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  • 99
    ATCC a549 cells
    Inhibition of JEV translation and replication by ZAP. (A and C) <t>A549-EGFP,</t> ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001.
    A549 Cells, supplied by ATCC, used in various techniques. Bioz Stars score: 99/100, based on 9741 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Millipore a549 cells
    Role of TFEB in A. baumannii internalization by <t>A549</t> cells. (A and C) Immunoblot analysis of A549 cells transfected with scrambled (SC) and TFEB siRNA and pEGFP-N1-TFEB for 48 and 24 h, respectively. Values in the bar graphs are the percentages of TFEB level in control (CTL) and transfected A549 cells. (B, D, and E) A549 cells were transfected with SC and TFEB siRNA and pEGFP-N1-TFEB and infected with 10 8 CFU/ml A. baumannii ATCC 17978 for 2, 4, or 8 h. An assay of adherence and invasion of A. baumannii ATCC 17978 into A549 cells was performed as described in Materials and Methods. The effect of TFEB siRNA and pEGFP-N1-TFEB mediated TFEB depletion and overexpression, respectively, on adherence or invasion of A. baumannii ATCC 17978. The percentages of total nontransfected A549 cells and A549 cells incubated with A. baumannii ATCC 17978 are shown for both adhesion and invasion. Results are from three independent experiments, and data are the means plus SEM (error bars). Values for untransfected and transfected groups in panels B and E that are significantly different ( P
    A549 Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 3802 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Thermo Fisher a549 cells
    β-catenin mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in <t>A549</t> cells, treated with different XAV939 concentrations. *P
    A549 Cells, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 94/100, based on 13422 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Becton Dickinson a549 cells
    Involvement of PI3K signaling in the reduction of the expression of TLR ligands and elements of TLR signaling pathway in U937 cells to Mtb H37Rv infection in the <t>A549</t> cell coculture model. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of TLR signaling cascade showed a reversed TLR signaling activity in U937 cells of the coinfection model in the presence of LY294002, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. The ability of A549 cell-mediated reduction of TLR signaling activity in U937 cells was reversed by the addition of LY294002. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA; the A549 cell-mediated reduction of cytokines in H37Rv-infected U937 cells was reversed in the presence of PI3K inhibitor. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p
    A549 Cells, supplied by Becton Dickinson, used in various techniques. Bioz Stars score: 94/100, based on 1558 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Corning Life Sciences a549 cells
    Effect of GV, SU, CIS, DTX, PRI-2191 and calcitriol on (A) p53 and (B) p21 expression in <t>A549</t> cells. Densitometric analysis was performed using ImageJ 1.46v software; the results were normalized to actin; bars represent the means ± SD, n=2–3 repeats. * p
    A549 Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 93/100, based on 809 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    99
    Beyotime a549 cells
    Inhibitive effects of BF or BF211 on the activity of porcine cortex Na+/K+-ATPase and the intracellular Ca2+ level of A4549 cells. (A) The activity of Na + /K + -ATPase extracted from porcine cerebral cortex in the presence of BF or BF211 at different concentrations was determined. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The level of intracellular Ca2+ level in <t>A549</t> cells treated with 50 nM BF or BF211 for different time periods. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments.
    A549 Cells, supplied by Beyotime, used in various techniques. Bioz Stars score: 99/100, based on 521 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    92
    Ribobio a549 cells
    Radiosensitizing effects of miR‐18a‐5p in vivo under irradiation. A, Growth curves of <t>A549</t> xenografts at day 10 (n = 6, P
    A549 Cells, supplied by Ribobio, used in various techniques. Bioz Stars score: 92/100, based on 181 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Inhibition of JEV translation and replication by ZAP. (A and C) A549-EGFP, ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Inhibition of JEV translation and replication by ZAP. (A and C) A549-EGFP, ZAP-L and ZAP-S cells absorbed with JEV (MOI = 10) on ice for 2 h were washed and then incubated at 37°C. (A) At the indicated time points, proteins were harvested for western blot with the indicated antibodies. (B) Relative NS3 protein levels normalized with actin from two independent experiments were quantified by ImageJ software. (C) RNA was collected for viral RNA determination by using RT-qPCR. Relative JEV RNA level was normalized by that of GAPDH. (D) Illustration of SP6 promoter-driven RdRP-dead JEV replicon (E) Western blot of 293T/17 cells expressing EGFP, ZAP-L-V5 and ZAP-S-V5 with the indicated antibodies. (F) 293T/17-EGFP and -ZAP-S cell were cotransfected with 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA for 1 and 2 h. At the indicated time points, cells were collected and separated into two portions for the measurements of luciferase activity and RNA level, respectively. The relative luciferase activity and RNA level of Renilla luciferase reporter normalized with transfection control firefly luciferase are shown as the percentage to that of EGFP at 1 h post transfection. (G) 5′-capped RdRP-dead JEV replicon RNA and control firefly luciferase RNA cotransfected 293T/17-EGFP and -ZAP-S cells were harvested at 3 and 9 h post-transfection to determine the replicon RNA level. The relative replicon RNA level normalized with that of firefly luciferase is shown. Representative data are shown as mean ± SD from 3 independent experiments and analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Inhibition, Incubation, Western Blot, Software, Quantitative RT-PCR, Expressing, Luciferase, Activity Assay, Transfection, Two Tailed Test

    Mapping of ZAP-responsive element (ZRE) in JEV 3′-UTR. (A) The CLIP-seq diagram of ZAP-S binding RNA mapped to JEV 3′-UTR. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. The JEV 3′-UTR is divided into domain I, II and III as indicated. The positions of CG dinucleotide are shown by dots. (B) Biotin-labeled JEV 3′-UTR RNA probes (full-length and individual domain I, II, III, and I+II) incubated with A549-ZAP-S cell extracts were pulled down with streptavidin beads and determined for ZAP-S-V5 protein by western blot (left panel). The intensity of ZAP-S was quantified by ImageJ software. Mean ± SD was calculated from 3 independent experiments and analyzed by two-tailed Student’s t test (right panel). (C) 5′-capped full-length and deleted Fluc/5′+3′-UTR RNA (left panel) and control Rluc RNA were cotransfected into 293T/17-EGFP and -ZAP-S cells. At 18 h post-transfection, cell lysates were collected to perform dual-luciferase assay (right panel). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Mapping of ZAP-responsive element (ZRE) in JEV 3′-UTR. (A) The CLIP-seq diagram of ZAP-S binding RNA mapped to JEV 3′-UTR. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. The JEV 3′-UTR is divided into domain I, II and III as indicated. The positions of CG dinucleotide are shown by dots. (B) Biotin-labeled JEV 3′-UTR RNA probes (full-length and individual domain I, II, III, and I+II) incubated with A549-ZAP-S cell extracts were pulled down with streptavidin beads and determined for ZAP-S-V5 protein by western blot (left panel). The intensity of ZAP-S was quantified by ImageJ software. Mean ± SD was calculated from 3 independent experiments and analyzed by two-tailed Student’s t test (right panel). (C) 5′-capped full-length and deleted Fluc/5′+3′-UTR RNA (left panel) and control Rluc RNA were cotransfected into 293T/17-EGFP and -ZAP-S cells. At 18 h post-transfection, cell lysates were collected to perform dual-luciferase assay (right panel). Representative data from three independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Cross-linking Immunoprecipitation, Binding Assay, Labeling, Incubation, Western Blot, Software, Two Tailed Test, Transfection, Luciferase

    ZAP-enhanced innate immune responses in JEV-infected cells. (A-C) A549 cells overexpressing EGFP, ZAP-L and ZAP-S were uninfected (Mock) or infected with JEV (MOI = 5) for 16 h. Total RNA was collected for IFN-β, TNF-α, and IL-6 mRNA detection by RT-qPCR. RT-qPCR results are presented relative to the expression of GAPDH. Representative data from repeated experiments with triplicate samples (mean ± SD, n = 3) were analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. (D) Western blot analysis for the knockdown effects of RIG-I and MDA5 in A549-shLacZ, -shRIG-I and -shMDA5 cells established by transduction with lentiviruses expressing the indicated shRNA. (E and F) A549-shLacZ, -shRIG-I and -shMDA5 cells transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5 were infected with JEV (MOI = 5) for 16 h. Western blots analysis was performed for the indicated proteins.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: ZAP-enhanced innate immune responses in JEV-infected cells. (A-C) A549 cells overexpressing EGFP, ZAP-L and ZAP-S were uninfected (Mock) or infected with JEV (MOI = 5) for 16 h. Total RNA was collected for IFN-β, TNF-α, and IL-6 mRNA detection by RT-qPCR. RT-qPCR results are presented relative to the expression of GAPDH. Representative data from repeated experiments with triplicate samples (mean ± SD, n = 3) were analyzed by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001. (D) Western blot analysis for the knockdown effects of RIG-I and MDA5 in A549-shLacZ, -shRIG-I and -shMDA5 cells established by transduction with lentiviruses expressing the indicated shRNA. (E and F) A549-shLacZ, -shRIG-I and -shMDA5 cells transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5 were infected with JEV (MOI = 5) for 16 h. Western blots analysis was performed for the indicated proteins.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Infection, Quantitative RT-PCR, Expressing, Two Tailed Test, Western Blot, Transduction, shRNA

    Inhibition of JEV but not DENV infection by human ZAP isoforms. A549 cells transduced with lentiviruses expressing EGFP (A549-EGFP), V5-tagged ZAP-L (A549-ZAP-L) and V5-tagged ZAP-S (A549-ZAP-S) were infected with JEV or DENV (MOI = 5). Culture supernatants, cell lysates and total RNA were harvested at the indicated time points. (A and D) Viral titration determined by plaque assay. Representative data from three independent experiments are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA. *** P ≤0.001; NS , not significant. (B and E) Protein levels of viral NS3, V5-tagged ZAP, EGFP and actin were analyzed by western blot. (C and F) RNA levels of viral genome and actin were performed by RT-PCR. hpi, hours post-infection.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Inhibition of JEV but not DENV infection by human ZAP isoforms. A549 cells transduced with lentiviruses expressing EGFP (A549-EGFP), V5-tagged ZAP-L (A549-ZAP-L) and V5-tagged ZAP-S (A549-ZAP-S) were infected with JEV or DENV (MOI = 5). Culture supernatants, cell lysates and total RNA were harvested at the indicated time points. (A and D) Viral titration determined by plaque assay. Representative data from three independent experiments are shown as mean ± SD (n = 3). Statistical significance was analyzed by two-way ANOVA. *** P ≤0.001; NS , not significant. (B and E) Protein levels of viral NS3, V5-tagged ZAP, EGFP and actin were analyzed by western blot. (C and F) RNA levels of viral genome and actin were performed by RT-PCR. hpi, hours post-infection.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Inhibition, Infection, Transduction, Expressing, Titration, Plaque Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction

    ZAP mainly targets JEV 3′-UTR. (A) Map of ZAP-S binding sites in full-length JEV genome by CLIP-seq of RNA isolated from JEV-infected ZAP-S overexpressing A549 cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (B) Schematic diagram of the reporter RNAs (left panel). 293T/17-EGFP and -ZAP-S cells were cotransfected with 5′-capped firefly luciferase (Fluc) flanked by JEV 5′-UTR 197 and/or 3′-UTR RNA and control Renilla luciferase (Rluc) RNA for 18 h. Relative luciferase activity was measured by dual-luciferase reporter assay (right panel). Representative data from two independent experiments are mean ± SD (n = 3) and analyzed by two-tailed Student’s t test. *** P ≤0.001; NS , not significant. (C) Biotin-labeled JEV 3′-UTR RNA (at the indicated amounts) was incubated with ZAP-S or ZAP-S-del4ZF overexpressing A549 cell extracts and then pulled down by using streptavidin beads. The co-precipitated ZAP-S-V5 (WT and del4ZFs) was assayed by western blot.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: ZAP mainly targets JEV 3′-UTR. (A) Map of ZAP-S binding sites in full-length JEV genome by CLIP-seq of RNA isolated from JEV-infected ZAP-S overexpressing A549 cells. Read coverage, the reads of each position normalized to the total number of reads mapping to the viral genome. (B) Schematic diagram of the reporter RNAs (left panel). 293T/17-EGFP and -ZAP-S cells were cotransfected with 5′-capped firefly luciferase (Fluc) flanked by JEV 5′-UTR 197 and/or 3′-UTR RNA and control Renilla luciferase (Rluc) RNA for 18 h. Relative luciferase activity was measured by dual-luciferase reporter assay (right panel). Representative data from two independent experiments are mean ± SD (n = 3) and analyzed by two-tailed Student’s t test. *** P ≤0.001; NS , not significant. (C) Biotin-labeled JEV 3′-UTR RNA (at the indicated amounts) was incubated with ZAP-S or ZAP-S-del4ZF overexpressing A549 cell extracts and then pulled down by using streptavidin beads. The co-precipitated ZAP-S-V5 (WT and del4ZFs) was assayed by western blot.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Binding Assay, Cross-linking Immunoprecipitation, Isolation, Infection, Luciferase, Activity Assay, Reporter Assay, Two Tailed Test, Labeling, Incubation, Western Blot

    The 3′-5′ RNA exosome-mediated, but not the 5′-3′ XRN1-mediated, RNA degradation is required for the anti-JEV activity of ZAP. A549 cells with shRNA targeting LacZ, XRN1, and EXOSC5 were transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5. After 10 h of JEV infection (MOI = 5), cells lysates were analyzed by western blot for the indicated proteins (A and D, upper panel). The relative quantification of NS3 normalized by actin was quantified by ImageJ software (A and D, lower panel). Data are mean ± SD (from four independent experiments). Total RNA and culture supernatants were harvested for the measurement of viral RNA by RT-PCR (B and E) and viral titer by plaque assay (C and F). Data are representative results from repeated experiments and shown as mean ± SD (n = 3). The statistical significance was estimated by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: The 3′-5′ RNA exosome-mediated, but not the 5′-3′ XRN1-mediated, RNA degradation is required for the anti-JEV activity of ZAP. A549 cells with shRNA targeting LacZ, XRN1, and EXOSC5 were transduced by lentiviruses expressing EGFP, ZAP-L-V5, and ZAP-S-V5. After 10 h of JEV infection (MOI = 5), cells lysates were analyzed by western blot for the indicated proteins (A and D, upper panel). The relative quantification of NS3 normalized by actin was quantified by ImageJ software (A and D, lower panel). Data are mean ± SD (from four independent experiments). Total RNA and culture supernatants were harvested for the measurement of viral RNA by RT-PCR (B and E) and viral titer by plaque assay (C and F). Data are representative results from repeated experiments and shown as mean ± SD (n = 3). The statistical significance was estimated by two-tailed Student’s t test. * P ≤0.05; ** P ≤0.01; *** P ≤0.001; NS , not significant.

