lung bronchial epithelium nl20 ta  (ATCC)

Journal: PLOS Pathogens

Article Title: Influenza virus infection reprograms cholesterol biosynthesis to facilitate virus replication by the TAK1-RORγ axis

doi: 10.1371/journal.ppat.1013646

Figure Lengend Snippet: (A-D) NL20 were infected with various MOIs of H5N1 (SY) or H1N1 (PR8) viruses for 24 h or infected with 0.1 MOI of H5N1 or H1N1 virus for the indicated length of time. Cell lysates were prepared and analyzed for the levels of the indicated proteins by Western blot (A and B). The results represent one of three independent experiments with similar results. (C and D) Total RNAs were extracted and analyzed for the mRNA levels of cholesterol biosynthesis-related genes by RT-qPCR. Data are the means ± standard deviation (SD) of three independent experiments. ns, non-significant; * p < 0.05, ** p < 0.01, compared to uninfected controls. (E and F) MDCK, NL20, Vero, and A549 cells were left uninfected or infected with 1 MOI of H5N1 (SY) or H1N1 (PR8) virus for 24 h. Cell lysates were prepared and analyzed for the levels of the indicated proteins by Western blot. (G and H) Male C57BL/6 mice (6-8-week-old, 3 mice/group) were intranasally infected with H5N1 (5 × 10 4 pfu/mouse) or H1N1 (100 pfu/mouse) for 3 days, the lungs were collected and homogenized in the radioimmunoprecipitation assay buffer (RIPA) buffer and then analyzed for the levels of cholesterol biosynthesis-related proteins by Western blot. GAPDH was detected as a loading control.

Article Snippet: NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line), A549 (a human lung cancer cell line of alveolar epithelial cell origin), MDCK (a Madin-Darby canine kidney cell line), Vero (an African green monkey kidney cell line), and 293T (a human embryonic kidney cell line) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA).

Techniques: Infection, Virus, Western Blot, Quantitative RT-PCR, Standard Deviation, Radio Immunoprecipitation, Control

Journal: PLOS Pathogens

Article Title: Influenza virus infection reprograms cholesterol biosynthesis to facilitate virus replication by the TAK1-RORγ axis

doi: 10.1371/journal.ppat.1013646

Figure Lengend Snippet: (A and B) NL20 cells pretreated with the indicated concentrations of GSK805 ( A ) or XY018 ( B ) for 8 h were infected with 0.01 MOI of two H5N1 strains (SY & CK10) or 1 MOI of two H1N1 strains (PR8 & CA09) and then incubated for 24 h in the presence of the same concentrations of GSK805 or XY018. (C) LET1 cells pretreated with the indicated concentrations of XY018 for 8 h were infected with 0.01 MOI of two H5N1 strains (SY & CK10) or 1 MOI of the H1N1 virus (PR8) and then incubated for 24 h in the presence of the same concentrations of XY018. Untreated control cells were treated with 0.1% dimethyl sulfoxide (DMSO). Cell lysates were prepared and analyzed for the levels of the indicated proteins by Western blot. β-actin was detected as a loading control. ( D-I ) NL20 and LET1 cells seeded in a 96-well plate (3.5 × 10 4 cells/well) were incubated in the absence or presence of the indicated concentrations of GSK805 or XY018 in triplicate for 48 h. Cell viability was measured by using the CellTiter-Glo kit. The CC 50 values were calculated based on the mean ± SD of three experiments. To determine the EC 50 values, NL20 and LET1 cells seeded in a 24-well plate were pretreated with the indicated concentrations of GSK805 or XY018 for 8 h. After infection with 0.01 MOI H5N1 (SY) or 1 MOI of the H1N1 (PR8) virus, the cells were incubated in the absence or presence of the same concentrations of GSK805 or XY018 for 24 h. The conditioned media were collected and analyzed for virus titers by measuring TCID 50 values. The results represent the mean ± SD of three independent experiments. The selective index (S.I.) values were calculated by dividing the CC 50 values with the EC 50 values.

Article Snippet: NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line), A549 (a human lung cancer cell line of alveolar epithelial cell origin), MDCK (a Madin-Darby canine kidney cell line), Vero (an African green monkey kidney cell line), and 293T (a human embryonic kidney cell line) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA).

