a549 cells  (Abcam)

 
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    A549 whole cell lysate
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    Structured Review

    Abcam a549 cells
    Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in <t>A549</t> cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P

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    Images

    1) Product Images from "MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer"

    Article Title: MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer

    Journal: Molecular Medicine Reports

    doi: 10.3892/mmr.2017.7517

    Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in A549 cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P
    Figure Legend Snippet: Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in A549 cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P

    Techniques Used: Expressing, Migration

    Expression of MACC-1 in non-small-cell lung cancer cells and tissues. (A) RT-qPCR and (B) western blot analysis of MACC-1 mRNA and protein expression levels in NCI-H520, A549 and H358 lung cancer cells, compared with MRC-5 normal lung cells. (C) RT-qPCR and (D) western blot analysis of the mRNA and protein expression level of MACC-1 in large cell carcinoma, squamous cell carcinoma, adenocarcinoma and normal lung tissues. Data are presented as the mean ± standard error of mean, from triplicate samples. **P
    Figure Legend Snippet: Expression of MACC-1 in non-small-cell lung cancer cells and tissues. (A) RT-qPCR and (B) western blot analysis of MACC-1 mRNA and protein expression levels in NCI-H520, A549 and H358 lung cancer cells, compared with MRC-5 normal lung cells. (C) RT-qPCR and (D) western blot analysis of the mRNA and protein expression level of MACC-1 in large cell carcinoma, squamous cell carcinoma, adenocarcinoma and normal lung tissues. Data are presented as the mean ± standard error of mean, from triplicate samples. **P

    Techniques Used: Expressing, Quantitative RT-PCR, Western Blot

    Therapeutic and metastasis-inhibitory effects of Chanti-MACC-1 in A549-bearing mice. (A) Tumor volume was analyzed following Chanti-MACC-1 treatment in a 24-day short term observation. (B) Long-term survival probability was performed in a 150-day observation between Chanti-MACC-1 and PBS treatment groups. (C) NSCLC metastasis was analyzed between Chanti-MACC-1 and PBS treatment animals. (D) MACC-1expression was analyzed via histological staining in tumors following treatment with Chanti-MACC-1. Student's t-test revealed a significant effect. *P
    Figure Legend Snippet: Therapeutic and metastasis-inhibitory effects of Chanti-MACC-1 in A549-bearing mice. (A) Tumor volume was analyzed following Chanti-MACC-1 treatment in a 24-day short term observation. (B) Long-term survival probability was performed in a 150-day observation between Chanti-MACC-1 and PBS treatment groups. (C) NSCLC metastasis was analyzed between Chanti-MACC-1 and PBS treatment animals. (D) MACC-1expression was analyzed via histological staining in tumors following treatment with Chanti-MACC-1. Student's t-test revealed a significant effect. *P

    Techniques Used: Mouse Assay, Staining

    Inhibitory effects of Chanti-MACC-1 on MACC-1 expression and non-small-cell lung cancer cell growth in vitro . (A) MACC-1 mRNA expression levels were decreased in A549 cells following treatment with Chanti-MACC-1. (B) MACC-1 expression alterations in A549 cells transfected with Chanti-MACC-1, determined via immunofluorescence. (C) MTT assays analyzed the inhibitory effects of Chanti-MACC-1 on A549 cells. (D) Migration analysis was performed to detect the efficacy of Chanti-MACC-1 on A549 cells. Student t-tests revealed a significant difference. **P
    Figure Legend Snippet: Inhibitory effects of Chanti-MACC-1 on MACC-1 expression and non-small-cell lung cancer cell growth in vitro . (A) MACC-1 mRNA expression levels were decreased in A549 cells following treatment with Chanti-MACC-1. (B) MACC-1 expression alterations in A549 cells transfected with Chanti-MACC-1, determined via immunofluorescence. (C) MTT assays analyzed the inhibitory effects of Chanti-MACC-1 on A549 cells. (D) Migration analysis was performed to detect the efficacy of Chanti-MACC-1 on A549 cells. Student t-tests revealed a significant difference. **P

    Techniques Used: Expressing, In Vitro, Transfection, Immunofluorescence, MTT Assay, Migration

    2) Product Images from "COP9 signalosome subunit 5 regulates cancer metastasis by deubiquitinating SNAIL"

    Article Title: COP9 signalosome subunit 5 regulates cancer metastasis by deubiquitinating SNAIL

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25060

    Inhibition of deubiquitinating enzymes suppressed cancer cell invasion ( A ) A549 or Panc-1 cells were treated with 50 µg/ml cycloheximide (CHX) for the indicated times and subjected to Western blotting. The band intensities were measured by ImageJ, normalized to that at 0 h of each cells, and shown below each panel. ( B ) A549 or Panc-1 cells were treated with 10 µM MG132 for the indicated times and subjected to Western blotting. Other conditions were similar to Figure 1A. ( C , D ) A549 or Panc-1 cells were treated with 75 μM PR-619 or DMSO for 3 h and subjected to Matrigel invasion assay (C) or Western blotting (D). Invaded cells were counted and normalized with the vehicle control (–). Data are represented as the mean ± S.D. of at least three independent experiments. * P
    Figure Legend Snippet: Inhibition of deubiquitinating enzymes suppressed cancer cell invasion ( A ) A549 or Panc-1 cells were treated with 50 µg/ml cycloheximide (CHX) for the indicated times and subjected to Western blotting. The band intensities were measured by ImageJ, normalized to that at 0 h of each cells, and shown below each panel. ( B ) A549 or Panc-1 cells were treated with 10 µM MG132 for the indicated times and subjected to Western blotting. Other conditions were similar to Figure 1A. ( C , D ) A549 or Panc-1 cells were treated with 75 μM PR-619 or DMSO for 3 h and subjected to Matrigel invasion assay (C) or Western blotting (D). Invaded cells were counted and normalized with the vehicle control (–). Data are represented as the mean ± S.D. of at least three independent experiments. * P

    Techniques Used: Inhibition, Western Blot, Invasion Assay

    COPS5 regulates the invasiveness and metastasis of lung cancer cells ( A – C ) A549 and Panc-1 cells transfected with the indicated siRNA for 96 h were subjected to Wound healing assay (A), Matrigel-invasion assay (B), and Western blotting (C). The band intensities were measured by ImageJ, normalized to that of siCNTL-transfected cells, and shown below each panel. * shows non-specific band using anti-SNAIL antibody. ( D ) Two lung adenocarcinoma cells (H1650 and H2228) transfected with the indicated siRNA for 96 h were subjected to Matrigel-invasion assay (left panel) or Western blotting (right panel). ( E ) A549/Luc2 cells transfected with the indicated siRNA for 96 h were subjected to In vivo invasion assay ( n = 3, each group). Data are represented as the mean ± S.D. of at least three independent experiments. * P
    Figure Legend Snippet: COPS5 regulates the invasiveness and metastasis of lung cancer cells ( A – C ) A549 and Panc-1 cells transfected with the indicated siRNA for 96 h were subjected to Wound healing assay (A), Matrigel-invasion assay (B), and Western blotting (C). The band intensities were measured by ImageJ, normalized to that of siCNTL-transfected cells, and shown below each panel. * shows non-specific band using anti-SNAIL antibody. ( D ) Two lung adenocarcinoma cells (H1650 and H2228) transfected with the indicated siRNA for 96 h were subjected to Matrigel-invasion assay (left panel) or Western blotting (right panel). ( E ) A549/Luc2 cells transfected with the indicated siRNA for 96 h were subjected to In vivo invasion assay ( n = 3, each group). Data are represented as the mean ± S.D. of at least three independent experiments. * P

    Techniques Used: Transfection, Wound Healing Assay, Invasion Assay, Western Blot, In Vivo

    COPS5 directly regulates SNAIL stability through deubiquitination ( A ) A549 cells transfected with Flag-tagged COPS5 (Flag/COPS5) or empty vector (–) were treated with cycloheximide (CHX) for the indicated time and subjected to Western blotting (upper panels). * shows non-specific band using anti-SNAIL antibody. The band intensities in biological triplicated experiments were measured by ImageJ and normalized to that at 0 h of each transfected cells (lower panel). Data are represented as the mean ± S.D. of three independent experiments. ** P
    Figure Legend Snippet: COPS5 directly regulates SNAIL stability through deubiquitination ( A ) A549 cells transfected with Flag-tagged COPS5 (Flag/COPS5) or empty vector (–) were treated with cycloheximide (CHX) for the indicated time and subjected to Western blotting (upper panels). * shows non-specific band using anti-SNAIL antibody. The band intensities in biological triplicated experiments were measured by ImageJ and normalized to that at 0 h of each transfected cells (lower panel). Data are represented as the mean ± S.D. of three independent experiments. ** P

    Techniques Used: Transfection, Plasmid Preparation, Western Blot

    3) Product Images from "Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus"

    Article Title: Distinct Patterns of IFITM-Mediated Restriction of Filoviruses, SARS Coronavirus, and Influenza A Virus

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001258

    MARV and EBOV GP 1,2 -mediated entry is restricted by IFITM1, 2, and 3. ( A ) A549, ( C ) Vero E6, ( E ) HUVEC, or ( G ) 293T cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were infected with MLV-GFP pseudotyped with the entry proteins of EBOV, MARV, IAV, MLV, or MACV, as indicated. Two days later, pseudovirus infection was determined by flow cytometry. Relative infectivity represents the percentage of GFP-positive cells, normalized to that of cells transduced with vector alone. Numbers underneath figures indicate percentage of infected cells in vector-transduced cells. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant ( P
    Figure Legend Snippet: MARV and EBOV GP 1,2 -mediated entry is restricted by IFITM1, 2, and 3. ( A ) A549, ( C ) Vero E6, ( E ) HUVEC, or ( G ) 293T cells transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone were infected with MLV-GFP pseudotyped with the entry proteins of EBOV, MARV, IAV, MLV, or MACV, as indicated. Two days later, pseudovirus infection was determined by flow cytometry. Relative infectivity represents the percentage of GFP-positive cells, normalized to that of cells transduced with vector alone. Numbers underneath figures indicate percentage of infected cells in vector-transduced cells. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant ( P

    Techniques Used: Plasmid Preparation, Infection, Flow Cytometry, Cytometry, Transduction, Expressing

    SARS-CoV S infection is restricted by IFITM1, 2, and 3. ( A ) A549 or ( D ) Vero E6 cells transduced to express ACE2 were subsequently transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone. Two days later, cells were infected with indicated pseudoviruses. Pseudovirus infection was determined by flow cytometry, and normalized to that of cells transduced with vector alone. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant ( P
    Figure Legend Snippet: SARS-CoV S infection is restricted by IFITM1, 2, and 3. ( A ) A549 or ( D ) Vero E6 cells transduced to express ACE2 were subsequently transduced to express the indicated c-myc-tagged IFITM proteins or with vector alone. Two days later, cells were infected with indicated pseudoviruses. Pseudovirus infection was determined by flow cytometry, and normalized to that of cells transduced with vector alone. Differences in pseudovirus entry between vector alone and IFITM expressing cells are significant ( P