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Activity Assay, shRNA, Expressing, Infection, Western Blot, Software, Reverse Transcription Polymerase Chain Reaction, Plaque Assay, Two Tailed Test

    Antiviral potential of endogenous ZAP against JEV infection. (A) Western blot analysis for the expression of ZAP in A549 cells transduced with lentiviruses carrying shRNA targeting LacZ and ZAP-L/S. (B and C) A549-shLacZ and -shZAP-L/S cells were infected with JEV (MOI = 5) for 24 h. Total RNA and culture supernatants were harvested for RT-qPCR (B) and viral titration (C), respectively. Relative JEV RNA level normalized by GAPDH was determined using RT-qPCR. Viral titer was measured using plaque assay. Representative data from two independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01. (D) Confocal microscopy of mock and JEV (MOI = 1) infected A549 cells at 16 hpi stained with anti-dsRNA and anti-ZAP antibodies. Cell nuclei were counterstained with DAPI. Scale bar = 20 μm. (E) Co-localization of dsRNA with ZAP was estimated by Pearson’s correlation coefficient (PCC). Mean ± SD was calculated from 30 cells each group and the statistical significance was analyzed by two-tailed Student’s t test. *** P ≤0.001. (F) Cell lysates from mock and JEV (MOI = 1) infected A549 cells after 16 h of infection were incubated with anti-ZC3HAV1 (ZAP) antibody or control IgG. Western blot analysis of the immunoprecipitated ZAP isoforms (upper panel). The ZAP-binding viral RNA pulled down by antibodies was amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input JEV viral RNA (lower panel).

    Journal: PLoS Pathogens

    Article Title: Inhibition of Japanese encephalitis virus infection by the host zinc-finger antiviral protein

    doi: 10.1371/journal.ppat.1007166

    Figure Lengend Snippet: Antiviral potential of endogenous ZAP against JEV infection. (A) Western blot analysis for the expression of ZAP in A549 cells transduced with lentiviruses carrying shRNA targeting LacZ and ZAP-L/S. (B and C) A549-shLacZ and -shZAP-L/S cells were infected with JEV (MOI = 5) for 24 h. Total RNA and culture supernatants were harvested for RT-qPCR (B) and viral titration (C), respectively. Relative JEV RNA level normalized by GAPDH was determined using RT-qPCR. Viral titer was measured using plaque assay. Representative data from two independent experiments shown as mean ± SD (n = 3) were analyzed by two-tailed Student’s t test. ** P ≤0.01. (D) Confocal microscopy of mock and JEV (MOI = 1) infected A549 cells at 16 hpi stained with anti-dsRNA and anti-ZAP antibodies. Cell nuclei were counterstained with DAPI. Scale bar = 20 μm. (E) Co-localization of dsRNA with ZAP was estimated by Pearson’s correlation coefficient (PCC). Mean ± SD was calculated from 30 cells each group and the statistical significance was analyzed by two-tailed Student’s t test. *** P ≤0.001. (F) Cell lysates from mock and JEV (MOI = 1) infected A549 cells after 16 h of infection were incubated with anti-ZC3HAV1 (ZAP) antibody or control IgG. Western blot analysis of the immunoprecipitated ZAP isoforms (upper panel). The ZAP-binding viral RNA pulled down by antibodies was amplified by RT-PCR with JEV 3′-UTR specific primers (middle panel). RT-PCR of input JEV viral RNA (lower panel).

    Article Snippet: Human lung carcinoma A549 cells (ATCC, CCL-185) were cultured in F-12 medium containing 10% FBS, 2 mM L-glutamine and 1% P/S.

    Techniques: Infection, Western Blot, Expressing, Transduction, shRNA, Quantitative RT-PCR, Titration, Plaque Assay, Two Tailed Test, Confocal Microscopy, Staining, Periodic Counter-current Chromatography, Incubation, Immunoprecipitation, Binding Assay, Amplification, Reverse Transcription Polymerase Chain Reaction

    Prdx6 levels are sensitive to oxidative stress. ( A ) Primary cultures of human CEnCs) were treated with tert-butyl hydroperoxide (tBHP) for 3 h. pCEnCs were biotinylated and cell surface proteins purified by immunoprecipitation. The flow-through, unbound fraction served as a control for membrane fractionation. ( B ) pCEnCs and ( C ) A549 cells were treated with tBHP for 3 h and plasma membrane was isolated by density dependent centrifugation. Data from three independent experiments ± SEM is shown. Prdx6 levels were normalized to Na + /K + -ATPase and expressed relative to untreated (−) controls. Student t -test was performed to evaluate statistical significance (** p -value

    Journal: Antioxidants

    Article Title: Regulation of Oxidative Stress in Corneal Endothelial Cells by Prdx6

    doi: 10.3390/antiox7120180

    Figure Lengend Snippet: Prdx6 levels are sensitive to oxidative stress. ( A ) Primary cultures of human CEnCs) were treated with tert-butyl hydroperoxide (tBHP) for 3 h. pCEnCs were biotinylated and cell surface proteins purified by immunoprecipitation. The flow-through, unbound fraction served as a control for membrane fractionation. ( B ) pCEnCs and ( C ) A549 cells were treated with tBHP for 3 h and plasma membrane was isolated by density dependent centrifugation. Data from three independent experiments ± SEM is shown. Prdx6 levels were normalized to Na + /K + -ATPase and expressed relative to untreated (−) controls. Student t -test was performed to evaluate statistical significance (** p -value

    Article Snippet: The SV40-transformed human corneal endothelial cell line (B4G12, [ ]), the human lung adenocarcinoma A549 cell line (ATCC; CCL-185), HEK293T, and the spontaneous arising retinal pigmented epithelial cell line ARPE19 were maintained at 37 °C, 5% CO2 in DMEM (high glucose) supplemented with 10% bovine serum (Gibco; Thermo Fisher Scientific, Waltham, MA, USA) together with antibiotics.

    Techniques: Purification, Immunoprecipitation, Flow Cytometry, Fractionation, Isolation, Centrifugation

    Positional profiles of phenotypic consequences of individual nucleotides at individual siRNA positions. ( A – C ). (A, B) Positional profile for cell count and infection phenotypes, respectively, in a genome-wide Brucella abortus screen in HeLa CCL2 cells conducted with a non-pooled siRNA library (Qiagen). (C) siRNA nucleotide positional profile for cell count in a genome-wide screen for Salmonella entry into MEFs (murine embryonic fibroblasts, unpublished). ( D ) Global comparison of siRNA nucleotide profiles, generated for three different pathogen screens ( Brucella abortus, Salmonella Typhimurium and Adenovirus ), across three different libraries of non-pooled siRNAs designed by Qiagen, Dharmacon and Ambion (QU, DU and AU, respectively) targeting human kinome genes (∼ 800) in HeLa CCL2 cells, together with another genome-wide screen carried out in human A549 cells and mouse embryonic fibroblast cells using non-pooled Dharmacon (DU) and Qiagen libraries (QUM), respectively. Each data point represents the entire nucleotide profile as seen in panels A–C. Black color denotes cell count readouts and gray color denotes infection readouts.

    Journal: Nucleic Acids Research

    Article Title: Growth-restricting effects of siRNA transfections: a largely deterministic combination of off-target binding and hybridization-independent competition

    doi: 10.1093/nar/gky798

    Figure Lengend Snippet: Positional profiles of phenotypic consequences of individual nucleotides at individual siRNA positions. ( A – C ). (A, B) Positional profile for cell count and infection phenotypes, respectively, in a genome-wide Brucella abortus screen in HeLa CCL2 cells conducted with a non-pooled siRNA library (Qiagen). (C) siRNA nucleotide positional profile for cell count in a genome-wide screen for Salmonella entry into MEFs (murine embryonic fibroblasts, unpublished). ( D ) Global comparison of siRNA nucleotide profiles, generated for three different pathogen screens ( Brucella abortus, Salmonella Typhimurium and Adenovirus ), across three different libraries of non-pooled siRNAs designed by Qiagen, Dharmacon and Ambion (QU, DU and AU, respectively) targeting human kinome genes (∼ 800) in HeLa CCL2 cells, together with another genome-wide screen carried out in human A549 cells and mouse embryonic fibroblast cells using non-pooled Dharmacon (DU) and Qiagen libraries (QUM), respectively. Each data point represents the entire nucleotide profile as seen in panels A–C. Black color denotes cell count readouts and gray color denotes infection readouts.

    Article Snippet: Secondly, despite being trained on HeLa CCL2 cells only, the model was able to predict cell counts not only in HeLa cells (with up to 70% accuracy, Figure ) but also in A549 (cancerous cells; adenocarcinomic human alveolar basal epithelial cells) and WI38 (normal diploid fibroblasts derived from lung tissue) cells to about 60% and 50% accuracy, respectively (Figure ).

    Techniques: Cell Counting, Infection, Genome Wide, Generated

    FF selectively impairs the transendothelial migration of fibroblastoid A549 cells. ( A ) A549 cells were seeded on microporous membranes covered by control or FF-pretreated (Pre-FF) HUVEC monolayers, cultivated for 24 h in control conditions or in the presence of 25 μM FF (FF), and the fraction of transmigrated cells was estimated (TransEndothelial penetration Index (TEI)). ( B ) Morphology of A549 microclones derived from the cells that transmigrated through HUVEC-covered microporous membranes in the same conditions as in ( A ). ( C ) Motility of A549 progenies derived from the cells that transmigrated through HUVEC-covered microporous membranes in the same conditions as in ( A ). ( D ) Progenies derived from A549 cells that most readily transmigrated through HUVEC monolayers were treated with FF, and their morphology and the fraction of epithelioid clones was estimated after 72 h. Error bars represent SEM. The statistical significance of the differences was tested with one-way ANOVA followed by post-hoc Tukey’s HSD ( A , B , D ) or non-parametric Dunnett comparison ( C ); * p

    Journal: Cancers

    Article Title: Fenofibrate Interferes with the Diapedesis of Lung Adenocarcinoma Cells through the Interference with Cx43/EGF-Dependent Intercellular Signaling

    doi: 10.3390/cancers10100363

    Figure Lengend Snippet: FF selectively impairs the transendothelial migration of fibroblastoid A549 cells. ( A ) A549 cells were seeded on microporous membranes covered by control or FF-pretreated (Pre-FF) HUVEC monolayers, cultivated for 24 h in control conditions or in the presence of 25 μM FF (FF), and the fraction of transmigrated cells was estimated (TransEndothelial penetration Index (TEI)). ( B ) Morphology of A549 microclones derived from the cells that transmigrated through HUVEC-covered microporous membranes in the same conditions as in ( A ). ( C ) Motility of A549 progenies derived from the cells that transmigrated through HUVEC-covered microporous membranes in the same conditions as in ( A ). ( D ) Progenies derived from A549 cells that most readily transmigrated through HUVEC monolayers were treated with FF, and their morphology and the fraction of epithelioid clones was estimated after 72 h. Error bars represent SEM. The statistical significance of the differences was tested with one-way ANOVA followed by post-hoc Tukey’s HSD ( A , B , D ) or non-parametric Dunnett comparison ( C ); * p

    Article Snippet: Human lung cancer A549 cells (ATCC CCL-185) were cultured in RPMI-1640 medium (Lonza) supplemented with 10% FBS and antibiotics [ ].

    Techniques: Migration, Derivative Assay, Clone Assay

    Effects of protease inhibitors and antioxidants on the activation of protein kinase C (PKC). a A549 or Beas2B cells were treated with medium (C), dust extract (1 %) (DE), α1-antitrypsin (25 μg/ml) (α1-AT) or soybean trypsin inhibitor (25 μg/ml) (SBTI) alone, or a combination of DE with α1-AT or SBTI for 5 min. A representative Western blot depicting the levels of phosphorylated PKC and tubulin is shown. b and c Quantification of the effects of α1-AT and SBTI on phosphorylated PKC levels. Levels of phosphorylated PKC and tubulin were quantified and phosphorylated PKC levels normalized to tubulin levels. Levels of phosphorylated PKC in cells treated with medium (C) were arbitrarily considered as 100, and data are shown as means ± SE ( n = 3). * P

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Effects of protease inhibitors and antioxidants on the activation of protein kinase C (PKC). a A549 or Beas2B cells were treated with medium (C), dust extract (1 %) (DE), α1-antitrypsin (25 μg/ml) (α1-AT) or soybean trypsin inhibitor (25 μg/ml) (SBTI) alone, or a combination of DE with α1-AT or SBTI for 5 min. A representative Western blot depicting the levels of phosphorylated PKC and tubulin is shown. b and c Quantification of the effects of α1-AT and SBTI on phosphorylated PKC levels. Levels of phosphorylated PKC and tubulin were quantified and phosphorylated PKC levels normalized to tubulin levels. Levels of phosphorylated PKC in cells treated with medium (C) were arbitrarily considered as 100, and data are shown as means ± SE ( n = 3). * P

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Activation Assay, Western Blot

    Dust extracts induce intracellular reactive oxygen species levels. a A549 and Beas2B cells grown on coverslips were incubated with 10 μM dihydroethidium for 1 h in the dark in serum-free medium and then exposed to medium alone (Control) or medium containing dust extract (1 %) (DE) for 10 min. After exposure, slides were viewed under a fluorescent microscope equipped with an Ultra-VIEW LCI scanning confocal system using 488 nm excitation and 568 nm emission filters. Fluorescent images are representative of three independent experiments. Red color indicates intracellular ROS production. b A549 cells grown on coverslips were treated with medium alone (Control), dust extract (DE) (1 %) or phorbol myristate acetate (PMA) (10 nM) for 1 h under serum-free conditions. Afterwards, hydroxynonenal conjugated proteins were visualized by immunostaining. Images shown are representative of two independent experiments. PMA was used as a positive control for the generation of intracellular reactive oxygen species (ROS). Magnification, 40×

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Dust extracts induce intracellular reactive oxygen species levels. a A549 and Beas2B cells grown on coverslips were incubated with 10 μM dihydroethidium for 1 h in the dark in serum-free medium and then exposed to medium alone (Control) or medium containing dust extract (1 %) (DE) for 10 min. After exposure, slides were viewed under a fluorescent microscope equipped with an Ultra-VIEW LCI scanning confocal system using 488 nm excitation and 568 nm emission filters. Fluorescent images are representative of three independent experiments. Red color indicates intracellular ROS production. b A549 cells grown on coverslips were treated with medium alone (Control), dust extract (DE) (1 %) or phorbol myristate acetate (PMA) (10 nM) for 1 h under serum-free conditions. Afterwards, hydroxynonenal conjugated proteins were visualized by immunostaining. Images shown are representative of two independent experiments. PMA was used as a positive control for the generation of intracellular reactive oxygen species (ROS). Magnification, 40×

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Incubation, Microscopy, Immunostaining, Positive Control

