viruses a549 cell line  (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