    Techniques Used: Infection, Plasmid Preparation, Flow Cytometry, Cytometry, Transduction, Expressing

    Murine and chicken IFITM orthologs differentially restrict infection mediated by MARV, EBOV, and IAV entry proteins. A549 cells were transduced to express the indicated c-myc-tagged ( A ) human, ( C ) mouse, or ( E ) chicken IFITM orthologs. Two days later, cells were infected with MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Pseudovirus infection was measured by flow cytometry, and normalized to that of cells transduced with vector alone. Expression of ( B ) human, ( D ) mouse, or ( F ) chicken IFITM protein orthologs in A549 cells assayed in (A), (C), and (E), respectively was measured by western blot using the anti-c-myc antibody (9E10). All differences in pseudovirus entry between control and IFITM-expressing cells are significant ( P
    Figure Legend Snippet: Murine and chicken IFITM orthologs differentially restrict infection mediated by MARV, EBOV, and IAV entry proteins. A549 cells were transduced to express the indicated c-myc-tagged ( A ) human, ( C ) mouse, or ( E ) chicken IFITM orthologs. Two days later, cells were infected with MLV-GFP pseudotyped with the indicated viral entry glycoproteins. Pseudovirus infection was measured by flow cytometry, and normalized to that of cells transduced with vector alone. Expression of ( B ) human, ( D ) mouse, or ( F ) chicken IFITM protein orthologs in A549 cells assayed in (A), (C), and (E), respectively was measured by western blot using the anti-c-myc antibody (9E10). All differences in pseudovirus entry between control and IFITM-expressing cells are significant ( P

    Techniques Used: Infection, Flow Cytometry, Cytometry, Transduction, Plasmid Preparation, Expressing, Western Blot

    4) Product Images from "Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform"

    Article Title: Cancer Associated Fibroblast-Derived Hepatocyte Growth Factor Inhibits the Paclitaxel-Induced Apoptosis of Lung Cancer A549 Cells by Up-Regulating the PI3K/Akt and GRP78 Signaling on a Microfluidic Platform

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0129593

    The percentages of apoptotic A549 cells. A549 cells were cultured in maintenance medium for 24h and continually cultured in triplicate in the same medium or mixture of maintenance medium and CAF matrix in the presence or absence of the PI3K or GRP78 inhibitor on the 3D chambers for 24h. Subsequently, the cells were stained with Hochest33342 and PI, imaged and the percentages of apoptotic A549 cells were counted. Data are representative images (Fig 4A) (magnification x 200) or expressed as the means ± SD of the percentages of apoptotic cells in individual groups of cells from three separate experiments (Fig 4B). * P
    Figure Legend Snippet: The percentages of apoptotic A549 cells. A549 cells were cultured in maintenance medium for 24h and continually cultured in triplicate in the same medium or mixture of maintenance medium and CAF matrix in the presence or absence of the PI3K or GRP78 inhibitor on the 3D chambers for 24h. Subsequently, the cells were stained with Hochest33342 and PI, imaged and the percentages of apoptotic A549 cells were counted. Data are representative images (Fig 4A) (magnification x 200) or expressed as the means ± SD of the percentages of apoptotic cells in individual groups of cells from three separate experiments (Fig 4B). * P

    Techniques Used: Cell Culture, Staining

    The design and validation of a 3D culture microfluidic chip. (a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and > 95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.
    Figure Legend Snippet: The design and validation of a 3D culture microfluidic chip. (a)The schematic design of the microfluidic chip with CGG and downstream cell chambers (the upper panel) and the fabricated chip with pumping machine (the lower panel). (b)The diffused Rh-123 in the 3D chamber within 30 min and > 95% cells were viable (green). Magnification ×100. (c) The morphological features of A549 cells in the 3D chamber without or with CAF matrix. The white arrows indicate apoptotic cells. (d)The α-SMA immunofluorescence assay of HFL1 cells. HFL1 cells induced by A549 medium showed a positive α-SMA staining (right) compared to the untreated HFL1 (left). Magnification ×400. (e) Immunohistochemistry assay for lung cancer tissues. The expression of α-SMA protein in the lung cancer tissues is higher than that in adjacent tissues. Magnification ×200.

    Techniques Used: Chromatin Immunoprecipitation, Immunofluorescence, Staining, Immunohistochemistry, Expressing

    Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. * P
    Figure Legend Snippet: Western blot analysis of the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. A549 cells were cultured in the condition as described above and the relative levels of phosphorylated Met, PI3Kp85, AKT and GRP78 expression in the different groups of cells were characterized by Western blot assays and quantified. Data are representative images and expressed as the means ± SD of each protein in individual groups of cells from three separate experiments. * P

    Techniques Used: Western Blot, Activation Assay, Expressing, Cell Culture

    Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip. A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.
    Figure Legend Snippet: Immunofluorescent analysis of the MET/PI3K/AKT activation and GRP78 expression on the microfluidic chip. A549 cells were cultured in triplicate in the maintenance medium alone, mixed with the CAF matrix in the presence or absence of anti-HGF or containing 40 ng/ml of human HGF in the 3D chambers for 48h. The cells were stained with the indicated FITC-conjugated antibodies, and examined under a fluorescent microscope. Furthermore, the cells were cultured in the mixture of maintenance medium and CAF matrix in the presence or absence of an inhibitor for c-Met, PI3K or GRP78 for 48h and stained as described above. Data are representative images (magnification x 200) from three separate experiments. (A)The CAF matrix or HGF enhances the c-Met/PI3K/AKT activation and GRP78 expression in A549 cells. (B)The effect of an inhibitor of c-Met, PI3K or GRP78 on the CAF-enhanced c-Met/PI3K/AKT activation and GRP78 expression in A549 cells.

    Techniques Used: Activation Assay, Expressing, Chromatin Immunoprecipitation, Cell Culture, Staining, Microscopy

    5) Product Images from "Regulation of Interferon-? by MAGI-1 and Its Interaction with Influenza A Virus NS1 Protein with ESEV PBM"

    Article Title: Regulation of Interferon-? by MAGI-1 and Its Interaction with Influenza A Virus NS1 Protein with ESEV PBM

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0041251

    ESEV PBM impairs activation of IRF3 during infection. A). A549 cells were infected at an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Cell lysates were prepared at the 6 hours post-infection and phosphorylated and total IRF3 levels were quantified in an immunoblot. Densitometry was performed with ImageJ software and the pIRF3 levels were normalized to total IRF3. B. A549 cells were infected with indicated viruses and at 6 hours post-infection cell lysates were prepared and separated into soluble and insoluble fractions as described in Materials and Methods ; levels of viral NP and NS1 proteins in cell fractions were quantified with ImageJ software and normalized to β-actin levels in the corresponding sample. C) A549 cells were infected an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting as described in Materials and Methods for three independent experiments. A minimum 200 cells were examined to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. D). A representative immunofluorescence images used for quantitation in panel C is shown.
    Figure Legend Snippet: ESEV PBM impairs activation of IRF3 during infection. A). A549 cells were infected at an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Cell lysates were prepared at the 6 hours post-infection and phosphorylated and total IRF3 levels were quantified in an immunoblot. Densitometry was performed with ImageJ software and the pIRF3 levels were normalized to total IRF3. B. A549 cells were infected with indicated viruses and at 6 hours post-infection cell lysates were prepared and separated into soluble and insoluble fractions as described in Materials and Methods ; levels of viral NP and NS1 proteins in cell fractions were quantified with ImageJ software and normalized to β-actin levels in the corresponding sample. C) A549 cells were infected an m.o.i. of 1 with an H3N2 influenza A virus that expresses either an H6N6 NS1protein with the wt ESEV PBM or mutant ESEA PBM virus. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting as described in Materials and Methods for three independent experiments. A minimum 200 cells were examined to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. D). A representative immunofluorescence images used for quantitation in panel C is shown.

    Techniques Used: Activation Assay, Infection, Mutagenesis, Software, Immunofluorescence, Quantitation Assay

    ESEV PBM impairs NS1 inhibition of phosphorylation and nuclear localization of IRF3. A). A549 cells were transfected with wt (ESEV) or PBM mutant (ESEA) NS1 expression plasmids. Cells were re-transfected with poly(I:C) 24 hours later, cell extracts were prepared after 24 hours and levels of phosphorylated IRF3 were evaluated in an immunoblot. Mock-transfected cells were also analyzed. Densitometry was performed with ImageJ software. Values shown are normalized to corresponding total IRF3 levels. B) Percentages of cells with nuclear localized-IRF3 were quantified in three independent experiments by manually counting as described in Materials and Methods and panel C below. A minimum 200 cells were examined in each experiment to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C). A549 cells were transfected with a wt or mutant ESEA NS1 expression plasmid (with intact CPSF30 binding site). After 24 hours of transfection, cells were activated by transfection of poly(I:C). After 20 hours of activation, cells were processed for immunofluorescence. Nuclei were stained with DAPI; IRF3 is shown as red, and NS1 is shown as green. A representative immunofluorescence images is shown from three independent experiments.
    Figure Legend Snippet: ESEV PBM impairs NS1 inhibition of phosphorylation and nuclear localization of IRF3. A). A549 cells were transfected with wt (ESEV) or PBM mutant (ESEA) NS1 expression plasmids. Cells were re-transfected with poly(I:C) 24 hours later, cell extracts were prepared after 24 hours and levels of phosphorylated IRF3 were evaluated in an immunoblot. Mock-transfected cells were also analyzed. Densitometry was performed with ImageJ software. Values shown are normalized to corresponding total IRF3 levels. B) Percentages of cells with nuclear localized-IRF3 were quantified in three independent experiments by manually counting as described in Materials and Methods and panel C below. A minimum 200 cells were examined in each experiment to quantify IRF3 nuclear localization. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C). A549 cells were transfected with a wt or mutant ESEA NS1 expression plasmid (with intact CPSF30 binding site). After 24 hours of transfection, cells were activated by transfection of poly(I:C). After 20 hours of activation, cells were processed for immunofluorescence. Nuclei were stained with DAPI; IRF3 is shown as red, and NS1 is shown as green. A representative immunofluorescence images is shown from three independent experiments.

    Techniques Used: Inhibition, Transfection, Mutagenesis, Expressing, Software, Plasmid Preparation, Binding Assay, Activation Assay, Immunofluorescence, Staining

    Effects of NS1 ESEA PBM on activation of IRF3 and IFN-β pre-mRNA levels during influenza A infection. A) A549 cells were transfected with IFN-β Luciferase and Renilla Luciferase plasmids. After 24 hours, cells were infected with wt or ESEA virus at an m.o.i. of 1.0. Samples were collected at the indicated times for Luciferase assays. IFN-β luciferase values were normalized to Renilla Luciferase. Error bars represent the standard error of the mean. B) A549 cells were infected with either wt or ESEA virus at an m.o.i. of 1.0. Total RNA was isolated at the indicated times post-infection and RT-PCR assays were performed for IFN-β pre-mRNA as described in Methods . IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean.
    Figure Legend Snippet: Effects of NS1 ESEA PBM on activation of IRF3 and IFN-β pre-mRNA levels during influenza A infection. A) A549 cells were transfected with IFN-β Luciferase and Renilla Luciferase plasmids. After 24 hours, cells were infected with wt or ESEA virus at an m.o.i. of 1.0. Samples were collected at the indicated times for Luciferase assays. IFN-β luciferase values were normalized to Renilla Luciferase. Error bars represent the standard error of the mean. B) A549 cells were infected with either wt or ESEA virus at an m.o.i. of 1.0. Total RNA was isolated at the indicated times post-infection and RT-PCR assays were performed for IFN-β pre-mRNA as described in Methods . IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean.