    Effects of α1-antitrypsin and CDDOIm on the induction of IL-8 promoter activity. a A549 cells were transfected with an IL-8 promoter plasmid containing −144/+44 bp of human IL-8 promoter sequence linked to luciferase reporter gene. Transfected cells were treated with medium (C), 0.25 % dust extract (DE), 10 μg/ml α1-antitrypsin alone (α1-AT), or 0.25 % dust extract in the presence of 10 μg/ml α1-antitrypsin for 6 h. Luciferase activities in cell lysates were measured and normalized to total cell protein. Luciferase activity in untreated cells (C) was arbitrarily set as 100. Data shown are means ± SE ( n = 4). * P

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Effects of α1-antitrypsin and CDDOIm on the induction of IL-8 promoter activity. a A549 cells were transfected with an IL-8 promoter plasmid containing −144/+44 bp of human IL-8 promoter sequence linked to luciferase reporter gene. Transfected cells were treated with medium (C), 0.25 % dust extract (DE), 10 μg/ml α1-antitrypsin alone (α1-AT), or 0.25 % dust extract in the presence of 10 μg/ml α1-antitrypsin for 6 h. Luciferase activities in cell lysates were measured and normalized to total cell protein. Luciferase activity in untreated cells (C) was arbitrarily set as 100. Data shown are means ± SE ( n = 4). * P

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Activity Assay, Transfection, Plasmid Preparation, Sequencing, Luciferase

    Protease activities in dust extract and the effects of protease inhibitors and heating on IL-8 mRNA and protein levels. a Trypsin and elastase activities in dust extract were measured using BAPNA and SAPNA substrates, respectively. Dust extract (5 μl) was mixed with BAPNA (0.92 mM) or SAPNA (0.37 mM) in a final volume of 200 μl of 0.1 M Tris-HCl 8.0 or 0.1 M Tris-HCl 8.3, incubated at room temperature and absorbance at 410 nm recorded at indicated times. Data shown are average of duplicate measurements. Similar results were obtained in a second independent experiment. b and c Trypsin and elastase activities were measured in the presence of protease inhibitor cocktail (0.5 ×) or α1-antitrypsin (10 μg) (α1-AT). Data shown are means ± SD of two independent experiments. d A549 cells were treated with medium (C), dust extract (0.25 %) (DE), dust extract (0.25 %) that was heated at 95 °C for 10 min, or dust extract (0.25 %) in the presence of 2 μl protease inhibitor cocktail (PIC), 10 μg/ml α1-antitrypsin (α1-AT), or 10 μg/ml soybean trypsin inhibitor (SBTI) for 3 h and IL-8 mRNA levels determined by qRT-PCR. IL-8 mRNA levels in dust extract treated cells were arbitrarily considered as 100, and relative IL-8 mRNA levels in other treatments are shown. Data shown are means ± SE ( n = 3). ** P

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Protease activities in dust extract and the effects of protease inhibitors and heating on IL-8 mRNA and protein levels. a Trypsin and elastase activities in dust extract were measured using BAPNA and SAPNA substrates, respectively. Dust extract (5 μl) was mixed with BAPNA (0.92 mM) or SAPNA (0.37 mM) in a final volume of 200 μl of 0.1 M Tris-HCl 8.0 or 0.1 M Tris-HCl 8.3, incubated at room temperature and absorbance at 410 nm recorded at indicated times. Data shown are average of duplicate measurements. Similar results were obtained in a second independent experiment. b and c Trypsin and elastase activities were measured in the presence of protease inhibitor cocktail (0.5 ×) or α1-antitrypsin (10 μg) (α1-AT). Data shown are means ± SD of two independent experiments. d A549 cells were treated with medium (C), dust extract (0.25 %) (DE), dust extract (0.25 %) that was heated at 95 °C for 10 min, or dust extract (0.25 %) in the presence of 2 μl protease inhibitor cocktail (PIC), 10 μg/ml α1-antitrypsin (α1-AT), or 10 μg/ml soybean trypsin inhibitor (SBTI) for 3 h and IL-8 mRNA levels determined by qRT-PCR. IL-8 mRNA levels in dust extract treated cells were arbitrarily considered as 100, and relative IL-8 mRNA levels in other treatments are shown. Data shown are means ± SE ( n = 3). ** P

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Incubation, Protease Inhibitor, Quantitative RT-PCR

    Effects of protease inhibitors on inflammatory gene induction. A549 ( a ), Beas2B ( b ) and SAE ( c ) cells were treated with medium, dust extract (DE) (0.25 %) alone or dust extract (0.25 %) in the presence of 25 μg/ml α1-antitrypsin (α1-AT) or 25 μg/ml soybean trypsin inhibitor for 3 h. Beas2B cells ( d ) were first treated with medium or medium containing metalloproteinase inhibitor GM6001 (10 μM) for 1 h and then exposed to medium or dust extract (DE) (0.25 %) for 3 h. Levels of mRNAs were determined by qRT-PCR. Levels of each mRNA in dust extract treated cells were arbitrarily considered as 100 and relative levels in cells treated with a combination of dust extract and inhibitors are shown. Data are means ± SE ( n = 3). * P

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Effects of protease inhibitors on inflammatory gene induction. A549 ( a ), Beas2B ( b ) and SAE ( c ) cells were treated with medium, dust extract (DE) (0.25 %) alone or dust extract (0.25 %) in the presence of 25 μg/ml α1-antitrypsin (α1-AT) or 25 μg/ml soybean trypsin inhibitor for 3 h. Beas2B cells ( d ) were first treated with medium or medium containing metalloproteinase inhibitor GM6001 (10 μM) for 1 h and then exposed to medium or dust extract (DE) (0.25 %) for 3 h. Levels of mRNAs were determined by qRT-PCR. Levels of each mRNA in dust extract treated cells were arbitrarily considered as 100 and relative levels in cells treated with a combination of dust extract and inhibitors are shown. Data are means ± SE ( n = 3). * P

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Quantitative RT-PCR

    Activation of PAR-1 and -2, and the effects of PAR-1 antagonist and siRNA knockdown of PAR-1 and PAR-2 on the induction of IL-8 mRNA and IL-8 protein levels. a A549 cells were transduced with adenovirus expressing recombinant PAR-1 or PAR-2 containing alkaline phosphatase linked at the amino terminus, and treated with medium (C), dust extract (DE), or DE in the presence of protease inhibitor cocktail (PIC) and alkaline phosphatase activity in the medium was measured by chemiluminiscent assay. Data shown are means of duplicate measurements. Similar results were obtained in two other independent experiments. b A549 cells were first incubated with medium (C) or PAR-1 antagonist BMS200261 (50 μM) for 1 h and then treated with dust extract (DE) (0.25 %) for 3 h. IL-8 levels in the medium were measured by ELISA. Data shown are means ± SE ( n = 4). * P

    Journal: Respiratory Research

    Article Title: Proteases and oxidant stress control organic dust induction of inflammatory gene expression in lung epithelial cells

    doi: 10.1186/s12931-016-0455-z

    Figure Lengend Snippet: Activation of PAR-1 and -2, and the effects of PAR-1 antagonist and siRNA knockdown of PAR-1 and PAR-2 on the induction of IL-8 mRNA and IL-8 protein levels. a A549 cells were transduced with adenovirus expressing recombinant PAR-1 or PAR-2 containing alkaline phosphatase linked at the amino terminus, and treated with medium (C), dust extract (DE), or DE in the presence of protease inhibitor cocktail (PIC) and alkaline phosphatase activity in the medium was measured by chemiluminiscent assay. Data shown are means of duplicate measurements. Similar results were obtained in two other independent experiments. b A549 cells were first incubated with medium (C) or PAR-1 antagonist BMS200261 (50 μM) for 1 h and then treated with dust extract (DE) (0.25 %) for 3 h. IL-8 levels in the medium were measured by ELISA. Data shown are means ± SE ( n = 4). * P

    Article Snippet: Cell culture A549 (ATCC CCL185) lung epithelial cells were grown on plastic culture dishes in F12 K medium containing 10 % fetal bovine serum and penicillin (100 U/ml), streptomycin (100 μg/ml), and amphotericin B (0.25 μg/ml).

    Techniques: Activation Assay, Transduction, Expressing, Recombinant, Protease Inhibitor, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay

    Cathepsin B plays an important role in lung cancer cell migration. a , The effect of the cathepsin B inhibitor CA-074Me on the EGF-stimulated lung cancer A549 cell migration determined by the wound healing assay. b , The effect of the cathepsin B inhibitor CA-074Me on the EGF-stimulated lung cancer A549 cell migration determined by the transwell assay. c , Quantification of the data from three independent transwell migration experiments. The statistics was performed with the treatment sample vs its control. ***, p

    Journal: Molecular Cancer

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    doi: 10.1186/s12943-018-0784-2

    Figure Lengend Snippet: Cathepsin B plays an important role in lung cancer cell migration. a , The effect of the cathepsin B inhibitor CA-074Me on the EGF-stimulated lung cancer A549 cell migration determined by the wound healing assay. b , The effect of the cathepsin B inhibitor CA-074Me on the EGF-stimulated lung cancer A549 cell migration determined by the transwell assay. c , Quantification of the data from three independent transwell migration experiments. The statistics was performed with the treatment sample vs its control. ***, p

    Article Snippet: The lung cancer cell lines A549 and H1650 were purchased from ATCC.

    Techniques: Migration, Wound Healing Assay, Transwell Assay

    NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells

    Journal: Molecular Cancer

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    doi: 10.1186/s12943-018-0784-2

    Figure Lengend Snippet: NEDD4 mediates EGFR-dependent lung cancer cell migration. a , Wound healing assay of A549 cell migration. Left top panel, the knockdown of NEDD4 by shNEDD4 (lane 2) and recovery of NEDD4 upon re-introducing NEDD4 cDNA in the knockdown cells (lane 3); NEDD4-HM, high molecular weight NEDD4; NEDD4-LM, low molecular weight NEDD4. Left bottom panel, the protein level of EGFR in the lung cancer cell lines A549 and H1650 shown by immunoblotting with the cell lysates. Middle panel, photo images of the cell migration. Right panel, quantification of the EGF-stimulated cell migration area occupied after 24 h from the data of three independent experiments using the imaging software Image J (NIH). The non-EGF-treated cell migration area was subtracted by the EGF-treated cell migration area to obtain the EGF-stimulated cell migration area. b , Transwell assay of A549 cell migration. Note that the small lightly-stained round dots are pores of the transwell plates (sh NEDD4 panels). c , Wound healing assay of H1650 cells

    Article Snippet: The lung cancer cell lines A549 and H1650 were purchased from ATCC.

    Techniques: Migration, Wound Healing Assay, Molecular Weight, Imaging, Software, Transwell Assay, Staining

    NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4

    Journal: Molecular Cancer

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    doi: 10.1186/s12943-018-0784-2

    Figure Lengend Snippet: NEDD4 is associated with activated EGFR. a , Co-immunoprecipitation of NEDD4 with activated EGFR in lung cancer cells. Lung cancer A549 or H358 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for indicated times. EGFR was immunoprecipitated with anti-EGFR (Mab528) and detected by immunoblotting with anti-EGFR (1005) (top panels). Co-immunoprecipitated NEDD4 was detected by immunoblotting with an anti-NEDD4 (second top panels). The level of EGFR and NEDD4 in the cell lysates was also detected by immunoblotting (middle and second bottom panels). Notice that EGFR in A549 and H358 cells has an EGF-induced degradation. b , Internalized EGFR is co-localized with NEDD4. A549 cells were serum-starved for 12 h followed by stimulation with EGF (50 ng/ml) for 0 or 60 min. The cells were immuno-stained with anti-EGFR (1005) (red) and anti-NEDD4 (green). Bar, 20 μM. c , Co-expression of NEDD4 with EGFR in lung adenocarcinoma tissue. The tissue microarray containing 63 lung adenocarcinoma section samples was immunohistochemically stained with anti-EGFR or anti-NEDD4

    Article Snippet: The lung cancer cell lines A549 and H1650 were purchased from ATCC.

    Techniques: Immunoprecipitation, Staining, Expressing, Microarray

    NEDD4 is required for EGF-stimulated lysosomal secretion of cathepsin B. a , Lysosomes function in lung cancer cell migration. A549 cells were resuspended in serum-free medium and used for transwell cell migration assay. The migration attractant was 10% fetal bovine serum plus or minus EGF (50 ng/ml). The lysosome inhibitors chloroquine (10 μM) was added in the medium with EGF. The cells migrated from the top well to the bottom well within 6 h. The migrated cells were stained and quantified as described in the section of Methods. b , Overexpression of the NEDD4 ligase-dead mutant NEDD4[C867A] eliminated the LAMP2-positive vesicles at cell edges. NEDD4 or the ligase-dead mutant was stably expressed in A549 cells. The cells were stimulated with EGF (50 ng/ml) for 30 min, followed by immunofluorescent staining. NEDD4 and LAMP2 were stained with anti-NEDD4 and anti-LAMP2. The white arrows indicate the putative lysosomal secretion vesicles. NEDD4-LD stands for the ligase-dead mutant of NEDD4, NEDD4[C867A]. Bar, 20 μM. c , The culture medium collected from the vector control or shNEDD4 cells treated with or without EGF for 12 h was used for detection of cathepsin B with a human cathepsin B ELISA assay kit. The assay was repeated three times. ***, p

    Journal: Molecular Cancer

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    doi: 10.1186/s12943-018-0784-2

    Figure Lengend Snippet: NEDD4 is required for EGF-stimulated lysosomal secretion of cathepsin B. a , Lysosomes function in lung cancer cell migration. A549 cells were resuspended in serum-free medium and used for transwell cell migration assay. The migration attractant was 10% fetal bovine serum plus or minus EGF (50 ng/ml). The lysosome inhibitors chloroquine (10 μM) was added in the medium with EGF. The cells migrated from the top well to the bottom well within 6 h. The migrated cells were stained and quantified as described in the section of Methods. b , Overexpression of the NEDD4 ligase-dead mutant NEDD4[C867A] eliminated the LAMP2-positive vesicles at cell edges. NEDD4 or the ligase-dead mutant was stably expressed in A549 cells. The cells were stimulated with EGF (50 ng/ml) for 30 min, followed by immunofluorescent staining. NEDD4 and LAMP2 were stained with anti-NEDD4 and anti-LAMP2. The white arrows indicate the putative lysosomal secretion vesicles. NEDD4-LD stands for the ligase-dead mutant of NEDD4, NEDD4[C867A]. Bar, 20 μM. c , The culture medium collected from the vector control or shNEDD4 cells treated with or without EGF for 12 h was used for detection of cathepsin B with a human cathepsin B ELISA assay kit. The assay was repeated three times. ***, p

    Article Snippet: The lung cancer cell lines A549 and H1650 were purchased from ATCC.