Techniques: Infection, Incubation, Virus, Control, Western Blot

Journal: PLOS Pathogens

Article Title: Influenza virus infection reprograms cholesterol biosynthesis to facilitate virus replication by the TAK1-RORγ axis

doi: 10.1371/journal.ppat.1013646

Figure Lengend Snippet: (A) The monolayers of NL20 cells seeded on coverslips were pre-incubated in the absence of presence of XY018 (2 μM) or cholesterol (5 μg/ml) for 4 h and then infected with H5N1 (SY) (1 MOI) or H1N1 (PR8) (5 MOI) virus. After incubation in serum-free media for 16 h, cells were stained for cholesterol with Filipin and for nuclei with Sytox Green as an internal control, and for NP with a monoclonal antibody. Cells were visualized and photographed under a fluorescent microscope. Scale bar, 10 μm. (B) The monolayers of NL20 cells grown in a 96-well plate were pre-incubated in the absence or presence of XY018 (2 μM) or cholesterol (5 μg/ml) for 4 h and then infected with H5N1 (SY) (1 MOI) or H1N1 (PR8) (5 MOI) virus. After incubation in serum-free media for 16 h, cells were stained for cholesterol with Filipin and for nuclei with Sytox Green as an internal control, and for NP with a monoclonal antibody. The plate was read in a microplate reader for the fluorescent signals of Filipin and Sytox Green. The arbitrary units of Filipin fluorescence intensity were normalized with that of Sytox Green intensity. The results represent the mean ± SD of three independent experiments. * p < 0.05; ** p < 0.01. (C) NL20 cells pretreated with the indicated concentrations of Atovastatin (ATV) for 8 h were infected with 0.01 MOI H5N1 (SY) and then incubated for 24 h in the presence of the same concentrations of ATV. Untreated control cells were treated with 0.1% DMSO. Cell lysates were prepared and analyzed for the levels of indicated proteins by Western blot. β-actin was detected as a loading control. (D) NL20 cells seeded in a 96-well plate (3.5 × 10 4 cells/well) were incubated in the absence or presence of the indicated concentrations of ATV in triplicate for 48 h. Cell viability was measured by using the CellTiter-Glo kit. The CC 50 values were calculated based on the mean ± SD of three independent experiments. To determine the EC 50 values, NL20 cells seeded in a 24-well plate were pretreated with the indicated concentrations of ATV for 8 h. After infection with 0.01 MOI H5N1 (SY) virus, the cells were incubated in the absence or presence of the same concentrations of ATV for 24 h. The conditioned media were collected and analyzed for virus titers by measuring TCID 50 values. The results represent the mean ± SD of three independent experiments. The S.I. values were calculated by dividing the CC 50 values with the EC 50 values. (E and F) Wild-type control and HMGCR-deficient (△HMGCR) NL20 cells infected with the indicated MOIs of H5N1 (SY) virus were incubated for 16 h. Cell lysates were analyzed for the expression of viral proteins. The results represent one of three independent experiments with similar results. Conditioned medial were collected and analyzed for virus titers by measuring TCID 50 values (F). Data are the mean ± SD of three independent experiments. ** p < 0.01. (G and H) NL20 cells pre-incubated in the absence or presence of cholesterol (5 or 10 μg/ml) minus or plus XY018 (2 μM) for 8 h were infected with 0.01 MOI of H5N1 (SY) virus and incubated for 24 h. Cell lysates were prepared and analyzed for the PB2, NP, and NS1 proteins. (H) Conditioned media were collected and analyzed for virus titers. Data are the mean ± SD of three independent experiments. ** p < 0.01. (I) NL20 cells pretreated in serum-free media containing XY018 (2 μM) for 8 h were infected with 2.5 MOI H5N1 (SY) virus and incubated at 4 °C for 1 h. After removing unattached viruses, total cellular RNAs were immediately extracted and analyzed for viral RNAs by RT-qPCR. Data are the mean ± SD of three independent experiments. ** p < 0.01. (J and K) NL20 cells were treated with XY018 (2 μM) for the indicated timepoints before or after H5N1 (SY) virus infection (0.01 MOI). Cell lysates were prepared and analyzed for viral proteins (J). Conditioned media were collected 24 h post infection and analyzed for virus titers (K). Data are the mean ± SD of three experiments. ** p < 0.01, compared to the untreated control.

Article Snippet: NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line), A549 (a human lung cancer cell line of alveolar epithelial cell origin), MDCK (a Madin-Darby canine kidney cell line), Vero (an African green monkey kidney cell line), and 293T (a human embryonic kidney cell line) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA).

Techniques: Incubation, Infection, Virus, Staining, Control, Microscopy, Fluorescence, Western Blot, Expressing, Quantitative RT-PCR