    Techniques Used: Activation Assay, Infection, Transfection, Luciferase, Isolation, Reverse Transcription Polymerase Chain Reaction

    Depletion of MAGI-1 activates IRF3. A) A549 cells were transfected with the indicated siRNAs against MAGI-1, Dlg-1, Scribble, and control siRNAs; lanes labeled “Cells” are non-transfected control cells. After 48 hours, cells extracted were prepared and the levels of indicated proteins were examined in immunoblots. The levels of proteins were measured by densitometry analysis using ImageJ software; the signal for each PDZ protein in non-transfected control cells was arbitrarily assigned a value of 1.0 and PDZ protein levels in transfected cells is shown relative to this 1.0 value. B) A549 cells were transfected with the indicated siRNAs. After 24 hours, cells were processed for immunofluorescence for total IRF3 as described in Methods for three independent experiments. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting of at least 200 cells (a representative image for this assay is shown in Figure 7C ). Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples.
    Figure Legend Snippet: Depletion of MAGI-1 activates IRF3. A) A549 cells were transfected with the indicated siRNAs against MAGI-1, Dlg-1, Scribble, and control siRNAs; lanes labeled “Cells” are non-transfected control cells. After 48 hours, cells extracted were prepared and the levels of indicated proteins were examined in immunoblots. The levels of proteins were measured by densitometry analysis using ImageJ software; the signal for each PDZ protein in non-transfected control cells was arbitrarily assigned a value of 1.0 and PDZ protein levels in transfected cells is shown relative to this 1.0 value. B) A549 cells were transfected with the indicated siRNAs. After 24 hours, cells were processed for immunofluorescence for total IRF3 as described in Methods for three independent experiments. Percentages of cells with nuclear localized-IRF3 were quantified by manually counting of at least 200 cells (a representative image for this assay is shown in Figure 7C ). Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples.

    Techniques Used: Transfection, Labeling, Western Blot, Software, Immunofluorescence

    NS1 with ESEV PBM co-localizes with MAGI-I and Scribble in cytoplasmic puncta. A) A549 cells were infected with wt or ESEA mutant virus at an m.o.i. of 1. Cells were processed for immunofluorescence at 24 hours post-infection. Arrows indicate perinuclear puncta where ESEV NS1 and MAGI-1 co-localize. B) Same image as panel A; NS1 is shown as green, MAGI-1 is shown as red, and Scribble is shown as red. Arrows indicate puncta containing co-localization of NS1, MAGI-1, and Scribble.
    Figure Legend Snippet: NS1 with ESEV PBM co-localizes with MAGI-I and Scribble in cytoplasmic puncta. A) A549 cells were infected with wt or ESEA mutant virus at an m.o.i. of 1. Cells were processed for immunofluorescence at 24 hours post-infection. Arrows indicate perinuclear puncta where ESEV NS1 and MAGI-1 co-localize. B) Same image as panel A; NS1 is shown as green, MAGI-1 is shown as red, and Scribble is shown as red. Arrows indicate puncta containing co-localization of NS1, MAGI-1, and Scribble.

    Techniques Used: Infection, Mutagenesis, Immunofluorescence

    Depletion of MAGI-1 activates IFN-β promoter. A) A549 cells were transfected in duplicate with siRNAs against MAGI-1, Dlg-1, Scribble, and control siRNAs; lanes labeled “Cells” are non-transfected control cells. Cells were re-transfected 24 hours later with IFN-β Luciferase and Renilla Luciferase plasmids and Luciferase expression was measured 24 hours later. IFN-β Luciferase expression was normalized to Renilla Luciferase. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. B) A549 cells were transfected in duplicate with the indicated siRNAs. At 24 hours post-transfection, total RNA was isolated and used in RT-PCR assays to quantify IFN-β pre-mRNA levels. IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples.
    Figure Legend Snippet: Depletion of MAGI-1 activates IFN-β promoter. A) A549 cells were transfected in duplicate with siRNAs against MAGI-1, Dlg-1, Scribble, and control siRNAs; lanes labeled “Cells” are non-transfected control cells. Cells were re-transfected 24 hours later with IFN-β Luciferase and Renilla Luciferase plasmids and Luciferase expression was measured 24 hours later. IFN-β Luciferase expression was normalized to Renilla Luciferase. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. B) A549 cells were transfected in duplicate with the indicated siRNAs. At 24 hours post-transfection, total RNA was isolated and used in RT-PCR assays to quantify IFN-β pre-mRNA levels. IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples.

    Techniques Used: Transfection, Labeling, Luciferase, Expressing, Isolation, Reverse Transcription Polymerase Chain Reaction

    ESEV PBM with intact CPSF30 binding site impairs NS1 inhibition of IFN-β promoter activation. A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids, IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C) A549 cells were transfected with wt and mutant ESEA NS1 plasmid with intact CPSF30 site. After 48 hours, cells were lysed and analyzed for expression of NS1 in an immunoblots. The NS1 protein levels were normalized to corresponding β-actin level. Densitometry analysis was performed by ImageJ software.
    Figure Legend Snippet: ESEV PBM with intact CPSF30 binding site impairs NS1 inhibition of IFN-β promoter activation. A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids, IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to protein concentration. Error bars represent the standard error of the mean. Statistical difference in effects of NS1 plasmids was determined by student t-test in all the experiments. C) A549 cells were transfected with wt and mutant ESEA NS1 plasmid with intact CPSF30 site. After 48 hours, cells were lysed and analyzed for expression of NS1 in an immunoblots. The NS1 protein levels were normalized to corresponding β-actin level. Densitometry analysis was performed by ImageJ software.

    Techniques Used: Binding Assay, Inhibition, Activation Assay, Transfection, Expressing, Luciferase, Plasmid Preparation, Protein Concentration, Mutagenesis, Western Blot, Software

    ESEV PBM impairs NS1 inhibition of IFN-β promoter activation. A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. Statistical differences in effects of NS1 plasmids were determined by student t-test in all the experiments. C) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site). At 24 hours post-transfection, cells were re-transfected with poly(I:C) and 24 hours later total RNA isolated and RT-PCR assays were performed for IFN-β pre-mRNA as described in Methods . IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. D) A549 cells were transfected with 500 ng of wt or ESEA mutant NS1 expression plasmids (containing inactivated CPSF30 binding site). Cell extracts were prepared 48 hours later and expression levels of the Flag-tagged NS1 proteins were evaluated in an immunoblot. Densitometry analysis was performed by using ImageJ software. Values shown were normalized to corresponding internal control β-actin protein.
    Figure Legend Snippet: ESEV PBM impairs NS1 inhibition of IFN-β promoter activation. A) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, and Renilla Luciferase plasmid. At 24 hours post-transfection, cells were re-transfected with poly(I:C) and Luciferase expression was measured 20 hours later. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. B) Cultures of A549 cells were co-transfected in duplicate with indicated amounts of NS1 expression plasmids (containing inactivated CPSF30 binding site), IFN-β promoter Luciferase plasmid, Renilla Luciferase plasmids and, full-length RIG-I expression plasmid. Luciferase expression from the IFN-β promoter plasmid was normalized to Renilla Luciferase expression. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. Statistical differences in effects of NS1 plasmids were determined by student t-test in all the experiments. C) Cultures of A549 cells were transfected in duplicate with indicated amounts of NS1 expression plasmids (containing intact CPSF30 binding site). At 24 hours post-transfection, cells were re-transfected with poly(I:C) and 24 hours later total RNA isolated and RT-PCR assays were performed for IFN-β pre-mRNA as described in Methods . IFN-β pre-mRNA levels were normalized to GAPDH mRNA levels. Error bars represent the standard error of the mean from three independent experiments, with each experiment containing duplicate samples. D) A549 cells were transfected with 500 ng of wt or ESEA mutant NS1 expression plasmids (containing inactivated CPSF30 binding site). Cell extracts were prepared 48 hours later and expression levels of the Flag-tagged NS1 proteins were evaluated in an immunoblot. Densitometry analysis was performed by using ImageJ software. Values shown were normalized to corresponding internal control β-actin protein.

    Techniques Used: Inhibition, Activation Assay, Transfection, Expressing, Binding Assay, Luciferase, Plasmid Preparation, Isolation, Reverse Transcription Polymerase Chain Reaction, Mutagenesis, Software

    6) Product Images from "β2-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence"

    Article Title: β2-Microglobulin participates in development of lung emphysema by inducing lung epithelial cell senescence

    Journal: American Journal of Physiology - Lung Cellular and Molecular Physiology

    doi: 10.1152/ajplung.00516.2016

    CSE-mediated proliferation inhibition and expression of β 2 M in A549 cells. A : images showed EdU staining (×200). B : quantification of percentages of EdU-positive cells. C : CSE exposure induced increased secretion of β 2 M by A549 cells. Data were expressed as means ± SE. * P
    Figure Legend Snippet: CSE-mediated proliferation inhibition and expression of β 2 M in A549 cells. A : images showed EdU staining (×200). B : quantification of percentages of EdU-positive cells. C : CSE exposure induced increased secretion of β 2 M by A549 cells. Data were expressed as means ± SE. * P

    Techniques Used: Inhibition, Expressing, Staining

    β 2 M induced cellular senescence in A549 cells. A : SA-β-Gal (blue, ×200)-positive cells increased after exposure of 100 μg/ml of β 2 M, compared with the negative control. B : quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE. * P
    Figure Legend Snippet: β 2 M induced cellular senescence in A549 cells. A : SA-β-Gal (blue, ×200)-positive cells increased after exposure of 100 μg/ml of β 2 M, compared with the negative control. B : quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE. * P

    Techniques Used: Negative Control

    Effect of anti-β 2 M antibody on CSE-induced cell proliferation. A : images show EdU staining of A549 cells under the indicated condition (×200). B : quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. * P
    Figure Legend Snippet: Effect of anti-β 2 M antibody on CSE-induced cell proliferation. A : images show EdU staining of A549 cells under the indicated condition (×200). B : quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. * P

    Techniques Used: Staining

    CSE induced cellular senescence in A549 cells. A : SA-β-Gal-positive cells (blue, × 200) increased after 5% CSE stimulation, compared with the positive controls. B : quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE * P
    Figure Legend Snippet: CSE induced cellular senescence in A549 cells. A : SA-β-Gal-positive cells (blue, × 200) increased after 5% CSE stimulation, compared with the positive controls. B : quantification of percentages of SA-β-Gal positive cells. Data were expressed as means ± SE * P

    Techniques Used:

    β 2 M inhibited proliferation of A549 cells. A : images showed EdU staining of A549 cells exposed to different concentrations of β 2 M (×200). B : quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. * P
    Figure Legend Snippet: β 2 M inhibited proliferation of A549 cells. A : images showed EdU staining of A549 cells exposed to different concentrations of β 2 M (×200). B : quantification of percentages of EdU-positive cells. Data were expressed as the means ± SE. * P

    Techniques Used: Staining

    7) Product Images from "Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing"

    Article Title: Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01559

    Human type II alveolar cells (A549) produce vitronectin after stimulation with bacterial OMV. Surface-bound vitronectin was detected on A549 epithelial cells by flow cytometry (A) . Cells were starved for 18 h prior to the experiment. Following the addition of OMV (5 μg) from P. aeruginosa (solid squares) or nontypeable H. influenzae (solid triangles), the cells were harvested at indicated time points. Untreated cells were used as controls (open circles). Mean values of two biological replicates are shown, each comprising three technical repetitions. ANOVA was used to calculate the p -values. A corresponding increase in vitronectin ( VTN ) mRNA levels was seen 1–3 h after challenge with 1 μg OMV from either bacterial species (B) . Symbols represent mean values and error bars represent standard error of the mean of three biological replicates. The difference in mean values between respective bacterial species and controls were assessed using a t -test.
    Figure Legend Snippet: Human type II alveolar cells (A549) produce vitronectin after stimulation with bacterial OMV. Surface-bound vitronectin was detected on A549 epithelial cells by flow cytometry (A) . Cells were starved for 18 h prior to the experiment. Following the addition of OMV (5 μg) from P. aeruginosa (solid squares) or nontypeable H. influenzae (solid triangles), the cells were harvested at indicated time points. Untreated cells were used as controls (open circles). Mean values of two biological replicates are shown, each comprising three technical repetitions. ANOVA was used to calculate the p -values. A corresponding increase in vitronectin ( VTN ) mRNA levels was seen 1–3 h after challenge with 1 μg OMV from either bacterial species (B) . Symbols represent mean values and error bars represent standard error of the mean of three biological replicates. The difference in mean values between respective bacterial species and controls were assessed using a t -test.

    Techniques Used: Flow Cytometry, Cytometry

    8) Product Images from "Investigation of Cytotoxicity Apoptotic and Inflammatory Responses of Biosynthesized Zinc Oxide Nanoparticles from Ocimum sanctum Linn in Human Skin Keratinocyte (Hacat) and Human Lung Epithelial (A549) Cells"

    Article Title: Investigation of Cytotoxicity Apoptotic and Inflammatory Responses of Biosynthesized Zinc Oxide Nanoparticles from Ocimum sanctum Linn in Human Skin Keratinocyte (Hacat) and Human Lung Epithelial (A549) Cells

    Journal: Oxidative Medicine and Cellular Longevity

    doi: 10.1155/2020/1835475

    Oxidative stress biomarkers after exposure to gZnNPs on HaCaT and A549 cells for 24 h. (a) LPO in HaCaT. ( b ) LPO in A549 cells (c). GSH in HaCaT cells. (d) GSH in A549 cells. Each value represents the mean ± SE of three experiments. ∗ p
    Figure Legend Snippet: Oxidative stress biomarkers after exposure to gZnNPs on HaCaT and A549 cells for 24 h. (a) LPO in HaCaT. ( b ) LPO in A549 cells (c). GSH in HaCaT cells. (d) GSH in A549 cells. Each value represents the mean ± SE of three experiments. ∗ p

    Techniques Used:

    Scatter diagram after treatments of gZnNPs to HaCaT and A549 cells for 24 h. (a) HaCaT cells at 0 μ g/ml. (b) HaCaT cells at 25 μ g/ml. (c) HaCaT cells at 35 μ g/ml. (d) A549 cells at 0 μ g/ml. (e) A549 cells at 25 μ g/ml. (f) A549 cells at 35 μ g/ml. (g) Percentage of early and late apoptotic cells after treatment of ZnNPs (0, 25, and 35 μ g/ml) for 24 h. (h) Caspase-3 activity after treatment of ZnNPs (0, 6, 25, and 35 μ g/ml) for 24 h. Data represents the mean ± SE of three experiments. ∗ p
    Figure Legend Snippet: Scatter diagram after treatments of gZnNPs to HaCaT and A549 cells for 24 h. (a) HaCaT cells at 0 μ g/ml. (b) HaCaT cells at 25 μ g/ml. (c) HaCaT cells at 35 μ g/ml. (d) A549 cells at 0 μ g/ml. (e) A549 cells at 25 μ g/ml. (f) A549 cells at 35 μ g/ml. (g) Percentage of early and late apoptotic cells after treatment of ZnNPs (0, 25, and 35 μ g/ml) for 24 h. (h) Caspase-3 activity after treatment of ZnNPs (0, 6, 25, and 35 μ g/ml) for 24 h. Data represents the mean ± SE of three experiments. ∗ p

    Techniques Used: Activity Assay

    Cytotoxicity of gZnNPs on HaCaT and A549 cells for 24 h, as measured by MTT assay. Each value represents the mean ± SE of three experiments. n = 3, ∗ p
    Figure Legend Snippet: Cytotoxicity of gZnNPs on HaCaT and A549 cells for 24 h, as measured by MTT assay. Each value represents the mean ± SE of three experiments. n = 3, ∗ p

    Techniques Used: MTT Assay

    ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. mRNA expression analysis was carried out to assess apoptosis and inflammatory response-related gene expression in HaCaT cells (a) and A549 cells (b). β -Actin was used as an internal reference. ZnNPs significantly increased the gene expression of Bax, caspase-3, and TNF- α and decreased the expression of Bcl-2 in a concentration-dependent manner (a, b). Data represents the mean ± SE of three experiments. ∗ p
    Figure Legend Snippet: ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. mRNA expression analysis was carried out to assess apoptosis and inflammatory response-related gene expression in HaCaT cells (a) and A549 cells (b). β -Actin was used as an internal reference. ZnNPs significantly increased the gene expression of Bax, caspase-3, and TNF- α and decreased the expression of Bcl-2 in a concentration-dependent manner (a, b). Data represents the mean ± SE of three experiments. ∗ p

    Techniques Used: Expressing, Concentration Assay

    (a) Photomicrograph of green fluorescence (DCF intensity) in HaCaT and A549 cells after exposure to gZnNPs for 24 h. (b) Intracellular intensity of ROS production in HaCaT cells (c). Intracellular intensity of ROS production in A549 cells. Each value represents the mean ± SE of three experiments. ∗ p
    Figure Legend Snippet: (a) Photomicrograph of green fluorescence (DCF intensity) in HaCaT and A549 cells after exposure to gZnNPs for 24 h. (b) Intracellular intensity of ROS production in HaCaT cells (c). Intracellular intensity of ROS production in A549 cells. Each value represents the mean ± SE of three experiments. ∗ p

    Techniques Used: Fluorescence

    ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. Western blot analysis was carried out to assess apoptosis and inflammatory response-related protein expression in HaCaT and A549 cells. β -Actin was used as an internal reference. ZnNPs significantly increased the expression of Bax, caspase-3, and TNF- α and decreased the expression of Bcl-2 at a concentration 35 μ g/ml (a, b). Data represents the mean ± SE of three experiments. ∗ p
    Figure Legend Snippet: ZnNPs induce apoptosis and inflammatory response of HaCaT and A549 cells. Western blot analysis was carried out to assess apoptosis and inflammatory response-related protein expression in HaCaT and A549 cells. β -Actin was used as an internal reference. ZnNPs significantly increased the expression of Bax, caspase-3, and TNF- α and decreased the expression of Bcl-2 at a concentration 35 μ g/ml (a, b). Data represents the mean ± SE of three experiments. ∗ p

    Techniques Used: Western Blot, Expressing, Concentration Assay

    9) Product Images from "Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus"

    Article Title: Expression of non-structural-1A binding protein in lung epithelial cells is modulated by miRNA-548an on exposure to influenza A virus

    Journal: Virology

    doi: 10.1016/j.virol.2013.08.031

    Effect of miRNA 548an mimic on viral replication. (A) A549 cells were transfected for 48 h with 25 nM miRNA-548an mimic or a scrambled oligonucleotide. After 48 h, the cells were infected with increasing MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Expression of influenza Matrix gene copies in cells transfected with the mimic is expressed relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **= p
    Figure Legend Snippet: Effect of miRNA 548an mimic on viral replication. (A) A549 cells were transfected for 48 h with 25 nM miRNA-548an mimic or a scrambled oligonucleotide. After 48 h, the cells were infected with increasing MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. Expression of influenza Matrix gene copies in cells transfected with the mimic is expressed relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **= p

    Techniques Used: Transfection, Infection, Reverse Transcription Polymerase Chain Reaction, Expressing

    Visualization of NS1ABP protein expression following modulation of miRNA-548an. A549 cells were transfected for 48 h with either the scrambled oligonucleotide (negative control) (top panel), miRNA-548an mimic (middle panel), or miRNA-548an inhibitor (third panel), and uninfected cells (bottom panel). Transfected cells were infected 1 MOI of influenza A for 3 h and expression of NS1ABP protein (green) and influenza nucleoprotein (red) was determined by immunofluorescence. DAPI (blue) represents the stained nucleus of the cells (not shown separately).
    Figure Legend Snippet: Visualization of NS1ABP protein expression following modulation of miRNA-548an. A549 cells were transfected for 48 h with either the scrambled oligonucleotide (negative control) (top panel), miRNA-548an mimic (middle panel), or miRNA-548an inhibitor (third panel), and uninfected cells (bottom panel). Transfected cells were infected 1 MOI of influenza A for 3 h and expression of NS1ABP protein (green) and influenza nucleoprotein (red) was determined by immunofluorescence. DAPI (blue) represents the stained nucleus of the cells (not shown separately).