    Techniques: Migration, Cell Migration Assay, Staining, Over Expression, Mutagenesis, Stable Transfection, Plasmid Preparation, Enzyme-linked Immunosorbent Assay

    NEDD4 does not ubiquitinate and downregulate PTEN. a , NEDD4 was co-expressed with flag-PTEN or Myc-ACK1 by transfection in HEK293 cells. Ubiquitinated ACK1 or PTEN was precipitated with bead-bound GST-Uba from the cell lysates followed by immunoblotting with anti-Myc or anti-flag antibodies. b , Lung cancer A549 cells were infected with lenti-viral vector pLKO.1 or the vector-loaded sh NEDD4 . NEDD4 in the cell lysates was detected by immunoblotting with anti-NEDD4 (second top panel). The effect of knockdown NEDD4 on expression of PTEN and activation of AKT was assessed by immunoblotting PTEN AKT or phospho-AKT in the cell lysates with their antibodies respectively. c , Immunohistochemical (IHC) staining of 63 human lung adenocarcinoma tumors with anti-NEDD4 and anti-PTEN antibodies. The positive tumor samples were assessed and counted under microscope and listed in the table

    Journal: Molecular Cancer

    Article Title: The E3 ubiquitin ligase NEDD4 mediates cell migration signaling of EGFR in lung cancer cells

    doi: 10.1186/s12943-018-0784-2

    Figure Lengend Snippet: NEDD4 does not ubiquitinate and downregulate PTEN. a , NEDD4 was co-expressed with flag-PTEN or Myc-ACK1 by transfection in HEK293 cells. Ubiquitinated ACK1 or PTEN was precipitated with bead-bound GST-Uba from the cell lysates followed by immunoblotting with anti-Myc or anti-flag antibodies. b , Lung cancer A549 cells were infected with lenti-viral vector pLKO.1 or the vector-loaded sh NEDD4 . NEDD4 in the cell lysates was detected by immunoblotting with anti-NEDD4 (second top panel). The effect of knockdown NEDD4 on expression of PTEN and activation of AKT was assessed by immunoblotting PTEN AKT or phospho-AKT in the cell lysates with their antibodies respectively. c , Immunohistochemical (IHC) staining of 63 human lung adenocarcinoma tumors with anti-NEDD4 and anti-PTEN antibodies. The positive tumor samples were assessed and counted under microscope and listed in the table

    Article Snippet: The lung cancer cell lines A549 and H1650 were purchased from ATCC.

    Techniques: Transfection, Infection, Plasmid Preparation, Expressing, Activation Assay, Immunohistochemistry, Staining, Microscopy

    IAV NS1 expression contributes to IAV-induced Mdm2 destabilization, and consecutively alters Mdm2/p53 interaction. ( A ) Stability assay in presence of NS1. A549 cells were transfected with either an empty plasmid or a plasmid expressing NS1 from A/Moscow/10/00 (H3N2), and Mdm2 stability was evaluated at 48 h post-transfection. Stability was assessed by monitoring relative protein levels (RPL) of Mdm2 during a 1 h period, after treatment with 50 µM cycloheximide (CHX). Mean values +/− standard deviation from three independent experiments are presented. *** for P-value

    Journal: Scientific Reports

    Article Title: Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

    doi: 10.1038/s41598-018-22139-6

    Figure Lengend Snippet: IAV NS1 expression contributes to IAV-induced Mdm2 destabilization, and consecutively alters Mdm2/p53 interaction. ( A ) Stability assay in presence of NS1. A549 cells were transfected with either an empty plasmid or a plasmid expressing NS1 from A/Moscow/10/00 (H3N2), and Mdm2 stability was evaluated at 48 h post-transfection. Stability was assessed by monitoring relative protein levels (RPL) of Mdm2 during a 1 h period, after treatment with 50 µM cycloheximide (CHX). Mean values +/− standard deviation from three independent experiments are presented. *** for P-value

    Article Snippet: Human lung epithelial A549 cells (ATCC CCL-185, wild type p53) and H1299 cells (ATCC CRL-5803, Homozygous partial deletion of TP53 gene) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Biowhittaker) supplemented with 100 units/ml penicillin, 200 μ g/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (Dutscher).

    Techniques: Expressing, Stability Assay, Transfection, Plasmid Preparation, Standard Deviation

    IAV infection modulates Mdm2 stability. Stability assay in IAV-infected cells at 4 hpi ( A ) and 24 hpi ( B ). Human lung A549 cells were mock-infected or infected with influenza A/Moscow/10/99 (H3N2) with an MOI of 4 or 0.1 and analyzed at 4 and 24 hpi, respectively. Stability was assessed by monitoring relative protein levels (RPL) of Mdm2 during a 1 h period, after treatment with 50 µM cycloheximide (CHX). Mean values +/− standard deviation from three independent experiments are presented. An asterisk indicates a significant difference compared with the results for mock-infected cells ( *** for P -value

    Journal: Scientific Reports

    Article Title: Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

    doi: 10.1038/s41598-018-22139-6

    Figure Lengend Snippet: IAV infection modulates Mdm2 stability. Stability assay in IAV-infected cells at 4 hpi ( A ) and 24 hpi ( B ). Human lung A549 cells were mock-infected or infected with influenza A/Moscow/10/99 (H3N2) with an MOI of 4 or 0.1 and analyzed at 4 and 24 hpi, respectively. Stability was assessed by monitoring relative protein levels (RPL) of Mdm2 during a 1 h period, after treatment with 50 µM cycloheximide (CHX). Mean values +/− standard deviation from three independent experiments are presented. An asterisk indicates a significant difference compared with the results for mock-infected cells ( *** for P -value

    Article Snippet: Human lung epithelial A549 cells (ATCC CCL-185, wild type p53) and H1299 cells (ATCC CRL-5803, Homozygous partial deletion of TP53 gene) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Biowhittaker) supplemented with 100 units/ml penicillin, 200 μ g/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (Dutscher).

    Techniques: Infection, Stability Assay, Standard Deviation

    An unexpected antiviral contribution of Mdm2 revealed by silencing/transient expression experiments. ( A ) Knock-down of p53 and/or Mdm2 mRNA expression in A549 cells differentially modulates levels of IAV production (Left panel). Forty-eight hours after si-RNA transfection using a control si-RNAs (si-Ctrl) or specific siRNAs (si-p53/si-Mdm2), A549 cells were infected by A/Moscow/10/99 (H3N2) at a MOI of 0.01 and the level of viral production at 24 h post-infection was assessed using three different experiments: (i) RT-qPCR (log10 RNA copies/mL, measured in three independent experiments. An asterisk indicates a significant difference compared with the results for si-Ctlr treated cells ( *** and **** for respectively P -values

    Journal: Scientific Reports

    Article Title: Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

    doi: 10.1038/s41598-018-22139-6

    Figure Lengend Snippet: An unexpected antiviral contribution of Mdm2 revealed by silencing/transient expression experiments. ( A ) Knock-down of p53 and/or Mdm2 mRNA expression in A549 cells differentially modulates levels of IAV production (Left panel). Forty-eight hours after si-RNA transfection using a control si-RNAs (si-Ctrl) or specific siRNAs (si-p53/si-Mdm2), A549 cells were infected by A/Moscow/10/99 (H3N2) at a MOI of 0.01 and the level of viral production at 24 h post-infection was assessed using three different experiments: (i) RT-qPCR (log10 RNA copies/mL, measured in three independent experiments. An asterisk indicates a significant difference compared with the results for si-Ctlr treated cells ( *** and **** for respectively P -values

    Article Snippet: Human lung epithelial A549 cells (ATCC CCL-185, wild type p53) and H1299 cells (ATCC CRL-5803, Homozygous partial deletion of TP53 gene) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Biowhittaker) supplemented with 100 units/ml penicillin, 200 μ g/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (Dutscher).

    Techniques: Expressing, Transfection, Infection, Quantitative RT-PCR

    Mdm2 expression is strongly impacted by IAV infection, mostly at post-transcriptional levels. ( A ) Human lung A549 cells were mock-infected or infected by influenza A/Moscow/10/99 (H3N2) at a MOI of 0.1 and cell lysates were analyzed by western blot for the expression of Mdm2 (SMP14 antibody), p53 and IAV NS1 at different time-points post infection. Ku80 was used as a loading control. Mdm2 relative protein levels (Mdm2 RPL) were measured by densitometry and calculated from data from three independent experiments. An asterisk indicates a significant difference compared with the results for mock-infected cells ( ** and *** for P -value

    Journal: Scientific Reports

    Article Title: Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

    doi: 10.1038/s41598-018-22139-6

    Figure Lengend Snippet: Mdm2 expression is strongly impacted by IAV infection, mostly at post-transcriptional levels. ( A ) Human lung A549 cells were mock-infected or infected by influenza A/Moscow/10/99 (H3N2) at a MOI of 0.1 and cell lysates were analyzed by western blot for the expression of Mdm2 (SMP14 antibody), p53 and IAV NS1 at different time-points post infection. Ku80 was used as a loading control. Mdm2 relative protein levels (Mdm2 RPL) were measured by densitometry and calculated from data from three independent experiments. An asterisk indicates a significant difference compared with the results for mock-infected cells ( ** and *** for P -value

    Article Snippet: Human lung epithelial A549 cells (ATCC CCL-185, wild type p53) and H1299 cells (ATCC CRL-5803, Homozygous partial deletion of TP53 gene) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Biowhittaker) supplemented with 100 units/ml penicillin, 200 μ g/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (Dutscher).

    Techniques: Expressing, Infection, Western Blot

    An unexpected antiviral contribution of Mdm2 revealed by small-molecules Mdm2 antagonists. ( A ) Impact of small molecule Mdm2 antagonists on viral production and IAV-induced apoptosis. Human lung epithelial A549 or H1299 cells were infected by influenza virus A/Moscow/10/99 (H3N2) at a MOI of 0.001, in presence of DMSO or small molecules Mdm2 antagonists (Nutlin-3 or NSC 66811) at different concentrations. The level of viral production at 48 h post-infection was evaluated by RT-qPCR (log10 RNA copies/mL measured in three independent experiments). An asterisk indicates a significant difference compared with the results for DMSO treated cells ( *, **, **** for P -values

    Journal: Scientific Reports

    Article Title: Influenza A viruses alter the stability and antiviral contribution of host E3-ubiquitin ligase Mdm2 during the time-course of infection

    doi: 10.1038/s41598-018-22139-6

    Figure Lengend Snippet: An unexpected antiviral contribution of Mdm2 revealed by small-molecules Mdm2 antagonists. ( A ) Impact of small molecule Mdm2 antagonists on viral production and IAV-induced apoptosis. Human lung epithelial A549 or H1299 cells were infected by influenza virus A/Moscow/10/99 (H3N2) at a MOI of 0.001, in presence of DMSO or small molecules Mdm2 antagonists (Nutlin-3 or NSC 66811) at different concentrations. The level of viral production at 48 h post-infection was evaluated by RT-qPCR (log10 RNA copies/mL measured in three independent experiments). An asterisk indicates a significant difference compared with the results for DMSO treated cells ( *, **, **** for P -values

    Article Snippet: Human lung epithelial A549 cells (ATCC CCL-185, wild type p53) and H1299 cells (ATCC CRL-5803, Homozygous partial deletion of TP53 gene) were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM, Lonza, Biowhittaker) supplemented with 100 units/ml penicillin, 200 μ g/ml streptomycin, 2 mM L-glutamine and 10% fetal calf serum (Dutscher).

    Techniques: Infection, Quantitative RT-PCR

    NETs induced tTG expression and extracellular release from A549 cells. Effects of NETs on A549 cells incubated with or without NE (a) or MPO (b) antibody. Significance is represented by ∗ P

    Journal: Mediators of Inflammation

    Article Title: Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    doi: 10.1155/2017/5675029

    Figure Lengend Snippet: NETs induced tTG expression and extracellular release from A549 cells. Effects of NETs on A549 cells incubated with or without NE (a) or MPO (b) antibody. Significance is represented by ∗ P

    Article Snippet: Cell Culture A549 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 Medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (50 μ g/mL).

    Techniques: Expressing, Incubation

    α -Enolase in A549 cells was degraded into small fragments by NET treatment. α -Enolase expression in cell lysates and culture supernatants was evaluated by Western blot.

    Journal: Mediators of Inflammation

    Article Title: Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    doi: 10.1155/2017/5675029

    Figure Lengend Snippet: α -Enolase in A549 cells was degraded into small fragments by NET treatment. α -Enolase expression in cell lysates and culture supernatants was evaluated by Western blot.

    Article Snippet: Cell Culture A549 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 Medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (50 μ g/mL).

    Techniques: Expressing, Western Blot

    Characterization of NETs isolated from peripheral blood neutrophils of SA. (a) Detection of NET production (a white arrow); scale bar, 10 μ m. (b) DNA bands (left panel) and concentration (right panel). (c) Protein profile (left panel) and concentration (right panel). (d) Western blot analysis of granule proteins. (e) Change in A549 cell morphology following NET treatment. (f) Cell viability assessed by Cell Counting Kit-8 (CCK8) assay. (g) Proinflammatory effects of NETs on A549 cells. ∗∗ P

    Journal: Mediators of Inflammation

    Article Title: Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    doi: 10.1155/2017/5675029

    Figure Lengend Snippet: Characterization of NETs isolated from peripheral blood neutrophils of SA. (a) Detection of NET production (a white arrow); scale bar, 10 μ m. (b) DNA bands (left panel) and concentration (right panel). (c) Protein profile (left panel) and concentration (right panel). (d) Western blot analysis of granule proteins. (e) Change in A549 cell morphology following NET treatment. (f) Cell viability assessed by Cell Counting Kit-8 (CCK8) assay. (g) Proinflammatory effects of NETs on A549 cells. ∗∗ P

    Article Snippet: Cell Culture A549 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 Medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (50 μ g/mL).

    Techniques: Isolation, Concentration Assay, Western Blot, Cell Counting, CCK-8 Assay

    NETs induced CK18 expression and extracellular release from A549 cells. Effects of NETs on A549 cells incubated with/without NE (a) or MPO (b) antibody. Significance is represented by ∗∗ P

    Journal: Mediators of Inflammation

    Article Title: Neutrophil Extracellular DNA Traps Induce Autoantigen Production by Airway Epithelial Cells

    doi: 10.1155/2017/5675029

    Figure Lengend Snippet: NETs induced CK18 expression and extracellular release from A549 cells. Effects of NETs on A549 cells incubated with/without NE (a) or MPO (b) antibody. Significance is represented by ∗∗ P

    Article Snippet: Cell Culture A549 cells (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI 1640 Medium supplemented with 10% FBS, penicillin (100 IU/mL), and streptomycin (50 μ g/mL).