Journal: PLOS Pathogens

Article Title: Influenza virus infection reprograms cholesterol biosynthesis to facilitate virus replication by the TAK1-RORγ axis

doi: 10.1371/journal.ppat.1013646

Figure Lengend Snippet: (A) NL20 cells were infected with 0.1 MOI of H5N1 (SY) virus and then treated with the indicated concentrations of the JNK inhibitor (SP600125, SP), the IKK inhibitor (BMS345541. BMS) and the TAK1 inhibitor (5Z-oxzeneonal, 5Z) for 24 h. Cell lysates were prepared and analyzed for the levels of the PB2 and NP proteins by Western blots. GAPDH was detected as a loading control. (B-E) NL20 cells were first infected with 0.1 MOI of H5N1 virus (SY). After incubation for 8 h, the cells were then incubated in the absence or presence of the indicated concentrations of SP, BMS, or 5Z alone ( B-D ) or SP plus BMS (E) for 16 h. Cell lysates were prepared and analyzed for the levels of the indicated proteins by Western blot. GAPDH was detected as a loading control. The experiments were repeated three times with similar results. (F) Female C57BL/6 mice were randomly divided into 4 groups (3 mice/group). The mice were either mock-infected or infected with H1N1 virus (1000 pfu/mouse). One day after infection, the mice were treated daily with the vehicle or 5Z at a dose of 2 mg/kg bodyweight for two consecutive days. On the third day post-infection, the mice received a last dose of 5Z at 8 hours prior to sacrifice. Lung tissue lysates were prepared and analyzed for the levels of the indicated proteins by Western blot. The band densities from 3 mice per group were analyzed using NIH Image-J software and normalized by the arbitrary units of their total protein bands or GAPDH levels. ns, non-significant, * p < 0.05; ** p < 0.01.

Article Snippet: NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line), A549 (a human lung cancer cell line of alveolar epithelial cell origin), MDCK (a Madin-Darby canine kidney cell line), Vero (an African green monkey kidney cell line), and 293T (a human embryonic kidney cell line) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA).

Techniques: Infection, Virus, Western Blot, Control, Incubation, Software

Journal: PLOS Pathogens

Article Title: Influenza virus infection reprograms cholesterol biosynthesis to facilitate virus replication by the TAK1-RORγ axis

doi: 10.1371/journal.ppat.1013646

Figure Lengend Snippet: (A and B) NL20 cells were transfected with the empty vector or the vector encoding TAK1. After incubation for 48 h, the cells were infected with the indicated MOIs of H5N1 and incubated for 16 h. Cell lysates were analyzed for the levels of the indicated proteins (A). Conditioned media were collected and analyzed for virus titers by measuring the TCID 50 assay (B). Data represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01. (C and D) Control and TAK1-deficient NL20 cells infected with the indicated MOIs of H5N1 (SY) were incubated for 16 h. Cell lysates were prepared and analyzed for the expression of cholesterol biosynthesis-related and viral proteins (C). Conditioned medial were collected and analyzed for virus titers by measuring TCID 50 values (D). △TAK1, TAK1 deficiency. Data are the mean ± SD of three experiments. * p < 0.05, ** p < 0.01. (E) Total cellular RNAs from control and TAK1-deficient cells were extracted and analyzed for the levels of RORC and HMGCR mRNA levels by RT-qPCR. Data represents the mean ± SD of three independent experiments. * p < 0.05, ** p < 0.01. (F-G) TAK1-deficient NL20 cells were infected with higher MOIs of H5N1 (SY) than control NL20 cells. After incubation for 16 h, cell lysates were analyzed for the expression of cholesterol biosynthesis-related and viral proteins (F). Conditioned medial were collected and analyzed for virus titers by measuring TCID 50 values (G). Data are the mean ± SD of three experiments. ns, non-significant.

Article Snippet: NL20 (an immortalized, nontumorigenic human bronchial epithelial cell line), A549 (a human lung cancer cell line of alveolar epithelial cell origin), MDCK (a Madin-Darby canine kidney cell line), Vero (an African green monkey kidney cell line), and 293T (a human embryonic kidney cell line) cells were obtained from the American Tissue Culture Collection (Manassas, VA, USA).

Techniques: Transfection, Plasmid Preparation, Incubation, Infection, Virus, Control, Expressing, Quantitative RT-PCR

Journal: Cancer Science

Article Title: Intrinsic PDL1 Signaling Modulates TGFBI ‐Mediated Growth Suppression in Lung Adenocarcinoma

doi: 10.1111/cas.70150

Figure Lengend Snippet: PDL1 regulates TGFBI to inhibit tumor growth. (A) qRT‐PCR of TGFBI in PDL1 ‐knockdown CL1‐5 cells. ***, p < 0.001. (B) Correlation of PDL1 and TGFBI in TCGA‐LUAD and GSE30219 datasets. ***, p < 0.001. (C) TGFBI knockdown in CL1‐5 cells: qRT‐PCR (left), clonogenic (right) assays. **, p < 0.01; ***, p < 0.001. (D) qRT‐PCR (upper) and immunoblotting (lower) of TGFBI expression in HEK293T cells with TGFBI overexpression. ***, p < 0.001. (E) Clonogenic assays of CL1‐5 and A549 cells treated with TGFBI‐conditioned medium. *, p < 0.05; **, p < 0.01.

Article Snippet: NL20, A549, H1650, PC9, H292, H358, and HEK293T cells were obtained from ATCC [ ].

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Expressing, Over Expression