    Techniques Used: Expressing, Transfection, Negative Control, Infection, Immunofluorescence, Staining

    Influenza A alters host miRNA expression. (A) PCR analysis of individual miRNAs that showed differential expression in microarray analysis. miRNA isolated after 3 h of exposure to influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), (MOI of 3). The fold chang e represents the change in miRNA expression in infected cells relative to that obtained in uninfected cells. (B) A549 cells were infected with 3MOIs of influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), for 3 h and matrix copy numbers were determined by RT-PCR. (C) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza. Data are expressed as ± standard error of the mean (SEM). n =4 (independent experiments) with triplicate replicates (*= p
    Figure Legend Snippet: Influenza A alters host miRNA expression. (A) PCR analysis of individual miRNAs that showed differential expression in microarray analysis. miRNA isolated after 3 h of exposure to influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), (MOI of 3). The fold chang e represents the change in miRNA expression in infected cells relative to that obtained in uninfected cells. (B) A549 cells were infected with 3MOIs of influenza A (A/WS/33 (H1N1), A/Aichi/2/68 (H3N2), A/Swine/1976/31 (H1N1), and A/Swine/Iowa/15/30 (H1N1)), for 3 h and matrix copy numbers were determined by RT-PCR. (C) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to 3MOI of influenza. Data are expressed as ± standard error of the mean (SEM). n =4 (independent experiments) with triplicate replicates (*= p

    Techniques Used: Expressing, Polymerase Chain Reaction, Microarray, Isolation, Infection, Reverse Transcription Polymerase Chain Reaction

    miRNA-548an and NS1ABP mRNA expressions in A549 cells infected with different MOIs of influenza A. Differentially expressed miRNAs identified in the microarray analysis were screened for a potential role in influenza propagation and miRNA-548an was selected for further analysis. A549 cells were infected for 3 h with increasing MOIs of influenza. (A) miRNA-548an expression in uninfected and infected A549 cells was normalized to expression of let-7 as a housekeeping miRNA. (B) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to increasing MOIs of influenza. p
    Figure Legend Snippet: miRNA-548an and NS1ABP mRNA expressions in A549 cells infected with different MOIs of influenza A. Differentially expressed miRNAs identified in the microarray analysis were screened for a potential role in influenza propagation and miRNA-548an was selected for further analysis. A549 cells were infected for 3 h with increasing MOIs of influenza. (A) miRNA-548an expression in uninfected and infected A549 cells was normalized to expression of let-7 as a housekeeping miRNA. (B) NS1ABP mRNA expression was normalized to expression of GAPDH mRNA in response to increasing MOIs of influenza. p

    Techniques Used: Infection, Microarray, Expressing

    Time course showing the expression levels of miRNA-548an and NS1ABP mRNA in cells infected with influenza A. (A) A549 cells were infected with 1 MOI of Influenza A and expression of miRNA-548an normalized to let-7 miRNA is shown over 0–6 h of infection. (B) A549 cells were infected with 1 MOI of Influenza A and expression of NS1ABP mRNA normalized to GAPDH mRNA is shown over 0–6 h of infection. The fold chang e represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (D) Protein expression by Western blot analysis in cells infected with influenza A for up to 3 h and α-tubulin was used as a loading control. Data are expressed as ± SEM. ***= p
    Figure Legend Snippet: Time course showing the expression levels of miRNA-548an and NS1ABP mRNA in cells infected with influenza A. (A) A549 cells were infected with 1 MOI of Influenza A and expression of miRNA-548an normalized to let-7 miRNA is shown over 0–6 h of infection. (B) A549 cells were infected with 1 MOI of Influenza A and expression of NS1ABP mRNA normalized to GAPDH mRNA is shown over 0–6 h of infection. The fold chang e represents the change in miRNA and mRNA expression in infected cells relative to that obtained in uninfected cells. (C) HBEpC cells were infected with 3 MOIs of influenza A for 3 h and matrix copy numbers were determined by RT-PCR. (D) Protein expression by Western blot analysis in cells infected with influenza A for up to 3 h and α-tubulin was used as a loading control. Data are expressed as ± SEM. ***= p

    Techniques Used: Expressing, Infection, Reverse Transcription Polymerase Chain Reaction, Western Blot

    Down or up-regulation of miRNA 548an alters the expression of NS1ABP mRNA. (A) Uninfected A549 cells were transfected for 48 h with 50 nM or 100 nM of either miRNA 548an inhibitor or a scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (B) After 48 h, transfected cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. n =3 (independent experiments) with triplicate replicates. (C) A549 cells were transfected for 48 h with 12.5 nM, 25 nM, or 50 nM of miRNA-548an mimic or scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (D) A549 cells were transfected for 48 h with miRNA-548an mimic or scrambled oligonucleotide. After 48 h, cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (E) NS1ABP expression profile after 30 and 48 h of transfection with mimics in A549 cells. (F) NS1ABP expression profile after 30 and 48 h of transfection with inhibitor in A549 cells. The fold chang e represents the change in NS1ABP mRNA expression and influenza A Matrix copy number in cells transfected with the inhibitor relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **= p
    Figure Legend Snippet: Down or up-regulation of miRNA 548an alters the expression of NS1ABP mRNA. (A) Uninfected A549 cells were transfected for 48 h with 50 nM or 100 nM of either miRNA 548an inhibitor or a scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (B) After 48 h, transfected cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. n =3 (independent experiments) with triplicate replicates. (C) A549 cells were transfected for 48 h with 12.5 nM, 25 nM, or 50 nM of miRNA-548an mimic or scrambled oligonucleotide as control. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (D) A549 cells were transfected for 48 h with miRNA-548an mimic or scrambled oligonucleotide. After 48 h, cells were infected with 1 MOI of influenza A for 3 h. Expression of NS1ABP mRNA normalized to GAPDH mRNA is shown. (E) NS1ABP expression profile after 30 and 48 h of transfection with mimics in A549 cells. (F) NS1ABP expression profile after 30 and 48 h of transfection with inhibitor in A549 cells. The fold chang e represents the change in NS1ABP mRNA expression and influenza A Matrix copy number in cells transfected with the inhibitor relative to that obtained in cells transfected with the scrambled oligonucleotide. Data are expressed as ± SEM, **= p

    Techniques Used: Expressing, Transfection, Infection

    Apoptotic pattern of cells transfected with miRNA-548an mimic or inhibitor and infected with influenza A. (A and C) A549 cells were transfected for 48 h with the scrambled oligonucleotide or (B) miRNA-548an mimic, or (D) miRNA-548an inhibitor. Transfected cells were then infected with 1 MOI of influenza A for 3 h. The cells were immune-fluorescently labeled with annexin V and apoptotic cells were analyzed by flow cytometry. Transfection did not change the proportion of necrotic cells detected by propidium iodide (PI) staining. Graph is a representative plot from 3 independent experiments and data presented as percentage of cells in each quadrant. Left upper quadrant=necrotic cells; left lower quadrant=viable cells; right lower quadrant=apoptotic cells; right upper quadrant=late apoptotic cells in necrotic state.
    Figure Legend Snippet: Apoptotic pattern of cells transfected with miRNA-548an mimic or inhibitor and infected with influenza A. (A and C) A549 cells were transfected for 48 h with the scrambled oligonucleotide or (B) miRNA-548an mimic, or (D) miRNA-548an inhibitor. Transfected cells were then infected with 1 MOI of influenza A for 3 h. The cells were immune-fluorescently labeled with annexin V and apoptotic cells were analyzed by flow cytometry. Transfection did not change the proportion of necrotic cells detected by propidium iodide (PI) staining. Graph is a representative plot from 3 independent experiments and data presented as percentage of cells in each quadrant. Left upper quadrant=necrotic cells; left lower quadrant=viable cells; right lower quadrant=apoptotic cells; right upper quadrant=late apoptotic cells in necrotic state.

    Techniques Used: Transfection, Infection, Labeling, Flow Cytometry, Cytometry, Staining

    NS1ABP protein expression is modulated by miRNA 548an. (A) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor or scrambled oligonucleotide. (B) Flow cytometry analysis of A549 cells transfected for 48 h with 25 nM miRNA-548an mimic or scrambled oligonucleotide. (C) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor and then infected with 1 MOI of influenza for 3 h. Expression of NS1ABP protein in cells transfected with the miRNA-548an inhibitor (green line), or scrambled oligonucleotide as control (black line) is shown. (D) Flow cytometry analysis in A549 cells transfected for 48 h with miRNA-548an mimic (green line) or scrambled oligonucleotide (black line) and then infected with 1 MOI of influenza for 3 h. The gray shaded or red shaded regions in the graph represent the isotype control. (E) Geometric mean fluorescent intensity (MFI) was measured from 3 independent experiments with triplicate replicates. ###= p
    Figure Legend Snippet: NS1ABP protein expression is modulated by miRNA 548an. (A) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor or scrambled oligonucleotide. (B) Flow cytometry analysis of A549 cells transfected for 48 h with 25 nM miRNA-548an mimic or scrambled oligonucleotide. (C) Flow cytometry analysis of A549 cells transfected for 48 h with 50 nM miRNA-548an inhibitor and then infected with 1 MOI of influenza for 3 h. Expression of NS1ABP protein in cells transfected with the miRNA-548an inhibitor (green line), or scrambled oligonucleotide as control (black line) is shown. (D) Flow cytometry analysis in A549 cells transfected for 48 h with miRNA-548an mimic (green line) or scrambled oligonucleotide (black line) and then infected with 1 MOI of influenza for 3 h. The gray shaded or red shaded regions in the graph represent the isotype control. (E) Geometric mean fluorescent intensity (MFI) was measured from 3 independent experiments with triplicate replicates. ###= p

    Techniques Used: Expressing, Flow Cytometry, Cytometry, Transfection, Infection

    10) Product Images from "Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy"

    Article Title: Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00033

    HBHA protein and rMS-HBHA infection could induce cell death on A549. (A) A549 cells were treated with HBHA of different concentrations for 24 h, and LDH release was detected. ( *** P
    Figure Legend Snippet: HBHA protein and rMS-HBHA infection could induce cell death on A549. (A) A549 cells were treated with HBHA of different concentrations for 24 h, and LDH release was detected. ( *** P

    Techniques Used: Infection

    Schematic model of the role of HBHA during mycobacterial infection . HBHA inhibited autophagy in mycobacterial infected A549 cells, thereby promoted intracellular bacterial survival and replication. Subsequently, HBHA induced apoptosis on A549 cells through activation of caspase-3 which may facilitate bacterial escape from lung epithelial cells and dissemination to the adjacent cells.
    Figure Legend Snippet: Schematic model of the role of HBHA during mycobacterial infection . HBHA inhibited autophagy in mycobacterial infected A549 cells, thereby promoted intracellular bacterial survival and replication. Subsequently, HBHA induced apoptosis on A549 cells through activation of caspase-3 which may facilitate bacterial escape from lung epithelial cells and dissemination to the adjacent cells.