    Techniques: Expressing, Incubation

    Sertraline enhanced the therapeutic efficacy of erlotinib in an orthotopic NSCLC mouse model. ( A ) Combining sertraline with erlotinib suppressed the growth of EGFR TKI–resistant NSCLC. After being injected with luciferase-labeled A549-luc2 cells, the mice were divided into 4 groups based on the initial bioluminescence ( n = 7 in each group): the vehicle control (PBS, p.o. daily), erlotinib (50 mg/kg, p.o. daily), sertraline (50 mg/kg, p.o. daily), and erlotinib combined with sertraline (50 mg/kg, p.o. daily; 50 mg/kg, p.o. daily). Bioluminescent images were recorded using a Xenogen IVIS 2000 Biophotonic Imager every 10 days. ( B and C ) Quantification of bioluminescence in different treatment groups, as described in A . P value was analyzed by 1-way ANOVA followed by Tukey’s multiple comparison test. ( D ) The mouse survival curve. P value was analyzed by the Log-rank test. ( E ) The body weight in mice. There was no significant difference in mouse body weight between the control group and treated groups. P

    Journal: JCI Insight

    Article Title: Repurposing sertraline sensitizes non–small cell lung cancer cells to erlotinib by inducing autophagy

    doi: 10.1172/jci.insight.98921

    Figure Lengend Snippet: Sertraline enhanced the therapeutic efficacy of erlotinib in an orthotopic NSCLC mouse model. ( A ) Combining sertraline with erlotinib suppressed the growth of EGFR TKI–resistant NSCLC. After being injected with luciferase-labeled A549-luc2 cells, the mice were divided into 4 groups based on the initial bioluminescence ( n = 7 in each group): the vehicle control (PBS, p.o. daily), erlotinib (50 mg/kg, p.o. daily), sertraline (50 mg/kg, p.o. daily), and erlotinib combined with sertraline (50 mg/kg, p.o. daily; 50 mg/kg, p.o. daily). Bioluminescent images were recorded using a Xenogen IVIS 2000 Biophotonic Imager every 10 days. ( B and C ) Quantification of bioluminescence in different treatment groups, as described in A . P value was analyzed by 1-way ANOVA followed by Tukey’s multiple comparison test. ( D ) The mouse survival curve. P value was analyzed by the Log-rank test. ( E ) The body weight in mice. There was no significant difference in mouse body weight between the control group and treated groups. P

    Article Snippet: NSCLC cell lines A549, H522, H1975, and PC9 were obtained from the American Type Culture Collection.

    Techniques: Injection, Luciferase, Labeling, Mouse Assay

    Drug combination of sertraline and erlotinib induces autophagy. ( A ) Sertraline increased the protein level of LC3-II in EGFR TKI–resistant NSCLC cell lines. A549, H522, PC9/R, and H1975 cells were treated with sertraline for 24 hours. The cell lysates were subjected to immunoblotting with indicated antibodies. ( B ) Sertraline in combination with erlotinib increased the level of LC3-II in EGFR TKI–resistant NSCLC cell lines. A549, H522, PC9/R, and H1975 cells were treated with sertraline, erlotinib, or drug combination for 24 hours. The cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting with indicated antibodies. ( C ) Increased GFP-LC3 puncta by different treatments. The representative images of GFP-LC3 puncta in A549 cells treated with vehicle, erlotinib, sertraline, or drug combination. Rapamycin (400 nM) served as the positive control. Scale bars: 20 µm. ( D ) Sertraline in combination with erlotinib downregulated intracellular expression of p62 in A549 and PC9/R cells. All experiments were performed independently in triplicate.

    Journal: JCI Insight

    Article Title: Repurposing sertraline sensitizes non–small cell lung cancer cells to erlotinib by inducing autophagy

    doi: 10.1172/jci.insight.98921

    Figure Lengend Snippet: Drug combination of sertraline and erlotinib induces autophagy. ( A ) Sertraline increased the protein level of LC3-II in EGFR TKI–resistant NSCLC cell lines. A549, H522, PC9/R, and H1975 cells were treated with sertraline for 24 hours. The cell lysates were subjected to immunoblotting with indicated antibodies. ( B ) Sertraline in combination with erlotinib increased the level of LC3-II in EGFR TKI–resistant NSCLC cell lines. A549, H522, PC9/R, and H1975 cells were treated with sertraline, erlotinib, or drug combination for 24 hours. The cell lysates were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by immunoblotting with indicated antibodies. ( C ) Increased GFP-LC3 puncta by different treatments. The representative images of GFP-LC3 puncta in A549 cells treated with vehicle, erlotinib, sertraline, or drug combination. Rapamycin (400 nM) served as the positive control. Scale bars: 20 µm. ( D ) Sertraline in combination with erlotinib downregulated intracellular expression of p62 in A549 and PC9/R cells. All experiments were performed independently in triplicate.

    Article Snippet: NSCLC cell lines A549, H522, H1975, and PC9 were obtained from the American Type Culture Collection.

    Techniques: Polyacrylamide Gel Electrophoresis, Positive Control, Expressing

    Shh+ cells produce Shh full-length protein (A) Immunoblot of supernatants from non-transfected Shh-sorted A549 cells probed for the Sonic Hedgehog (Shh) protein, with secreted MMP2 as a loading control. (B) Schematic representation of Shh constructs showing the sizes and locations of N-term HA and C-term FLAG tags stably expressed in NSCLC cells. (C) Immunofluorescence analysis of H838 cells showing cytosolic and membrane staining of N-term, C-term, wild-type Shh and C198A Shh constructs probed for the presence of HA (red) and FLAG (yellow). (D) NSCLC cell lines (A549 and H838) used in (C), analyzed for increases in viability (MTS assay) relative to the vector control after 4 days ( ** p

    Journal: Oncotarget

    Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

    doi: 10.18632/oncotarget.21781

    Figure Lengend Snippet: Shh+ cells produce Shh full-length protein (A) Immunoblot of supernatants from non-transfected Shh-sorted A549 cells probed for the Sonic Hedgehog (Shh) protein, with secreted MMP2 as a loading control. (B) Schematic representation of Shh constructs showing the sizes and locations of N-term HA and C-term FLAG tags stably expressed in NSCLC cells. (C) Immunofluorescence analysis of H838 cells showing cytosolic and membrane staining of N-term, C-term, wild-type Shh and C198A Shh constructs probed for the presence of HA (red) and FLAG (yellow). (D) NSCLC cell lines (A549 and H838) used in (C), analyzed for increases in viability (MTS assay) relative to the vector control after 4 days ( ** p

    Article Snippet: NSCLC cell lines and culture conditions All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

    Techniques: Transfection, Construct, Stable Transfection, Immunofluorescence, Staining, MTS Assay, Plasmid Preparation

    Shh+ cells are resistant to cisplatin but sensitive to GDC0449 (A) Correlation between the percentage of Shh+ cells (%) and cisplatin IC 50 in NSCLC cell lines. (B) Percentage of Shh+ cells (%) in A549 cells treated with cisplatin (1 mM, 72h). †dead cells; ‡Shh- cells; ₮Shh+ cells (5% of live cells). (C) Percentage of Shh+ cells (%) in A549 cells treated by cisplatin (600 μM and 1 mM, 72h). * p

    Journal: Oncotarget

    Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

    doi: 10.18632/oncotarget.21781

    Figure Lengend Snippet: Shh+ cells are resistant to cisplatin but sensitive to GDC0449 (A) Correlation between the percentage of Shh+ cells (%) and cisplatin IC 50 in NSCLC cell lines. (B) Percentage of Shh+ cells (%) in A549 cells treated with cisplatin (1 mM, 72h). †dead cells; ‡Shh- cells; ₮Shh+ cells (5% of live cells). (C) Percentage of Shh+ cells (%) in A549 cells treated by cisplatin (600 μM and 1 mM, 72h). * p

    Article Snippet: NSCLC cell lines and culture conditions All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

    Techniques:

    Shh+ cells produce Shh and are rare in vitro (A-C) Immunofluorescence (IF) analysis of A549 cells relative to the unstained controls (A: only secondary antibody) without membrane permeabilization (B) shows positive Shh (green) and nuclear staining (blue, DAPI). White arrows: positive membranous Shh staining in relatively few cells. (C) IF analysis of A549 cells with membrane permeabilization (Triton X-100) shows positive Shh (green) and nuclear staining (blue, DAPI) in a majority of the cells probed. (D) Flow cytometric analysis of A549 cells without membrane permeabilization probed for Shh (0.18%). (E) Flow cytometric analysis of A549 cells with membrane permeabilization (Tween 20) probed for Shh (70.12%). (F) IF analysis of sorted A549 cells without membrane permeabilization shows strong positive membranous Shh staining (green, white arrows) in Shh+ cells and low/no staining in Shh- cells/controls without the primary antibody. Red: membranous staining (lipophilic dye); blue: nuclear staining (DAPI). (G) Shh gene expression analysis by ddPCR in A549 Shh+ and Shh- cells. (H) Percentage of Shh+ cells (%, mean ±SD) in several NSCLC cell lines.

    Journal: Oncotarget

    Article Title: Membrane-bound full-length Sonic Hedgehog identifies cancer stem cells in human non-small cell lung cancer

    doi: 10.18632/oncotarget.21781

    Figure Lengend Snippet: Shh+ cells produce Shh and are rare in vitro (A-C) Immunofluorescence (IF) analysis of A549 cells relative to the unstained controls (A: only secondary antibody) without membrane permeabilization (B) shows positive Shh (green) and nuclear staining (blue, DAPI). White arrows: positive membranous Shh staining in relatively few cells. (C) IF analysis of A549 cells with membrane permeabilization (Triton X-100) shows positive Shh (green) and nuclear staining (blue, DAPI) in a majority of the cells probed. (D) Flow cytometric analysis of A549 cells without membrane permeabilization probed for Shh (0.18%). (E) Flow cytometric analysis of A549 cells with membrane permeabilization (Tween 20) probed for Shh (70.12%). (F) IF analysis of sorted A549 cells without membrane permeabilization shows strong positive membranous Shh staining (green, white arrows) in Shh+ cells and low/no staining in Shh- cells/controls without the primary antibody. Red: membranous staining (lipophilic dye); blue: nuclear staining (DAPI). (G) Shh gene expression analysis by ddPCR in A549 Shh+ and Shh- cells. (H) Percentage of Shh+ cells (%, mean ±SD) in several NSCLC cell lines.

    Article Snippet: NSCLC cell lines and culture conditions All 12 NSCLC cell lines (A549, H322, H441, H460, H522, H838, H1650, H1975, H2228, HCC2935, H1703, H2170) were purchased from American Type Culture Collection (ATCC).

    Techniques: In Vitro, Immunofluorescence, Staining, Flow Cytometry, Expressing

    Immunofluorescence Assay (IFA). A549 cells were pretreated with NITD008, infected with indicated viruses for 24 h. Cells were fixed for 15 min in 10% formalin supplemented with 0.2% Triton-X detergent, then stained with a primary anti-flavivirus HMAF and a secondary donkey anti-mouse antibody conjugated with DyLight 488 (green). Cells were counterstained with DAPI (blue), and images were captured at 40 × magnification using a Nikon Eclipse Ti inverted fluorescence microscope. White bar indicates length of 100 μm.

    Journal: Antiviral research

    Article Title: In vitro antiviral activity of adenosine analog NITD008 against tick-borne flaviviruses

    doi: 10.1016/j.antiviral.2016.03.013

    Figure Lengend Snippet: Immunofluorescence Assay (IFA). A549 cells were pretreated with NITD008, infected with indicated viruses for 24 h. Cells were fixed for 15 min in 10% formalin supplemented with 0.2% Triton-X detergent, then stained with a primary anti-flavivirus HMAF and a secondary donkey anti-mouse antibody conjugated with DyLight 488 (green). Cells were counterstained with DAPI (blue), and images were captured at 40 × magnification using a Nikon Eclipse Ti inverted fluorescence microscope. White bar indicates length of 100 μm.

    Article Snippet: Briefly, 2 × 104 human lung carcinoma (A549) cells (ATCC, Manassas, VA, USA) in 96-well opaque white plates (Costar, Corning, NY, USA) were pre-treated for 1 h with 3-fold serial dilutions of NITD008 (starting concentration was 100 μM) in quadruplicate and then mock-infected or infected with one of the above mentioned viruses at a multiplicity of infection (MOI) of 0.5.

    Techniques: Immunofluorescence, Infection, Staining, Fluorescence, Microscopy

    NITD008 inhibits tick-borne flavivirus replication. A549 cells were infected with (A) AHFV, (B) KFDV, (C) OHFV, or (D) TBEV at an MOI of 0.5 for 1 h before being treated with NITD008. Seventy-two h post-infection, the supernatants were harvested and were subjected to one freeze–thaw cycle before median tissue culture infective dose were determined (TCID 50 /Ml). Mean titers from four biological replicates are depicted, and error bars indicate standard error of the means. Dotted lines indicate the limit of detection.

    Journal: Antiviral research

    Article Title: In vitro antiviral activity of adenosine analog NITD008 against tick-borne flaviviruses

    doi: 10.1016/j.antiviral.2016.03.013

    Figure Lengend Snippet: NITD008 inhibits tick-borne flavivirus replication. A549 cells were infected with (A) AHFV, (B) KFDV, (C) OHFV, or (D) TBEV at an MOI of 0.5 for 1 h before being treated with NITD008. Seventy-two h post-infection, the supernatants were harvested and were subjected to one freeze–thaw cycle before median tissue culture infective dose were determined (TCID 50 /Ml). Mean titers from four biological replicates are depicted, and error bars indicate standard error of the means. Dotted lines indicate the limit of detection.

    Article Snippet: Briefly, 2 × 104 human lung carcinoma (A549) cells (ATCC, Manassas, VA, USA) in 96-well opaque white plates (Costar, Corning, NY, USA) were pre-treated for 1 h with 3-fold serial dilutions of NITD008 (starting concentration was 100 μM) in quadruplicate and then mock-infected or infected with one of the above mentioned viruses at a multiplicity of infection (MOI) of 0.5.

    Techniques: Infection

    Cell potency of CAT192 mutants. Inhibitory effects by the CAT192 IgG4 (A) light chain insertion mutants and (B) heavy chain mutants/combination mutant on TGFβ1-stimulated IL-11 production in an A549 cell potency assay.

    Journal: mAbs

    Article Title: Structure-based engineering to restore high affinity binding of an isoform-selective anti-TGFβ1 antibody

    doi: 10.1080/19420862.2018.1426421

    Figure Lengend Snippet: Cell potency of CAT192 mutants. Inhibitory effects by the CAT192 IgG4 (A) light chain insertion mutants and (B) heavy chain mutants/combination mutant on TGFβ1-stimulated IL-11 production in an A549 cell potency assay.

    Article Snippet: TGFβ-induced interleukin-11 (IL-11) release from A549 human lung epithelial cells (ATCC) was measured by ELISA in this bioassay.

