAPDH, cytoplasm). (C) TUFM knockout (TUFM-KO) or wild type (WT) A549 cells were transfected with the indicated plasmids for 24 h and analyzed for mitophagosome formation. In the zoomed images, yellow color indicated the colocalization of GFP-LC3 and TOMM20 (as a mitochondrial marker), white color indicated the colocalization of GFP-LC3, mitochondria, and PB1-F2. Scale bar: 10 μm. It was the representative of 20 cells (**p < 0.01; ***p < 0.001)

Article Snippet: Cells and viruses Human lung epithelial cell line A549 cells (ATCC CCL-185TM), human embryonic kidney (HEK) 293T cells (ATCC, CRL-3216TM) and Madin–Darby canine kidney (MDCK) cells (ATCC, ATCCPTA-7909) were cultured in HAM’S/F-12 (HyClone, SH30026.01), RPMI 1640 medium (HyClone, SH30809.01) and DMEM/HIGH GLUCOSE (HyClone, SH30022.01), respectively, and supplemented with 10% heat-inactivated fetal bovine serum (FBS; PAN biotech, P30-3302) at 37°C with 5% CO 2 .

Techniques: Transfection, Negative Control, Purification, Western Blot, Knock-Out, Marker

Journal: Virology

Article Title: Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

doi: 10.1016/j.virol.2017.09.007

Figure Lengend Snippet: (A) The relative amount of viral RNA detected in supernatants of A549 cells infected with RSV A2 in real time (RT) PCR and represented as inverse cycle threshold (Ct) values. Ct levels reflect the number of cycles required to exceed the background level; inverse Ct levels (1/Ct) are proportional to the amount of target nucleic acid in the sample. RT-PCR underwent 40 cycles of amplification. The data are represented as average ± SD from a representative experiment. (B) A549 cells were infected with indicated MOI of RSV A2 for 72 h. RNA was extracted from the cells and the expression levels of IDO mRNA were quantitated by quantitative SYBR Green real-time PCR. GAPDH mRNA levels were used as internal controls. The ΔΔCt method was applied to calculate the fold change. (C) IDO expression using two different RSV strains is shown. (D) IDO expression in lung tissue homogenate of BALB/c mice challenged with RSV A2 or 19F is shown.

Article Snippet: Human airway epithelial cells and viruses A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin.

Techniques: Infection, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Amplification, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

doi: 10.1016/j.virol.2017.09.007

Figure Lengend Snippet: (A) Steps in Kynurenine pathway (modified from Ref 30). (B) Representative heat map with CT values (red = high expression, blue = low expression). A549 cells were infected with indicated MOI of RSV A2 for 72 h. RNA was extracted from the cells and the expression levels of different tryptophan degrading enzyme mRNA were quantitated by quantitative SYBR Green real-time PCR.

Article Snippet: Human airway epithelial cells and viruses A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin.

Techniques: Modification, Expressing, Infection, SYBR Green Assay, Real-time Polymerase Chain Reaction

Journal: Virology

Article Title: Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

doi: 10.1016/j.virol.2017.09.007

Figure Lengend Snippet: (A) A549 cells were transfected with control and IDO siRNA for 12 hours followed by infection with 1MOI of RSV A2. RNA was extracted from the cells 72h post infection and the expression level of IDO mRNA was quantitated by quantitative SYBR Green real-time PCR. GAPDH mRNA levels were used as internal controls. The ΔΔCt method was applied to calculate the fold change. (B) The relative amount of viral RNA was detected in supernatants of transfected/infected A549 cells in real time (RT) PCR and represented as inverse cycle threshold (Ct) values. The data are represented as average ± SD from two independent experiments that were performed in duplicates. (C) A549 cells were treated with 25ng/ml IFN-γ followed by inoculation with 1MOI of RSV A2. RNA was extracted from the supernatant and the relative amount of viral RNA was detected in real time (RT) PCR. (D) IDO mRNA expression was detected from the cells that were harvested three days post infection. (E) RT PCR showing viral RNA in control and IDO knock out A549 cells that were treated with IFN-γ. Cumulative data are shown from two independent experiments that were performed in duplicates.

Article Snippet: Human airway epithelial cells and viruses A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin.

Techniques: Transfection, Control, Infection, Expressing, SYBR Green Assay, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Reverse Transcription Polymerase Chain Reaction, Knock-Out

Journal: Virology

Article Title: Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

doi: 10.1016/j.virol.2017.09.007

Figure Lengend Snippet: Cytokine response upon exposure of RSV to IDO and Control knock down A549 Cells with or without IFN-γ treatment

Article Snippet: Human airway epithelial cells and viruses A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin.

Techniques: Control, Knockdown

Journal: Virology

Article Title: Protective role of Indoleamine 2,3 dioxygenase in Respiratory Syncytial Virus associated immune response in airway epithelial cells

doi: 10.1016/j.virol.2017.09.007

Figure Lengend Snippet: Levels of cytokines and chemokines (pg/ml) in the supernatant of control and IDO knock down A549 cells and RSV infection with or without IFN-γ treatment. Data are average ±SD from duplicate infection from two independent experiments.

Article Snippet: Human airway epithelial cells and viruses A549 cells were obtained from American Type Culture Collection (ATCC) and grown in minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS), 1mM L-Glutamine and 1× penicillin streptomycin.

Techniques: Control, Knockdown, Infection