    Techniques Used: Infection, Activation Assay

    HBHA inhibited the maturation of autophagosome in A549 cells. (A) A549 cells were starved for 1 h and subsequently treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml) for 4 h. The cells were then stained with monodansylcadaverine (MDC), and MDC-labeled autophagic vacuoles were detected by confocal microscopy. Scale bars: 20 μm. (B) MDC-positive autophagic vacuoles were quantified in each group. ( *** P
    Figure Legend Snippet: HBHA inhibited the maturation of autophagosome in A549 cells. (A) A549 cells were starved for 1 h and subsequently treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml) for 4 h. The cells were then stained with monodansylcadaverine (MDC), and MDC-labeled autophagic vacuoles were detected by confocal microscopy. Scale bars: 20 μm. (B) MDC-positive autophagic vacuoles were quantified in each group. ( *** P

    Techniques Used: Staining, Labeling, Confocal Microscopy

    Recombinant Mycobacterium smegmatis ( MS ) expressing HBHA ( rMS-HBHA ) inhibited autophagy among starved A549 cells. (A) Total proteins of MC 2 155 and rMS-HBHA were extracted, and Western blot was carried out to detect HBHA using anti-HBHA antibody. (B) A549 cells were infected with the wild-type strain MC 2 155 or rMS-HBHA at the multiplicity of infection (MOI) of 10:1 for indicated time or treated with HBHA (8 μg/ml) for 90 min. The expression levels of LC3 were detected by Western blot. (C) The intensities of LC3II bands were normalized to the intensity of β-actin. ( *** P
    Figure Legend Snippet: Recombinant Mycobacterium smegmatis ( MS ) expressing HBHA ( rMS-HBHA ) inhibited autophagy among starved A549 cells. (A) Total proteins of MC 2 155 and rMS-HBHA were extracted, and Western blot was carried out to detect HBHA using anti-HBHA antibody. (B) A549 cells were infected with the wild-type strain MC 2 155 or rMS-HBHA at the multiplicity of infection (MOI) of 10:1 for indicated time or treated with HBHA (8 μg/ml) for 90 min. The expression levels of LC3 were detected by Western blot. (C) The intensities of LC3II bands were normalized to the intensity of β-actin. ( *** P

    Techniques Used: Recombinant, Mass Spectrometry, Expressing, Western Blot, Infection

    Inhibition of autophagy could promote survival of rMS-HBHA within A549 cells. (A) Growth curves of MS and rMS-HBHA were measured by OD 600 absorbance. (B) A549 cells were infected with MS or rMS-HBHA at the MOI of 10:1. The cells were lysed at 1, 10, or 18 h post-infection, and lysates were diluted and plated on agar plates to determine the number of viable intracellular bacteria. ( *** P
    Figure Legend Snippet: Inhibition of autophagy could promote survival of rMS-HBHA within A549 cells. (A) Growth curves of MS and rMS-HBHA were measured by OD 600 absorbance. (B) A549 cells were infected with MS or rMS-HBHA at the MOI of 10:1. The cells were lysed at 1, 10, or 18 h post-infection, and lysates were diluted and plated on agar plates to determine the number of viable intracellular bacteria. ( *** P

    Techniques Used: Inhibition, Mass Spectrometry, Infection

    HBHA treatment induced apoptosis on A549 cells through activation of caspase-3. (A) A549 cells were treated with HBHA (8 μg/ml) for 24 h in the presence or absence of RIPK1 inhibitor Nec-1 (30 μM), and LDH release was detected. ( *** P
    Figure Legend Snippet: HBHA treatment induced apoptosis on A549 cells through activation of caspase-3. (A) A549 cells were treated with HBHA (8 μg/ml) for 24 h in the presence or absence of RIPK1 inhibitor Nec-1 (30 μM), and LDH release was detected. ( *** P

    Techniques Used: Activation Assay

    Heparin-binding hemagglutinin (HBHA) inhibited the expression of LC3 and Beclin-1 in A549 cells. (A–D) A549 cells were starved for 1 h, and HBHA proteins of different concentrations were subsequently added to the cells for 90 min. LC3 (A) and ATG5/Beclin-1 (C) expression was detected by Western blot. The intensities of LC3II (B) and Beclin-1 (D) bands were normalized to the intensity of β-actin. ( * P
    Figure Legend Snippet: Heparin-binding hemagglutinin (HBHA) inhibited the expression of LC3 and Beclin-1 in A549 cells. (A–D) A549 cells were starved for 1 h, and HBHA proteins of different concentrations were subsequently added to the cells for 90 min. LC3 (A) and ATG5/Beclin-1 (C) expression was detected by Western blot. The intensities of LC3II (B) and Beclin-1 (D) bands were normalized to the intensity of β-actin. ( * P

    Techniques Used: Binding Assay, Expressing, Western Blot

    11) Product Images from "Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity"

    Article Title: Selective small-chemical inhibitors of protein arginine methyltransferase 5 with anti-lung cancer activity

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0181601

    Identified compounds inhibited cellular targets of PRMT5. A, The cellular permeability of identified compounds. Lung cancer A549 cells were cultured in the presence of various concentrations of compounds for 2h and the compound concentrations in the cell were determined by HPLC chromatography with a C18 column. B, The cellular stability of identified compounds. Lung cancer A549 cells were cultured in the presence of compounds (20 μM) for 2 h and then the medium was removed. Cells were washed with the medium and cultured for additional 5 or 10 h after the compounds were removed. The compound concentrations in the cell were determined by HPLC chromatography with a C18 column. C, Identified compounds inhibited arginine methylation of histones and B-Raf in A549 cells. A549 cells were grown in the presence of DMSO or compound C5, C9 or C11 (20 μM). Histones were purified and analyzed by SDS-PAGE stained with Commassie blue (lanes 1–4, bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (lanes 1–4, top). B-Raf was immunoprecipitated with anti-B-Raf antibody from whole cell lysates and submitted for Western blot analysis with anti-B-Raf (lanes 5 and 6, bottom) or anti-symmetric dimethyl arginine (lanes 5 and 6, top) antibody.
    Figure Legend Snippet: Identified compounds inhibited cellular targets of PRMT5. A, The cellular permeability of identified compounds. Lung cancer A549 cells were cultured in the presence of various concentrations of compounds for 2h and the compound concentrations in the cell were determined by HPLC chromatography with a C18 column. B, The cellular stability of identified compounds. Lung cancer A549 cells were cultured in the presence of compounds (20 μM) for 2 h and then the medium was removed. Cells were washed with the medium and cultured for additional 5 or 10 h after the compounds were removed. The compound concentrations in the cell were determined by HPLC chromatography with a C18 column. C, Identified compounds inhibited arginine methylation of histones and B-Raf in A549 cells. A549 cells were grown in the presence of DMSO or compound C5, C9 or C11 (20 μM). Histones were purified and analyzed by SDS-PAGE stained with Commassie blue (lanes 1–4, bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (lanes 1–4, top). B-Raf was immunoprecipitated with anti-B-Raf antibody from whole cell lysates and submitted for Western blot analysis with anti-B-Raf (lanes 5 and 6, bottom) or anti-symmetric dimethyl arginine (lanes 5 and 6, top) antibody.

    Techniques Used: Permeability, Cell Culture, High Performance Liquid Chromatography, Chromatography, Methylation, Purification, SDS Page, Staining, Western Blot, Immunoprecipitation

    The compound C9a inhibited growth of lung tumor xenografts in nude mice. A, Lungs derived from mice injected with A549 cells treated with the vehicle or C9a at the dosage of 100 mg/kg for 21 days. B, Mean size of tumors. C, The C9a treatment inhibited histone 2A (H2A) arginine methylation in lung tumor xenografts. Histones were isolated from tumors and analyzed by SDS-PAGE stained with Commassie blue R250 (bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (top).
    Figure Legend Snippet: The compound C9a inhibited growth of lung tumor xenografts in nude mice. A, Lungs derived from mice injected with A549 cells treated with the vehicle or C9a at the dosage of 100 mg/kg for 21 days. B, Mean size of tumors. C, The C9a treatment inhibited histone 2A (H2A) arginine methylation in lung tumor xenografts. Histones were isolated from tumors and analyzed by SDS-PAGE stained with Commassie blue R250 (bottom) or by Western blot with anti-symmetric dimethyl arginine antibody (SYM10) (top).

    Techniques Used: Mouse Assay, Derivative Assay, Injection, Methylation, Isolation, SDS Page, Staining, Western Blot

    Identified compounds inhibited growth of lung cancer cells. A, Lung cancer A549 cells were cultured in the presence of various concentrations (μM) of identified compounds and growth inhibition was determined by cell counting at various time points. B, Lung cancer PC14 cells were cultured in the presence of DMSO or various concentrations of identified compounds and growth inhibition was determined by cell counting 2 days post the compound treatment. C, The serum concentrations of compound C9 after oral administration. Mice were sacrificed and blood samples were collected immediately before (0 h) and at 2, 4, 6 and 8h after compound administration.
    Figure Legend Snippet: Identified compounds inhibited growth of lung cancer cells. A, Lung cancer A549 cells were cultured in the presence of various concentrations (μM) of identified compounds and growth inhibition was determined by cell counting at various time points. B, Lung cancer PC14 cells were cultured in the presence of DMSO or various concentrations of identified compounds and growth inhibition was determined by cell counting 2 days post the compound treatment. C, The serum concentrations of compound C9 after oral administration. Mice were sacrificed and blood samples were collected immediately before (0 h) and at 2, 4, 6 and 8h after compound administration.

    Techniques Used: Cell Culture, Inhibition, Cell Counting, Mouse Assay

    The C9 analogue has improved PRMT5-inhibitory efficacy and oral drug availability. A, The chemical structure of the C9 analogue. B, Compound C9a inhibited methylation of SmD3 by PRMT5. The recombinant SmD3 (2 μg) was used as the substrate in the absence (lane 5) or presence of 1, 10 or 100 μM compounds as indicated. Lane 1 shows the product of the reaction without the enzyme. C, C9a has improved efficacy to inhibit lung cancer growth. A549 cells were cultured in the presence of various concentrations of C9 or C9a and growth inhibition was determined by cell counting 2 days post the compound treatment. D, C9a has improved oral drug availability in the mouse. Male BALB/c mice were randomized and administrated C9a by oral gavage and compound concentrations in blood samples were determined.
    Figure Legend Snippet: The C9 analogue has improved PRMT5-inhibitory efficacy and oral drug availability. A, The chemical structure of the C9 analogue. B, Compound C9a inhibited methylation of SmD3 by PRMT5. The recombinant SmD3 (2 μg) was used as the substrate in the absence (lane 5) or presence of 1, 10 or 100 μM compounds as indicated. Lane 1 shows the product of the reaction without the enzyme. C, C9a has improved efficacy to inhibit lung cancer growth. A549 cells were cultured in the presence of various concentrations of C9 or C9a and growth inhibition was determined by cell counting 2 days post the compound treatment. D, C9a has improved oral drug availability in the mouse. Male BALB/c mice were randomized and administrated C9a by oral gavage and compound concentrations in blood samples were determined.

    Techniques Used: Methylation, Recombinant, Cell Culture, Inhibition, Cell Counting, Mouse Assay

    12) Product Images from "Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions"

    Article Title: Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67488-3

    Effects of iNOS and endocytosis inhibitors on indium-induced 8-nitroG formation. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. The cells were co-treated with 1400 W, Bay, MBCD, MDC and CytoD and 8-nitroG formation was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitive image analysis for the effects of iNOS and endocytosis inhibitors on indium-exposed A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of iNOS and endocytosis inhibitors on indium-induced 8-nitroG formation. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. The cells were co-treated with 1400 W, Bay, MBCD, MDC and CytoD and 8-nitroG formation was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitive image analysis for the effects of iNOS and endocytosis inhibitors on indium-exposed A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Immunocytochemistry, Staining, Software

    8-NitroG formation in indium-treated cells. A549 cells were incubated with the indicated concentrations of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. Positive control was prepared by incubating A549 cells with culture supernatant of MNCNT-exposed cells as described in “ Methods ” section. 8-NitroG formation was detected by immunocytochemistry as described in “ Methods ” section. ( A ) Fluorescent images of indium-induced 8-nitroG formation in A549 cells. The red fluorescence shows 8-nitroG formation and the blue fluorescence shows the nucleus stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis for indium-induced 8-nitroG formation in A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 4–8 independent experiments. * p
    Figure Legend Snippet: 8-NitroG formation in indium-treated cells. A549 cells were incubated with the indicated concentrations of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. Positive control was prepared by incubating A549 cells with culture supernatant of MNCNT-exposed cells as described in “ Methods ” section. 8-NitroG formation was detected by immunocytochemistry as described in “ Methods ” section. ( A ) Fluorescent images of indium-induced 8-nitroG formation in A549 cells. The red fluorescence shows 8-nitroG formation and the blue fluorescence shows the nucleus stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis for indium-induced 8-nitroG formation in A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 4–8 independent experiments. * p

    Techniques Used: Incubation, Positive Control, Immunocytochemistry, Fluorescence, Staining, Software

    Proposed mechanism of indium-induced DNA damage in A549 cells.
    Figure Legend Snippet: Proposed mechanism of indium-induced DNA damage in A549 cells.