    Techniques: Mutagenesis, Potency Assay

    Role of TFEB in A. baumannii internalization by A549 cells. (A and C) Immunoblot analysis of A549 cells transfected with scrambled (SC) and TFEB siRNA and pEGFP-N1-TFEB for 48 and 24 h, respectively. Values in the bar graphs are the percentages of TFEB level in control (CTL) and transfected A549 cells. (B, D, and E) A549 cells were transfected with SC and TFEB siRNA and pEGFP-N1-TFEB and infected with 10 8 CFU/ml A. baumannii ATCC 17978 for 2, 4, or 8 h. An assay of adherence and invasion of A. baumannii ATCC 17978 into A549 cells was performed as described in Materials and Methods. The effect of TFEB siRNA and pEGFP-N1-TFEB mediated TFEB depletion and overexpression, respectively, on adherence or invasion of A. baumannii ATCC 17978. The percentages of total nontransfected A549 cells and A549 cells incubated with A. baumannii ATCC 17978 are shown for both adhesion and invasion. Results are from three independent experiments, and data are the means plus SEM (error bars). Values for untransfected and transfected groups in panels B and E that are significantly different ( P

    Journal: mSphere

    Article Title: Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

    doi: 10.1128/mSphere.00106-18

    Figure Lengend Snippet: Role of TFEB in A. baumannii internalization by A549 cells. (A and C) Immunoblot analysis of A549 cells transfected with scrambled (SC) and TFEB siRNA and pEGFP-N1-TFEB for 48 and 24 h, respectively. Values in the bar graphs are the percentages of TFEB level in control (CTL) and transfected A549 cells. (B, D, and E) A549 cells were transfected with SC and TFEB siRNA and pEGFP-N1-TFEB and infected with 10 8 CFU/ml A. baumannii ATCC 17978 for 2, 4, or 8 h. An assay of adherence and invasion of A. baumannii ATCC 17978 into A549 cells was performed as described in Materials and Methods. The effect of TFEB siRNA and pEGFP-N1-TFEB mediated TFEB depletion and overexpression, respectively, on adherence or invasion of A. baumannii ATCC 17978. The percentages of total nontransfected A549 cells and A549 cells incubated with A. baumannii ATCC 17978 are shown for both adhesion and invasion. Results are from three independent experiments, and data are the means plus SEM (error bars). Values for untransfected and transfected groups in panels B and E that are significantly different ( P

    Article Snippet: Control and transfected A549 cells (transfected with TFEB siRNA or pEGFP-N1-TFEB) (1 × 106 cells) were collected, homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, Spain) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g for 20 min at 4°C.

    Techniques: Transfection, CTL Assay, Infection, Over Expression, Incubation

    Evaluation of the role of the autophagosome-lysosome system in A. baumannii intracellular trafficking. (A) The lysosomes in A549 cells were incubated with A. baumannii ATCC 17978 for 2 h, immunostained, and imaged by immunofluorescence microscopy. Acidic organelles were detected with LysoTracker red (75 nM), and mitochondria were detected with MitoTracker green (250 nM). The values for labeling of lysosomes in infected A549 cells in the bar graph are percentages compared to the value for noninfected cells. Values that are significantly different ( P

    Journal: mSphere

    Article Title: Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

    doi: 10.1128/mSphere.00106-18

    Figure Lengend Snippet: Evaluation of the role of the autophagosome-lysosome system in A. baumannii intracellular trafficking. (A) The lysosomes in A549 cells were incubated with A. baumannii ATCC 17978 for 2 h, immunostained, and imaged by immunofluorescence microscopy. Acidic organelles were detected with LysoTracker red (75 nM), and mitochondria were detected with MitoTracker green (250 nM). The values for labeling of lysosomes in infected A549 cells in the bar graph are percentages compared to the value for noninfected cells. Values that are significantly different ( P

    Article Snippet: Control and transfected A549 cells (transfected with TFEB siRNA or pEGFP-N1-TFEB) (1 × 106 cells) were collected, homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, Spain) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g for 20 min at 4°C.

    Techniques: Incubation, Immunofluorescence, Microscopy, Labeling, Infection

    A. baumannii activates the autophagosome-lysosome system. (A and B) Additive effect of autophagy and TFEB on A. baumannii internalization by A549 cells. A549 cells were transfected with scrambled (SC) or TFEB siRNA or pEGFP-N1-TFEB and treated with pepstatin (20 µg/ml), bafilomycin (0.8 µM), or wortmannin (1 µM), and infected with 10 8 CFU/ml A. baumannii ATCC 17978 for 2 h to study bacterial adherence and invasion to host cells. Results are representative of three independent experiments, and data are the means plus SEM. Values for treated and untreated groups that are significantly different ( P

    Journal: mSphere

    Article Title: Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

    doi: 10.1128/mSphere.00106-18

    Figure Lengend Snippet: A. baumannii activates the autophagosome-lysosome system. (A and B) Additive effect of autophagy and TFEB on A. baumannii internalization by A549 cells. A549 cells were transfected with scrambled (SC) or TFEB siRNA or pEGFP-N1-TFEB and treated with pepstatin (20 µg/ml), bafilomycin (0.8 µM), or wortmannin (1 µM), and infected with 10 8 CFU/ml A. baumannii ATCC 17978 for 2 h to study bacterial adherence and invasion to host cells. Results are representative of three independent experiments, and data are the means plus SEM. Values for treated and untreated groups that are significantly different ( P

    Article Snippet: Control and transfected A549 cells (transfected with TFEB siRNA or pEGFP-N1-TFEB) (1 × 106 cells) were collected, homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, Spain) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g for 20 min at 4°C.

    Techniques: Transfection, Infection

    A. baumannii stimulates the autophagy. (A) Expression of human autophagy genes after A. baumannii infection. Total RNA was isolated from A549 cells infected with A. baumannii ATCC 17978 and uninfected cells. cDNAs were synthesized by reverse transcription of the total RNA. A real-time PCR analysis was performed by using the Stratagene Mx3005p system. Samples were normalized to beta-2-microglobulin. Human autophagy gene expression after infection is represented by the heat map. Results are representative of two independent experiments. (B) Western blot analysis of LC3B in A549 cells infected with A. baumannii ATCC 17978 for 2 h. The blots were part of the same internally controlled experiment in Fig. 3C. Results are representative of three independent experiments. The solid white line separates the spliced portions between control and infected cells.

    Journal: mSphere

    Article Title: Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

    doi: 10.1128/mSphere.00106-18

    Figure Lengend Snippet: A. baumannii stimulates the autophagy. (A) Expression of human autophagy genes after A. baumannii infection. Total RNA was isolated from A549 cells infected with A. baumannii ATCC 17978 and uninfected cells. cDNAs were synthesized by reverse transcription of the total RNA. A real-time PCR analysis was performed by using the Stratagene Mx3005p system. Samples were normalized to beta-2-microglobulin. Human autophagy gene expression after infection is represented by the heat map. Results are representative of two independent experiments. (B) Western blot analysis of LC3B in A549 cells infected with A. baumannii ATCC 17978 for 2 h. The blots were part of the same internally controlled experiment in Fig. 3C. Results are representative of three independent experiments. The solid white line separates the spliced portions between control and infected cells.

    Article Snippet: Control and transfected A549 cells (transfected with TFEB siRNA or pEGFP-N1-TFEB) (1 × 106 cells) were collected, homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, Spain) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g for 20 min at 4°C.

    Techniques: Expressing, Infection, Isolation, Synthesized, Real-time Polymerase Chain Reaction, Western Blot

    Expression of TFEB in A549 cells by A. baumannii . (A) Western blot analysis of TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5, 2, and 6 h. The solid black lines in the blots separate the spliced portions of the blots between 2 and 6 h. Values shown in the bar graph are the percentage of TFEB expression in control (CTL) and infected A549 cells. (B) TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5 and 2 h, immunostaining, and imaging by immunofluorescence microscopy. TFEB detected with rabbit anti-TFEB antibodies and labeled with Alexa Fluor 594-tagged secondary antibodies (red). Blue staining with DAPI shows the location of nuclei of A549 cells. The percentage of TFEB expression in the nuclei of A549 cells was calculated as follows: (number of A549 cells that expressed TFEB in the nuclei of A549 cells/total number of A549 cells) × 100. Results are from three independent experiments, and data are means plus standard errors of the means (SEM) (error bars). Values that are significantly different ( P

    Journal: mSphere

    Article Title: Intracellular Trafficking and Persistence of Acinetobacter baumannii Requires Transcription Factor EB

    doi: 10.1128/mSphere.00106-18

    Figure Lengend Snippet: Expression of TFEB in A549 cells by A. baumannii . (A) Western blot analysis of TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5, 2, and 6 h. The solid black lines in the blots separate the spliced portions of the blots between 2 and 6 h. Values shown in the bar graph are the percentage of TFEB expression in control (CTL) and infected A549 cells. (B) TFEB in A549 cells after incubation with A. baumannii ATCC 17978 for 0.5 and 2 h, immunostaining, and imaging by immunofluorescence microscopy. TFEB detected with rabbit anti-TFEB antibodies and labeled with Alexa Fluor 594-tagged secondary antibodies (red). Blue staining with DAPI shows the location of nuclei of A549 cells. The percentage of TFEB expression in the nuclei of A549 cells was calculated as follows: (number of A549 cells that expressed TFEB in the nuclei of A549 cells/total number of A549 cells) × 100. Results are from three independent experiments, and data are means plus standard errors of the means (SEM) (error bars). Values that are significantly different ( P

    Article Snippet: Control and transfected A549 cells (transfected with TFEB siRNA or pEGFP-N1-TFEB) (1 × 106 cells) were collected, homogenized in radioimmunoprecipitation assay (RIPA) buffer (Sigma, Spain) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10% cocktail of protease inhibitors (Sigma, Spain), and centrifuged at 13,000 × g for 20 min at 4°C.

    Techniques: Expressing, Western Blot, Incubation, CTL Assay, Infection, Immunostaining, Imaging, Immunofluorescence, Microscopy, Labeling, Staining

    β-catenin mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: β-catenin mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    MTT assays. A549 cells were treated with increasing concentration of XAV939 (0.1, 0.5, 1,5 and 10 µM) for 24, 4, 72 and 96 h. The result was shown as relative cell viability per concentration at each time point.

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: MTT assays. A549 cells were treated with increasing concentration of XAV939 (0.1, 0.5, 1,5 and 10 µM) for 24, 4, 72 and 96 h. The result was shown as relative cell viability per concentration at each time point.

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: MTT Assay, Concentration Assay

    Localization of β-catenin immunofluorescence in response to treatment with NaCl (control), 0., 1 and 10 µmol/l XAV939 in A549 cells (magnification, ×400).

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: Localization of β-catenin immunofluorescence in response to treatment with NaCl (control), 0., 1 and 10 µmol/l XAV939 in A549 cells (magnification, ×400).

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: Immunofluorescence

    c-Myc mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: c-Myc mRNA expression at different XAV939 concentrations as detected by RT-sqPCR. (A) RT-PCR gel (B) and relative expression of β-catenin mRNA in A549 cells, treated with different XAV939 concentrations. *P

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction

    Εffect of XAV939 on A549 cell migration was detected via wound-healing assay. The wound widths in the different XAV939 treatment groups (0.1, 0.5, 1, 5, 10 µmol/l) after 24 h were all significantly wider than those of the control group (magnification, ×200).

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: Εffect of XAV939 on A549 cell migration was detected via wound-healing assay. The wound widths in the different XAV939 treatment groups (0.1, 0.5, 1, 5, 10 µmol/l) after 24 h were all significantly wider than those of the control group (magnification, ×200).

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: Migration, Wound Healing Assay

    Expression of TNKS in three lung adenocarcinoma cell lines. (A) Western blot analysis and (B) quantification of this analysis demonstrated that the level of TNKS protein expression in A549 cells was significantly higher compared with that in Calu-3 and SK-LU-1 cells. *P

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: Expression of TNKS in three lung adenocarcinoma cell lines. (A) Western blot analysis and (B) quantification of this analysis demonstrated that the level of TNKS protein expression in A549 cells was significantly higher compared with that in Calu-3 and SK-LU-1 cells. *P

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: Expressing, Western Blot

    (A) XAV939 inhibits the clonogenicity of A549 cells in vitro . (B) Treatment of the A549 cell line with 0.1, 1 and 10 µmol/l XAV939 significantly inhibited colony formation compared with the control. *P

    Journal: Oncology Letters

    Article Title: XAV939 inhibits the proliferation and migration of lung adenocarcinoma A549 cells through the WNT pathway

    doi: 10.3892/ol.2018.8491

    Figure Lengend Snippet: (A) XAV939 inhibits the clonogenicity of A549 cells in vitro . (B) Treatment of the A549 cell line with 0.1, 1 and 10 µmol/l XAV939 significantly inhibited colony formation compared with the control. *P

    Article Snippet: Owing to the higher TNKS protein expression levels observed in A549 cells compared with Calu-3 and SK-LU-1 cells, A549 cells were selected for subsequent experiments.

    Techniques: In Vitro

    Involvement of PI3K signaling in the reduction of the expression of TLR ligands and elements of TLR signaling pathway in U937 cells to Mtb H37Rv infection in the A549 cell coculture model. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of TLR signaling cascade showed a reversed TLR signaling activity in U937 cells of the coinfection model in the presence of LY294002, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. The ability of A549 cell-mediated reduction of TLR signaling activity in U937 cells was reversed by the addition of LY294002. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA; the A549 cell-mediated reduction of cytokines in H37Rv-infected U937 cells was reversed in the presence of PI3K inhibitor. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Involvement of PI3K signaling in the reduction of the expression of TLR ligands and elements of TLR signaling pathway in U937 cells to Mtb H37Rv infection in the A549 cell coculture model. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of TLR signaling cascade showed a reversed TLR signaling activity in U937 cells of the coinfection model in the presence of LY294002, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. The ability of A549 cell-mediated reduction of TLR signaling activity in U937 cells was reversed by the addition of LY294002. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA; the A549 cell-mediated reduction of cytokines in H37Rv-infected U937 cells was reversed in the presence of PI3K inhibitor. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Expressing, Infection, Activity Assay, Software, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Impact of PI3K signaling in U937 cells in response to H37Rv infection. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis by RT-PCR assay. Inductions of indicated transcripts of U937 cells infected with Mtb H37Rv in different conditions. The data was presented as the fold of changes of indicated transcripts over the noninfected cells. The ability of A549 cell-mediated reduction of TLR-mediated inflammations in U937 cells was reversed by the addition of LY294002. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Impact of PI3K signaling in U937 cells in response to H37Rv infection. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis by RT-PCR assay. Inductions of indicated transcripts of U937 cells infected with Mtb H37Rv in different conditions. The data was presented as the fold of changes of indicated transcripts over the noninfected cells. The ability of A549 cell-mediated reduction of TLR-mediated inflammations in U937 cells was reversed by the addition of LY294002. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Mtb H37Rv-infected A549 cells inhibited the expression of TLR signaling of U937 cells in response to Mycobacteria infection. The coculture model of A549/U937 cells was infected with H37Rv Mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the U937 cells were harvested for analysis by RT-PCR assay. (a–g) Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. (a) Fold of changes of TLR-2 transcripts over the noninfected cells; (B) fold of changes of TLR-4 transcripts over the noninfected cells; (c) fold of changes of TLR-6 transcripts over the noninfected cells; (d) fold of changes of TLR-8 transcripts over the noninfected cells; (e) fold of changes of MyD88 transcripts over the noninfected cells; (f) fold of changes of TRAF6 transcripts over the noninfected cells; (g) fold of changes of NF- κ B transcripts over the noninfected cells. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Mtb H37Rv-infected A549 cells inhibited the expression of TLR signaling of U937 cells in response to Mycobacteria infection. The coculture model of A549/U937 cells was infected with H37Rv Mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the U937 cells were harvested for analysis by RT-PCR assay. (a–g) Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. (a) Fold of changes of TLR-2 transcripts over the noninfected cells; (B) fold of changes of TLR-4 transcripts over the noninfected cells; (c) fold of changes of TLR-6 transcripts over the noninfected cells; (d) fold of changes of TLR-8 transcripts over the noninfected cells; (e) fold of changes of MyD88 transcripts over the noninfected cells; (f) fold of changes of TRAF6 transcripts over the noninfected cells; (g) fold of changes of NF- κ B transcripts over the noninfected cells. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Scheme showing a possible mechanism of the alleviated TLR-mediated inflammatory responses of macrophage U937 cells to the mycobacterial infection by coinfected A549 epithelial cells. In a coinfected condition, the undefined signaling from H37Rv mycobacteria-infected epithelial cells could reduce TLR-mediated inflammation of macrophages via the PI3K/Akt/mTOR signaling axis, but not the PI3K/Akt/GSK3 β pathway. Upon a coinfection of epithelial cells and macrophages to Mtb , the inflammatory responses in both host cell types were triggered in alveoli. In order to maintain the homeostasis of the alveolar microenvironment, the Mtb infection-induced epithelial cells subsequently alleviated the inflammation of the alveolar environment to secret soluble cytokines or mediators, which in turn inhibited the TLR-mediated inflammatory responses in macrophages to Mtb via the PI3K/Akt/mTOR pathway.

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Scheme showing a possible mechanism of the alleviated TLR-mediated inflammatory responses of macrophage U937 cells to the mycobacterial infection by coinfected A549 epithelial cells. In a coinfected condition, the undefined signaling from H37Rv mycobacteria-infected epithelial cells could reduce TLR-mediated inflammation of macrophages via the PI3K/Akt/mTOR signaling axis, but not the PI3K/Akt/GSK3 β pathway. Upon a coinfection of epithelial cells and macrophages to Mtb , the inflammatory responses in both host cell types were triggered in alveoli. In order to maintain the homeostasis of the alveolar microenvironment, the Mtb infection-induced epithelial cells subsequently alleviated the inflammation of the alveolar environment to secret soluble cytokines or mediators, which in turn inhibited the TLR-mediated inflammatory responses in macrophages to Mtb via the PI3K/Akt/mTOR pathway.

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection

    Impact of mTOR on the expression of TLR ligands and elements of the TLR signaling pathway in U937 cells to H37Rv infection in the A549 cell coculture model. In the presence or absence of mTOR inhibitor rapamycin, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade showed an increased expression of TLRs and MyD88 but a reduced expression of NF- κ B in U937 cells of the coinfection model in the presence of rapamycin, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software from three independent experiments. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA. An increased TNF- α was observed in the presence of mTOR inhibitor rapamycin. The A549 cell-mediated reduction of cytokines in the Mtb H37Rv-infected U937 cells was reversed in the presence of mTOR inhibitor rapamycin. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Impact of mTOR on the expression of TLR ligands and elements of the TLR signaling pathway in U937 cells to H37Rv infection in the A549 cell coculture model. In the presence or absence of mTOR inhibitor rapamycin, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade showed an increased expression of TLRs and MyD88 but a reduced expression of NF- κ B in U937 cells of the coinfection model in the presence of rapamycin, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software from three independent experiments. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA. An increased TNF- α was observed in the presence of mTOR inhibitor rapamycin. The A549 cell-mediated reduction of cytokines in the Mtb H37Rv-infected U937 cells was reversed in the presence of mTOR inhibitor rapamycin. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Expressing, Infection, Software, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Impact of GSK3 β on the expression of TLR ligands and elements of the TLR signaling pathway in U937 cells to Mtb H37Rv infection in the A549 cell coculture model. In the presence or absence of GSK3 β inhibitor LiCl, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade showed an activated TLR signaling in U937 cells of the coinfection model in the presence of LiCl, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA. An augmented cytokine production was observed in the presence of GSK3 β inhibitor LiCl, but the trend of a reduced TLR-mediated inflammatory response was not altered by the addition of LiCl. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Impact of GSK3 β on the expression of TLR ligands and elements of the TLR signaling pathway in U937 cells to Mtb H37Rv infection in the A549 cell coculture model. In the presence or absence of GSK3 β inhibitor LiCl, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade showed an activated TLR signaling in U937 cells of the coinfection model in the presence of LiCl, in comparison with the absence of an inhibitor. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments. (c) Concentrations of TNF- α , IL-10, and IL-6 in culture media determined by ELISA. An augmented cytokine production was observed in the presence of GSK3 β inhibitor LiCl, but the trend of a reduced TLR-mediated inflammatory response was not altered by the addition of LiCl. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Expressing, Infection, Software, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Impact of GSK3 β signaling in U937 cells in response to H37Rv infection. In the presence or absence of GSK3 β inhibitor LiCl, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis by RT-PCR assay. Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. The data was presented as the fold of changes of indicated transcripts over the noninfected cells. An increased abundance of indicated TLR signaling and cytokines was observed in the presence of GSK3 β inhibitor LiCl, but the trend of a reduction of TLR-mediated inflammatory responses was not altered by the addition of LiCl. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Impact of GSK3 β signaling in U937 cells in response to H37Rv infection. In the presence or absence of GSK3 β inhibitor LiCl, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis by RT-PCR assay. Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. The data was presented as the fold of changes of indicated transcripts over the noninfected cells. An increased abundance of indicated TLR signaling and cytokines was observed in the presence of GSK3 β inhibitor LiCl, but the trend of a reduction of TLR-mediated inflammatory responses was not altered by the addition of LiCl. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Mtb H37Rv-infected A549 cells reduced the expression of cytokines of U937 cells in response to mycobacterial infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the U937 cells were harvested for analysis by a RT-PCR assay. (a–g) Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. (a) Fold of changes of IL-1 β transcripts over the noninfected cells; (b) fold of changes of IL-2 transcripts over the noninfected cells; (c) fold of changes of IL-6 transcripts over the noninfected cells; (d) fold of changes of IL-8 transcripts over the noninfected cells; (e) fold of changes of IL-10 transcripts over the noninfected cells; (f) fold of changes of IL-12 β transcripts over the noninfected cells; (g) fold of changes of TNF- α transcripts over the noninfected cells. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Mtb H37Rv-infected A549 cells reduced the expression of cytokines of U937 cells in response to mycobacterial infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the U937 cells were harvested for analysis by a RT-PCR assay. (a–g) Inductions of indicated transcripts of U937 cells infected with H37Rv in different conditions. (a) Fold of changes of IL-1 β transcripts over the noninfected cells; (b) fold of changes of IL-2 transcripts over the noninfected cells; (c) fold of changes of IL-6 transcripts over the noninfected cells; (d) fold of changes of IL-8 transcripts over the noninfected cells; (e) fold of changes of IL-10 transcripts over the noninfected cells; (f) fold of changes of IL-12 β transcripts over the noninfected cells; (g) fold of changes of TNF- α transcripts over the noninfected cells. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Expressing, Reverse Transcription Polymerase Chain Reaction, Standard Deviation

    Mtb H37Rv-infected A549 cells reduced TLR signaling activity and cytokine production of U937 cells in response to mycobacterial infection. (a) Illustration of cell culture models used for infection in this study. A549 cells were cultured on the apical surface of transwells at an air-liquid interface state for 24 hours; then the transwell insert was transferred to a well containing PMA-stimulated U937 cells for infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (b) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade (left panel) and fold of changes of proteins of interest in U937 cells semiquantitatively determined by densitometric assay using ImageJ software Fiji (right panel). H37Rv-infected A549 cells showed an ability to reduce TLR signaling activity in U937 cells in response to mycobacterial infection. Concentrations of TNF- α (c), IL-10 (d), and IL-6 (e) in culture media determined by ELISA. H37Rv-infected A549 cells led a reduction of cytokine production in U937 cells in response to Mycobacteria infection. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Mtb H37Rv-infected A549 cells reduced TLR signaling activity and cytokine production of U937 cells in response to mycobacterial infection. (a) Illustration of cell culture models used for infection in this study. A549 cells were cultured on the apical surface of transwells at an air-liquid interface state for 24 hours; then the transwell insert was transferred to a well containing PMA-stimulated U937 cells for infection. The coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (b) Representative blots of immunoblotting assay for indicated components of TLR signaling cascade (left panel) and fold of changes of proteins of interest in U937 cells semiquantitatively determined by densitometric assay using ImageJ software Fiji (right panel). H37Rv-infected A549 cells showed an ability to reduce TLR signaling activity in U937 cells in response to mycobacterial infection. Concentrations of TNF- α (c), IL-10 (d), and IL-6 (e) in culture media determined by ELISA. H37Rv-infected A549 cells led a reduction of cytokine production in U937 cells in response to Mycobacteria infection. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Activity Assay, Cell Culture, Software, Enzyme-linked Immunosorbent Assay, Standard Deviation

    Impact of PI3K/Akt/GSK3 β /mTOR signaling in U937 cells in response to H37Rv infection. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of PI3K/Akt/GSK3 β /mTOR signaling showed an involvement of Akt, GSK3 β , and mTOR signaling in U937 cells of the coinfection model. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments; the ability of A549 cell-mediated reduction of Akt, GSK3 β , and mTOR signaling activity in U937 cells was lost in the presence of LY294002. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Journal: Mediators of Inflammation

    Article Title: Epithelial Cells Attenuate Toll-Like Receptor-Mediated Inflammatory Responses in Monocyte-Derived Macrophage-Like Cells to Mycobacterium tuberculosis by Modulating the PI3K/Akt/mTOR Signaling Pathway

    doi: 10.1155/2018/3685948

    Figure Lengend Snippet: Impact of PI3K/Akt/GSK3 β /mTOR signaling in U937 cells in response to H37Rv infection. In the presence or absence of PI3K inhibitor LY294002, the coculture model of A549/U937 cells was infected with H37Rv mycobacteria from the upper chamber (A549 cells, AI), lower chamber (U937 cells, UI), or both chambers (A549 and U937 cells, CI) at a MOI of 3 for 18 h before the culture medium and U937 cells were harvested for analysis. (a) Representative blots of immunoblotting assay for the indicated components of PI3K/Akt/GSK3 β /mTOR signaling showed an involvement of Akt, GSK3 β , and mTOR signaling in U937 cells of the coinfection model. (b) The fold of changes of proteins of interest in (a) semiquantitatively determined by densitometric assay using ImageJ software Fiji from three independent experiments; the ability of A549 cell-mediated reduction of Akt, GSK3 β , and mTOR signaling activity in U937 cells was lost in the presence of LY294002. Error bars represent the standard deviation (SD) from three independent experiments. Compared to noninfection (NI) control, ∗ p

    Article Snippet: At the time of seeding of A549 cells, 5 × 106 cells/well of U937 cells were separately cultured in another 6-well plate (Primera; Becton-Dickinson Labware, Franklin Lakes, NJ, USA) in RPMI 1640 medium supplemented with 10% FBS containing 0.2 μ g/mL phorbol-12-myristate-13-acetate (PMA) to derive macrophages overnight.

    Techniques: Infection, Software, Activity Assay, Standard Deviation

    Effect of GV, SU, CIS, DTX, PRI-2191 and calcitriol on (A) p53 and (B) p21 expression in A549 cells. Densitometric analysis was performed using ImageJ 1.46v software; the results were normalized to actin; bars represent the means ± SD, n=2–3 repeats. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Effect of GV, SU, CIS, DTX, PRI-2191 and calcitriol on (A) p53 and (B) p21 expression in A549 cells. Densitometric analysis was performed using ImageJ 1.46v software; the results were normalized to actin; bars represent the means ± SD, n=2–3 repeats. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Expressing, Software

    Inhibition of the proliferation of A549 cells after 72 h of incubation with GV, SU, CIS, DTX, PRI-2191 and/or calcitriol, followed by 48 h of culture in medium without FBS. Bars represent the means ± SD. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Inhibition of the proliferation of A549 cells after 72 h of incubation with GV, SU, CIS, DTX, PRI-2191 and/or calcitriol, followed by 48 h of culture in medium without FBS. Bars represent the means ± SD. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Inhibition, Incubation

    Effect of SU, CIS, DTX and PRI-2191 and their combinations on p53 expression and localization in A549 cells. A549 cells cultured on a coverglass were incubated with the tested combinations for 72 h, then fixed, permeabilized and stained for p53 (FITC) and nuclear staining (DAPI). Representative images of the combinations of SU with CIS and DTX and PRI-2191 are shown. SU, sunitinib; CIS, cisplatin; DTX, docetaxel; PRI-2191, 1,24(OH) 2 D 3 .

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Effect of SU, CIS, DTX and PRI-2191 and their combinations on p53 expression and localization in A549 cells. A549 cells cultured on a coverglass were incubated with the tested combinations for 72 h, then fixed, permeabilized and stained for p53 (FITC) and nuclear staining (DAPI). Representative images of the combinations of SU with CIS and DTX and PRI-2191 are shown. SU, sunitinib; CIS, cisplatin; DTX, docetaxel; PRI-2191, 1,24(OH) 2 D 3 .