    Techniques Used:

    Time course of 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of indium-treated A549 cells at different incubation times. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 at 37 °C for indicated durations. 8-NitroG was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis of 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control at 2 h was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. * p
    Figure Legend Snippet: Time course of 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of indium-treated A549 cells at different incubation times. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 at 37 °C for indicated durations. 8-NitroG was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis of 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control at 2 h was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. * p

    Techniques Used: Incubation, Immunocytochemistry, Staining, Software

    Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Expressing, Transfection, Western Blot, Negative Control, Immunocytochemistry, Staining, Software

    Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Immunocytochemistry, Staining, Software

    13) Product Images from "microRNA-802/Rnd3 pathway imposes on carcinogenesis and metastasis of fine particulate matter exposure"

    Article Title: microRNA-802/Rnd3 pathway imposes on carcinogenesis and metastasis of fine particulate matter exposure

    Journal: Oncotarget

    doi: 10.18632/oncotarget.9019

    Long-term PM 2.5 exposure regulates in vitro and in vivo A549 cell carcinogenesis and metastases A. Cell migration and invasion capacity. B. Colony formation ability of A549 cells was significantly increased following long-term PM 2.5 exposure. C. Flank tumor and lung metastatic burden measured by luciferase activity. D. miR-802 expression levels were down regulated. E. Rnd3 expression was enhanced in A549 cells and flank tumor after long-term PM 2.5 treatment. * P
    Figure Legend Snippet: Long-term PM 2.5 exposure regulates in vitro and in vivo A549 cell carcinogenesis and metastases A. Cell migration and invasion capacity. B. Colony formation ability of A549 cells was significantly increased following long-term PM 2.5 exposure. C. Flank tumor and lung metastatic burden measured by luciferase activity. D. miR-802 expression levels were down regulated. E. Rnd3 expression was enhanced in A549 cells and flank tumor after long-term PM 2.5 treatment. * P

    Techniques Used: In Vitro, In Vivo, Migration, Luciferase, Activity Assay, Expressing

    Functional analysis of the potential targets and validation of the significantly modulated miRNAs A. The GO enrichment analysis of biological processes showed that most categories were involved in actin-dependent processes and apoptosis. B. Nine genes and their related miRNAs were involved in BP enrichment. C. The miRNA expression levels were validated by qRT-PCR in A549 cells. The expression of miR-1469, −1322, and −802 were modulated in a dose-dependent manner. * P
    Figure Legend Snippet: Functional analysis of the potential targets and validation of the significantly modulated miRNAs A. The GO enrichment analysis of biological processes showed that most categories were involved in actin-dependent processes and apoptosis. B. Nine genes and their related miRNAs were involved in BP enrichment. C. The miRNA expression levels were validated by qRT-PCR in A549 cells. The expression of miR-1469, −1322, and −802 were modulated in a dose-dependent manner. * P

    Techniques Used: Functional Assay, Expressing, Quantitative RT-PCR

    Cell-based assays confirmed the regulation to cellular phenotypes by miR-802 A. Immunodetection of F-actin expression in A549 cells. B. PM 2.5 treatment induced A549 cell actin cytoskeletal reorganization and miR-802 mimic partially rescued actin reorganization. C and D. PM 2.5 significantly increased the proportion of cells that underwent apoptosis and cell death, and the miR-802 mimic partially protected the cells against apoptosis and fully rescued the cell death phenotype in A549 cells. * P
    Figure Legend Snippet: Cell-based assays confirmed the regulation to cellular phenotypes by miR-802 A. Immunodetection of F-actin expression in A549 cells. B. PM 2.5 treatment induced A549 cell actin cytoskeletal reorganization and miR-802 mimic partially rescued actin reorganization. C and D. PM 2.5 significantly increased the proportion of cells that underwent apoptosis and cell death, and the miR-802 mimic partially protected the cells against apoptosis and fully rescued the cell death phenotype in A549 cells. * P

    Techniques Used: Immunodetection, Expressing

    The regulation of miR-802 to Rnd2, LIMCH1, CALD1, and the associated cellular phenotype A. The expression of Rnd3 and LIMCH1 increased significantly after the transfection of miR-802 inhibitor. B. mRNA expression level of miR-802 target gene increased in A549 cells following PM 2.5 treatment; after miR-802 mimic transfection, expression of Rnd3 and C. LIMCH1 attenuated to control levels. D. The expression of CALD1 was not affected by miR-802 mimic. E. miR-802 target Rnd3 and LIMCH1 were knocked down in A549 cells, and their protein expression levels were not affected by PM 2.5 exposure. F. After knocking down of Rnd3 or LIMCH1 in A549 cells, actin organization was not regulated by PM 2.5 treatment. * P
    Figure Legend Snippet: The regulation of miR-802 to Rnd2, LIMCH1, CALD1, and the associated cellular phenotype A. The expression of Rnd3 and LIMCH1 increased significantly after the transfection of miR-802 inhibitor. B. mRNA expression level of miR-802 target gene increased in A549 cells following PM 2.5 treatment; after miR-802 mimic transfection, expression of Rnd3 and C. LIMCH1 attenuated to control levels. D. The expression of CALD1 was not affected by miR-802 mimic. E. miR-802 target Rnd3 and LIMCH1 were knocked down in A549 cells, and their protein expression levels were not affected by PM 2.5 exposure. F. After knocking down of Rnd3 or LIMCH1 in A549 cells, actin organization was not regulated by PM 2.5 treatment. * P

    Techniques Used: Expressing, Transfection

    14) Product Images from "Potential Combinational Anti-Cancer Therapy in Non-Small Cell Lung Cancer with Traditional Chinese Medicine Sun-Bai-Pi Extract and Cisplatin"

    Article Title: Potential Combinational Anti-Cancer Therapy in Non-Small Cell Lung Cancer with Traditional Chinese Medicine Sun-Bai-Pi Extract and Cisplatin

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155469

    SBPE induced cancer cell autophagy. SBPE 5 mg/mL (a) and 10 mg/mL (b) were respectively added the cells and incubated for 2, 4, 6, 12, and 24 hours. Immunostaining was used to label the autophagy protein LC3 (green), LAMP-1 (red), and DAPI (blue) cancer cells. Magnification was 400X. (c) Magnified the cells incubated for six hours at 630X to observe the autophagy distribution. (d) Overlay of the green fluorescence (LC3) and the red fluorescence (LAMP-1), and calculated the number of cells with autophagic lysosomes. The vertical axis represents the total cell number per unit area divided by the number of cells with autophagic lysosomes. (e) Western blots were used to observe the effects SBPE had on the regulation of A549 cell autophagy related proteins, and found that the SBPE induced cell autophagy, which increased over time and peaked at eight hours. All the gels have been run under the same experimental conditions. (f) Statistical analyses for proteins performances.
    Figure Legend Snippet: SBPE induced cancer cell autophagy. SBPE 5 mg/mL (a) and 10 mg/mL (b) were respectively added the cells and incubated for 2, 4, 6, 12, and 24 hours. Immunostaining was used to label the autophagy protein LC3 (green), LAMP-1 (red), and DAPI (blue) cancer cells. Magnification was 400X. (c) Magnified the cells incubated for six hours at 630X to observe the autophagy distribution. (d) Overlay of the green fluorescence (LC3) and the red fluorescence (LAMP-1), and calculated the number of cells with autophagic lysosomes. The vertical axis represents the total cell number per unit area divided by the number of cells with autophagic lysosomes. (e) Western blots were used to observe the effects SBPE had on the regulation of A549 cell autophagy related proteins, and found that the SBPE induced cell autophagy, which increased over time and peaked at eight hours. All the gels have been run under the same experimental conditions. (f) Statistical analyses for proteins performances.

    Techniques Used: Incubation, Immunostaining, Fluorescence, Western Blot

    Cisplatin and SBPE combination therapy can reduce cancer cell survival rates. A549 tumor cell survival rate analyses after 1 μM, 5 μM, 10 μM, and 25 μM of Cisplatin was administered and incubated for 24 and 48 hours. The MTS reagent was used to detect the survival rates. Pre-treatment with using SBPE 5 mg/mL (a) and SBPE 10 mg/mL (b) doses of Cisplatin was added and incubated for 24 and 48 hours. The MTS reagent was used to detect the survival rates.
    Figure Legend Snippet: Cisplatin and SBPE combination therapy can reduce cancer cell survival rates. A549 tumor cell survival rate analyses after 1 μM, 5 μM, 10 μM, and 25 μM of Cisplatin was administered and incubated for 24 and 48 hours. The MTS reagent was used to detect the survival rates. Pre-treatment with using SBPE 5 mg/mL (a) and SBPE 10 mg/mL (b) doses of Cisplatin was added and incubated for 24 and 48 hours. The MTS reagent was used to detect the survival rates.

    Techniques Used: Incubation

    SBPE induced apoptosis. (a) After administration of SBPE 10 mg/mL; the Caspase3/7 reagent was used to detect their performances. (b) The effects that SBPE has on the protein control factors related to A549 cell apoptosis and anti-apoptosis. All the gels have been run under the same experimental conditions. (c) Statistical chart for protein performance. (d) FACS analyzes for cell cycle distribution, and use of the ModFit LT software to analyze cell cycle distribution.
    Figure Legend Snippet: SBPE induced apoptosis. (a) After administration of SBPE 10 mg/mL; the Caspase3/7 reagent was used to detect their performances. (b) The effects that SBPE has on the protein control factors related to A549 cell apoptosis and anti-apoptosis. All the gels have been run under the same experimental conditions. (c) Statistical chart for protein performance. (d) FACS analyzes for cell cycle distribution, and use of the ModFit LT software to analyze cell cycle distribution.

    Techniques Used: FACS, Software

    Cell viability. (a) Performed cell viability tests by administering different concentrations of SBPE to WI38 and A549 cells. (b) Administered 5 mg/mL and 10 mg/mL of SBPE to A549 and incubated the cells for various time points. Then, we used the MTS reagent to test their viabilities.
    Figure Legend Snippet: Cell viability. (a) Performed cell viability tests by administering different concentrations of SBPE to WI38 and A549 cells. (b) Administered 5 mg/mL and 10 mg/mL of SBPE to A549 and incubated the cells for various time points. Then, we used the MTS reagent to test their viabilities.