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Expressing, Cell Culture, Incubation, Staining

    Effect of GV, SU, CIS, DTX, PRI-2191 and calcitriol on (A) VDR and (B) CYP24 expression in A549 cells. Densitometric analysis was performed using ImageJ 1.46v software; the results were normalized to actin; bars represent the means ± SD, n=3 repeats. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Effect of GV, SU, CIS, DTX, PRI-2191 and calcitriol on (A) VDR and (B) CYP24 expression in A549 cells. Densitometric analysis was performed using ImageJ 1.46v software; the results were normalized to actin; bars represent the means ± SD, n=3 repeats. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Expressing, Software

    (A) Vitamin D receptor (VDR), (B) 1α-hydroxylase (CYP27B1), (C) 24-hydroxylase of vitamin D (CYP24), (D) NF-κB, (E) IκB, (F) PTEN, (G) Bcl-2 and (H) Bax expression in A549 tumor lysates harvested from mice treated with SU, DTX and vitamin D analog PRI-2191 at the end of experiment (D51). Densitometric analysis was performed in ImageJ 1.46v software; blots were normalized to actin; means ± SD, n=3–4 lysates in each group; (B) * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: (A) Vitamin D receptor (VDR), (B) 1α-hydroxylase (CYP27B1), (C) 24-hydroxylase of vitamin D (CYP24), (D) NF-κB, (E) IκB, (F) PTEN, (G) Bcl-2 and (H) Bax expression in A549 tumor lysates harvested from mice treated with SU, DTX and vitamin D analog PRI-2191 at the end of experiment (D51). Densitometric analysis was performed in ImageJ 1.46v software; blots were normalized to actin; means ± SD, n=3–4 lysates in each group; (B) * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Expressing, Mouse Assay, Software

    (A) VEGF-A and (B) PDGF-BB expression in A549 tumor lysates harvested from mice treated with SU, DTX, and PRI-2191 at the end of the experiment (D51). VEGF-A and PDGF-BB levels were normalized to the total protein concentration; bars represent the means ± SD; * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: (A) VEGF-A and (B) PDGF-BB expression in A549 tumor lysates harvested from mice treated with SU, DTX, and PRI-2191 at the end of the experiment (D51). VEGF-A and PDGF-BB levels were normalized to the total protein concentration; bars represent the means ± SD; * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Expressing, Mouse Assay, Protein Concentration

    Caspase-3 activity in A549 cells after 72 h of incubation with the tested combinations of TKIs, CYT and vitamin D compounds. Bars represent the means ± SD. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Caspase-3 activity in A549 cells after 72 h of incubation with the tested combinations of TKIs, CYT and vitamin D compounds. Bars represent the means ± SD. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Activity Assay, Incubation

    Real-time PCR analysis of (A) TP53 , (B) VEGFA and (C) MYC levels in A549 cells after 72 h of incubation with GV, SU, CIS, DTX, PRI-2191 and calcitriol and their combinations. Bars represent relative quantification (RQ) calculated in Expression Suite v1.0.3 software with the ΔΔCt method. (A) * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Real-time PCR analysis of (A) TP53 , (B) VEGFA and (C) MYC levels in A549 cells after 72 h of incubation with GV, SU, CIS, DTX, PRI-2191 and calcitriol and their combinations. Bars represent relative quantification (RQ) calculated in Expression Suite v1.0.3 software with the ΔΔCt method. (A) * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Real-time Polymerase Chain Reaction, Incubation, Expressing, Software

    Antitumor activity of SU, DTX and vitamin D analog PRI-2191 in an A549 lung cancer model in vivo. SU was administered at the dose of 40 mg/kg/body weight daily, DTX at 5 mg/kg/body weight once a week, and PRI-2191 at 1 μ g/kg/body weight 3 times a week. (A) Kinetics of A549 tumor growth in all experimental groups; (B) comparison of groups receiving SU alone or in combination with DTX and/or PRI-2191; (C) body weight loss of mice bearing A549 tumors treated with SU, DTX, and/or vitamin D analog PRI-219; (D) comparison of tumor mass on the final day of the experiment [day (D)51]; * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Antitumor activity of SU, DTX and vitamin D analog PRI-2191 in an A549 lung cancer model in vivo. SU was administered at the dose of 40 mg/kg/body weight daily, DTX at 5 mg/kg/body weight once a week, and PRI-2191 at 1 μ g/kg/body weight 3 times a week. (A) Kinetics of A549 tumor growth in all experimental groups; (B) comparison of groups receiving SU alone or in combination with DTX and/or PRI-2191; (C) body weight loss of mice bearing A549 tumors treated with SU, DTX, and/or vitamin D analog PRI-219; (D) comparison of tumor mass on the final day of the experiment [day (D)51]; * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Activity Assay, In Vivo, Mouse Assay

    Cell cycle analysis of A549 lung cancer cells after 72 h of incubation with GV, SU, CIS, DTX and/or PRI-2191 or calcitriol. Bars represent the means ± SD. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Cell cycle analysis of A549 lung cancer cells after 72 h of incubation with GV, SU, CIS, DTX and/or PRI-2191 or calcitriol. Bars represent the means ± SD. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Cell Cycle Assay, Incubation

    Antiproliferative activity of GV, SU, CIS, DTX, PRI-2191 and/or calcitriol against A549 lung cancer cells. A549 cells were exposed to combinations of the test compounds at different concentrations for 72 h: N, TKI and CYT were used at their respective IC 50 concentrations; 0.5 N, half of IC 50 ; 0.25 N, a quarter of IC 50 ; PRI-2191 and calcitriol were used at constant concentration of 100 nM. Bars represent the means ± SD; * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Antiproliferative activity of GV, SU, CIS, DTX, PRI-2191 and/or calcitriol against A549 lung cancer cells. A549 cells were exposed to combinations of the test compounds at different concentrations for 72 h: N, TKI and CYT were used at their respective IC 50 concentrations; 0.5 N, half of IC 50 ; 0.25 N, a quarter of IC 50 ; PRI-2191 and calcitriol were used at constant concentration of 100 nM. Bars represent the means ± SD; * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Activity Assay, Concentration Assay

    VEGF-A secretion by A549 lung cancer cells in vitro following treatment with GV, SU, CIS, DTX, PRI-2191 and/or calcitriol. Bars represent the means ± SD. VEGF-A level was normalized to the number of cells assessed indirectly using sulforhodamine B (SRB) assay. * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: VEGF-A secretion by A549 lung cancer cells in vitro following treatment with GV, SU, CIS, DTX, PRI-2191 and/or calcitriol. Bars represent the means ± SD. VEGF-A level was normalized to the number of cells assessed indirectly using sulforhodamine B (SRB) assay. * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: In Vitro, Sulforhodamine B Assay

    Anti-proliferative activity of GV and SU with DTX and/or with PRI-2191 against A549 lung cancer cells. DTX was used at concentration a 10-fold lower concentration than the calculated IC 50 value. Bars represent the means ± SD; * p

    Journal: International Journal of Oncology

    Article Title: Vitamin D derivatives potentiate the anticancer and anti-angiogenic activity of tyrosine kinase inhibitors in combination with cytostatic drugs in an A549 non-small cell lung cancer model

    doi: 10.3892/ijo.2017.4228

    Figure Lengend Snippet: Anti-proliferative activity of GV and SU with DTX and/or with PRI-2191 against A549 lung cancer cells. DTX was used at concentration a 10-fold lower concentration than the calculated IC 50 value. Bars represent the means ± SD; * p

    Article Snippet: Cell cycle and cell death analysis The A549 cells and HLMECs were seeded in 24-well plates (Corning Inc., Corning NY, USA) in culture medium.

    Techniques: Activity Assay, Concentration Assay

    Inhibitive effects of BF or BF211 on the activity of porcine cortex Na+/K+-ATPase and the intracellular Ca2+ level of A4549 cells. (A) The activity of Na + /K + -ATPase extracted from porcine cerebral cortex in the presence of BF or BF211 at different concentrations was determined. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The level of intracellular Ca2+ level in A549 cells treated with 50 nM BF or BF211 for different time periods. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments.

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: Inhibitive effects of BF or BF211 on the activity of porcine cortex Na+/K+-ATPase and the intracellular Ca2+ level of A4549 cells. (A) The activity of Na + /K + -ATPase extracted from porcine cerebral cortex in the presence of BF or BF211 at different concentrations was determined. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The level of intracellular Ca2+ level in A549 cells treated with 50 nM BF or BF211 for different time periods. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments.

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: Activity Assay

    Inhibiting effects of BF and BF211 on the proliferation of A549 cells. (A) Chemical structures of BF and BF211. (B) The cell viability (MTT assay result) of A549 cells treated with various concentrations of BF or BF211 for 24 or 48 h. The data comprise the statistical results of three independent experiments. (B) A representative of the flow cytometry analysis results of apoptosis induced by 24 h treatment of BF or BF211 at different concentrations. (D) The statistical analysis results of the percentage of apoptotic cells after treatment with BF or BF211 at different concentrations for 24 h. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: Inhibiting effects of BF and BF211 on the proliferation of A549 cells. (A) Chemical structures of BF and BF211. (B) The cell viability (MTT assay result) of A549 cells treated with various concentrations of BF or BF211 for 24 or 48 h. The data comprise the statistical results of three independent experiments. (B) A representative of the flow cytometry analysis results of apoptosis induced by 24 h treatment of BF or BF211 at different concentrations. (D) The statistical analysis results of the percentage of apoptotic cells after treatment with BF or BF211 at different concentrations for 24 h. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. * p

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: MTT Assay, Flow Cytometry, Cytometry

    Effects of BF or BF211 on activation of Src in A549 cells. (A) The time-dependent effects of BF on the activation of Src in A549 cells treated with 50 nM BF for different time periods. Both representative western blot assay results and quantification analysis results were shown. The quantification data are the statistical results (n = 3, mean ± SEM) of three independent experiments. *p

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: Effects of BF or BF211 on activation of Src in A549 cells. (A) The time-dependent effects of BF on the activation of Src in A549 cells treated with 50 nM BF for different time periods. Both representative western blot assay results and quantification analysis results were shown. The quantification data are the statistical results (n = 3, mean ± SEM) of three independent experiments. *p

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: Activation Assay, Western Blot

    In vivo anti-cancer effects of BF and BF211 in nude mice inoculated with A549 cells. (A) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), BF or BF211 at similar concentrations as indicated. (B) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), and BF211 at concentrations of 2, 4, and 6 mg/kg.

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: In vivo anti-cancer effects of BF and BF211 in nude mice inoculated with A549 cells. (A) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), BF or BF211 at similar concentrations as indicated. (B) The tumor growth curve of nude mice treated with vehicle control, rapamycin (positive control), and BF211 at concentrations of 2, 4, and 6 mg/kg.

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: In Vivo, Mouse Assay, Positive Control

    Effects of BF or BF211 on caspase-3 activation and PARP cleavage in A549 cells. (A) The r epresentative western blot assay results probing the levels of caspase-3 and PARP in cells treated with BF or BF211 at different concentrations for 24 h. (B) The quantification of the western blot assay results of the activation of caspase-3 (percentage of cleaved caspase-3 from the total caspase-3) and cleavage of PARP (percentage of cleaved PARP from the total PARP) in cells treated with BF or BF211 at different concentrations for 24 h. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. * p

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: Effects of BF or BF211 on caspase-3 activation and PARP cleavage in A549 cells. (A) The r epresentative western blot assay results probing the levels of caspase-3 and PARP in cells treated with BF or BF211 at different concentrations for 24 h. (B) The quantification of the western blot assay results of the activation of caspase-3 (percentage of cleaved caspase-3 from the total caspase-3) and cleavage of PARP (percentage of cleaved PARP from the total PARP) in cells treated with BF or BF211 at different concentrations for 24 h. The data are the statistical results (n = 3, mean ± SEM) of three independent experiments. * p

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: Activation Assay, Western Blot

    Expression of Na+/K+-ATPase α subunits in A549 cells and binding affinity of BF or BF211 to Na+/K+-ATPase α subunits. (A) The results of the RT-PCR analysis of the expression levels of the α subunits of Na+/K+-ATPase in A549 cells. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The representative results of the western blot assay probing the protein levels of the α1 and α3 subunits of Na+/K+-ATPase in A549 cells. (C) The real time binding affinity measurements of BF or BF211 to the recombinant Na+/K+-ATPase α subunits. The binding ability between the compound and the recombinant protein is reflected in recorded response unit (RU) values.

    Journal: PLoS ONE

    Article Title: A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice

    doi: 10.1371/journal.pone.0159789

    Figure Lengend Snippet: Expression of Na+/K+-ATPase α subunits in A549 cells and binding affinity of BF or BF211 to Na+/K+-ATPase α subunits. (A) The results of the RT-PCR analysis of the expression levels of the α subunits of Na+/K+-ATPase in A549 cells. The data comprise the statistical results (n = 3, mean ± SEM) of three independent experiments. (B) The representative results of the western blot assay probing the protein levels of the α1 and α3 subunits of Na+/K+-ATPase in A549 cells. (C) The real time binding affinity measurements of BF or BF211 to the recombinant Na+/K+-ATPase α subunits. The binding ability between the compound and the recombinant protein is reflected in recorded response unit (RU) values.

    Article Snippet: The time-dependent effects of BF and BF211 on the free intracellular Ca2+ level of A549 cells The level of intracellular free Ca2+ in A549 cells was determined using the fluorescent dye Fluo-3 AM (S1056, Beyotime Biotechnology, Shanghai, China), which can cross the cell membrane and be cleaved into Fluo-3 by intracellular esterase.

    Techniques: Expressing, Binding Assay, Reverse Transcription Polymerase Chain Reaction, Western Blot, Recombinant

    Radiosensitizing effects of miR‐18a‐5p in vivo under irradiation. A, Growth curves of A549 xenografts at day 10 (n = 6, P

    Journal: Cancer Medicine

    Article Title: Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α, et al. Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α

    doi: 10.1002/cam4.1527

    Figure Lengend Snippet: Radiosensitizing effects of miR‐18a‐5p in vivo under irradiation. A, Growth curves of A549 xenografts at day 10 (n = 6, P

    Article Snippet: 3.1 Radiosensitizing effects of miR‐18a‐5p on A549 cells The expressions of miR‐18a‐5p in HBE and A549 cells before and after irradiation were detected by qRT‐PCR.

    Techniques: In Vivo, Irradiation

    Radiosensitizing effects of miR‐18a‐5p on A549 cells. A, miR‐18a‐5p expression in HBE and A549 cells (** P

    Journal: Cancer Medicine

    Article Title: Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α, et al. Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α

    doi: 10.1002/cam4.1527

    Figure Lengend Snippet: Radiosensitizing effects of miR‐18a‐5p on A549 cells. A, miR‐18a‐5p expression in HBE and A549 cells (** P

    Article Snippet: 3.1 Radiosensitizing effects of miR‐18a‐5p on A549 cells The expressions of miR‐18a‐5p in HBE and A549 cells before and after irradiation were detected by qRT‐PCR.

    Techniques: Expressing

    Radiosensitizing effects of miR‐18a‐5p on CD 133 + cells. A, The proportion of CD 133 + stem‐like cells in parent A549 cells and A549 cells induced into spheres. B, mRNA expression of stemness factors (Oct4, Nestin, Nanog) in CD 133‐ and stem‐like CD 133 + cells (** P

    Journal: Cancer Medicine

    Article Title: Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α, et al. Radiosensitizing effects of miR‐18a‐5p on lung cancer stem‐like cells via downregulating both ATM and HIF‐1α

    doi: 10.1002/cam4.1527

    Figure Lengend Snippet: Radiosensitizing effects of miR‐18a‐5p on CD 133 + cells. A, The proportion of CD 133 + stem‐like cells in parent A549 cells and A549 cells induced into spheres. B, mRNA expression of stemness factors (Oct4, Nestin, Nanog) in CD 133‐ and stem‐like CD 133 + cells (** P

    Article Snippet: 3.1 Radiosensitizing effects of miR‐18a‐5p on A549 cells The expressions of miR‐18a‐5p in HBE and A549 cells before and after irradiation were detected by qRT‐PCR.

    Techniques: Expressing