    Techniques Used: Incubation

    The effects that combining SBPE to chemotherapy drugs Gemcitabine and Paclitaxel have on cancer cell viability. (a) A549 tumor cell viability analysis after the administration of Gemcitabine. After 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM of Gemcitabine was administered for 24 and 48 hours, the MTS reagent was used to detect their activities. Secondly, after 6 hours of treatment with 10 mg/mL of SBPE; Gemcitabine was added for 24 hours (b), 48 hours (c) and 72 hours (d). (e) A549 tumor cell viability analysis after treatment with 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM of Paclitaxel and incubated for 24 and 48 hours. The MTS reagent was used to detect their activities. Secondly, after 6 hours of treatment with 10 mg/mL of SBPE; Paclitaxel treatment was added for 24 hours (f), 48 hours (g) and 72 hours (h).
    Figure Legend Snippet: The effects that combining SBPE to chemotherapy drugs Gemcitabine and Paclitaxel have on cancer cell viability. (a) A549 tumor cell viability analysis after the administration of Gemcitabine. After 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM of Gemcitabine was administered for 24 and 48 hours, the MTS reagent was used to detect their activities. Secondly, after 6 hours of treatment with 10 mg/mL of SBPE; Gemcitabine was added for 24 hours (b), 48 hours (c) and 72 hours (d). (e) A549 tumor cell viability analysis after treatment with 1 μM, 5 μM, 10 μM, 50 μM, and 100 μM of Paclitaxel and incubated for 24 and 48 hours. The MTS reagent was used to detect their activities. Secondly, after 6 hours of treatment with 10 mg/mL of SBPE; Paclitaxel treatment was added for 24 hours (f), 48 hours (g) and 72 hours (h).

    Techniques Used: Incubation

    15) Product Images from "Neuregulin1 acts as a suppressor in human lung adenocarcinoma via AKT and ERK1/2 pathway"

    Article Title: Neuregulin1 acts as a suppressor in human lung adenocarcinoma via AKT and ERK1/2 pathway

    Journal: Journal of Thoracic Disease

    doi: 10.21037/jtd.2018.05.175

    Detection of cell transfection efficacy. (A) The transfection of fluorescent siRNA into cells was detected by immunofluorescence; (B) the expression of NRG1 was detected by gel electrophoresis assay after siRNA was transfected into the cells; (C) the transfection efficacy was detected by qRT-PCR in A549 cells; (D) the transfection efficacy was detected by qRT-PCR in H1975 cells. The transfection efficiency was 70.3% in A549 and 73.7% in H1975 cells. The two transfections were effective. NRG1, neuregulin1; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; NC, non-specific control.
    Figure Legend Snippet: Detection of cell transfection efficacy. (A) The transfection of fluorescent siRNA into cells was detected by immunofluorescence; (B) the expression of NRG1 was detected by gel electrophoresis assay after siRNA was transfected into the cells; (C) the transfection efficacy was detected by qRT-PCR in A549 cells; (D) the transfection efficacy was detected by qRT-PCR in H1975 cells. The transfection efficiency was 70.3% in A549 and 73.7% in H1975 cells. The two transfections were effective. NRG1, neuregulin1; siRNA, small interfering RNA; qRT-PCR, quantitative real-time polymerase chain reaction; NC, non-specific control.

    Techniques Used: Transfection, Immunofluorescence, Expressing, Nucleic Acid Electrophoresis, Quantitative RT-PCR, Small Interfering RNA, Real-time Polymerase Chain Reaction

    Effect of NRG1 on the cell invasion ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the cell invasion of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the cell invasion of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell invasion of A549 cells; (D) the effect of downregulated NRG1 expression on the cell invasion of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell invasion (P
    Figure Legend Snippet: Effect of NRG1 on the cell invasion ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the cell invasion of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the cell invasion of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell invasion of A549 cells; (D) the effect of downregulated NRG1 expression on the cell invasion of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell invasion (P

    Techniques Used: Expressing

    Effects of NRG1 on the cell cycle of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell cycle of A549 cells; (B) the effect of exposure to Re-NRG1 on the cell cycle of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell cycle of A549 cells; (D) the effect of downregulated NRG1 expression on the cell cycle of H1975 cells. These results showed that the upregulated NRG1 expression did not affect the cell cycle of these two cell lines (P=0.753, 0.599). Moreover, the downregulated NRG1 expression by gene knockdown did not alter the cell cycle of A549 and H1975 cells (P=0.532, 0.500). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.
    Figure Legend Snippet: Effects of NRG1 on the cell cycle of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell cycle of A549 cells; (B) the effect of exposure to Re-NRG1 on the cell cycle of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell cycle of A549 cells; (D) the effect of downregulated NRG1 expression on the cell cycle of H1975 cells. These results showed that the upregulated NRG1 expression did not affect the cell cycle of these two cell lines (P=0.753, 0.599). Moreover, the downregulated NRG1 expression by gene knockdown did not alter the cell cycle of A549 and H1975 cells (P=0.532, 0.500). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.

    Techniques Used: Expressing, Small Interfering RNA

    Effects of NRG1 on the proliferation of A549 and H1975 cells. (A) The effects of seven different Re-NRG1 concentrations on the proliferation of A549 cell line at four time points; (B) the effect of exogenous NRG1 on the proliferation of H1975 cells; (C) the effect of NRG1 gene knockdown on the proliferation of A549 cells; (D) the effect of NRG1 gene knockdown on the proliferation of H1975 cells. Furthermore, NRG1 played an inhibitory role in the proliferation of A549 and H1975 cells at same doses when reacted for 72 h (P
    Figure Legend Snippet: Effects of NRG1 on the proliferation of A549 and H1975 cells. (A) The effects of seven different Re-NRG1 concentrations on the proliferation of A549 cell line at four time points; (B) the effect of exogenous NRG1 on the proliferation of H1975 cells; (C) the effect of NRG1 gene knockdown on the proliferation of A549 cells; (D) the effect of NRG1 gene knockdown on the proliferation of H1975 cells. Furthermore, NRG1 played an inhibitory role in the proliferation of A549 and H1975 cells at same doses when reacted for 72 h (P

    Techniques Used:

    Effects of NRG1 on the cell apoptosis status of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell apoptosis of A549 cells; (B) the effect of exposure in Re-NRG1 on the cell apoptosis of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell apoptosis of A549 cells; (D) the effect of downregulated NRG1 expression on the cell apoptosis in H1975 cells. The upregulated NRG1 expression did not affect the cell apoptosis of these two cell lines (P=0.753, 0.858). Moreover, the downregulated NRG1 expression by knockdown of the gene could not alter the cell apoptosis status of A549 and H1975 cells (P=0.831, 0.332). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.
    Figure Legend Snippet: Effects of NRG1 on the cell apoptosis status of A549 and H1975 cells. (A) The effect of upregulated NRG1 expression on the cell apoptosis of A549 cells; (B) the effect of exposure in Re-NRG1 on the cell apoptosis of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell apoptosis of A549 cells; (D) the effect of downregulated NRG1 expression on the cell apoptosis in H1975 cells. The upregulated NRG1 expression did not affect the cell apoptosis of these two cell lines (P=0.753, 0.858). Moreover, the downregulated NRG1 expression by knockdown of the gene could not alter the cell apoptosis status of A549 and H1975 cells (P=0.831, 0.332). NRG1, neuregulin1; siRNA, small interfering RNA; NC, non-specific control.

    Techniques Used: Expressing, Small Interfering RNA

    Effects of NRG1 on the cell migration ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the migration of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the migration of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell migration of A549 cells; (D) the effect of downregulated NRG1 expression on the cell migration of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell migration (P
    Figure Legend Snippet: Effects of NRG1 on the cell migration ability of A549 and H1975 cells. (A) The role of different doses of exogenous Re-NRG1 on the migration of A549 cells; (B) the role of different doses of exogenous Re-NRG1 on the migration of H1975 cells; (C) the effect of downregulated NRG1 expression on the cell migration of A549 cells; (D) the effect of downregulated NRG1 expression on the cell migration of H1975 cells. These effects displayed an inhibitory effect of NRG1 on the cell migration (P

    Techniques Used: Migration, Expressing

    16) Product Images from "Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection"

    Article Title: Leukemia inhibitory factor protects the lung during respiratory syncytial viral infection

    Journal: BMC Immunology

    doi: 10.1186/s12865-014-0041-4

    RSV induces multiple pathogen recognition receptors in A549 and SAE cells. (A) Representative immunofluorescence staining of SAE and A549 cells treated with mock or RSV and stained 24 hours later with RSV F-specific monoclonal antibody. Cell lysates were examined for viable viruses by plaque assays. (B) qPCR and (C) immunblots were performed to examine levels of pathogen recognition receptors and downstream signaling in cells 1-day post RSV infection. Graphs are represented as (B) PFU (Log 10 ) or (C) RQ of the mean ± S.E.M, where n = 10 replicates/group and each assay was performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to mock treated mice.
    Figure Legend Snippet: RSV induces multiple pathogen recognition receptors in A549 and SAE cells. (A) Representative immunofluorescence staining of SAE and A549 cells treated with mock or RSV and stained 24 hours later with RSV F-specific monoclonal antibody. Cell lysates were examined for viable viruses by plaque assays. (B) qPCR and (C) immunblots were performed to examine levels of pathogen recognition receptors and downstream signaling in cells 1-day post RSV infection. Graphs are represented as (B) PFU (Log 10 ) or (C) RQ of the mean ± S.E.M, where n = 10 replicates/group and each assay was performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to mock treated mice.

    Techniques Used: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Infection, Mouse Assay

    Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.
    Figure Legend Snippet: Silencing RIG-I subdues RSV induced cytokine release from A549 cells. (A-B) Multiplex or ELISA analysis was performed on secreted cytokines from A549 cells transfected with siRNA targeting RIG-I (DDX58), LGP2 (DHX58), MDA5 (IFIH1), Trif or a negative sequence control. Graphs are represented as mean cytokine concentration ± S.E.M, where n = 10 replicates/group and each assay were performed in triplicate. Each experiment was performed on samples obtained from 3 experiments from separate days. *Represents a p value less than 0.05 compared to control siRNA treated cells following RSV infection.

    Techniques Used: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Transfection, Sequencing, Concentration Assay, Infection

    RSV replication induces host cytokines production in airway epithelial cells. A549 cells were treated with mock, RSV or UV-inactivated RSV (UV-RSV) for 24 hours. (A-B) Secreted cytokines were analyzed by multiplex or ELISA. Graphs are represented as mean cytokine concentration (pg/ml) ± S.E.M, with each measurement performed 3 times on 6 replicates/group. Samples were collected from 3 experiments performed on separate days. * and ** represent a p value less than 0.05 compared to mock or RSV treated cells for each cytokine target, respectively.
    Figure Legend Snippet: RSV replication induces host cytokines production in airway epithelial cells. A549 cells were treated with mock, RSV or UV-inactivated RSV (UV-RSV) for 24 hours. (A-B) Secreted cytokines were analyzed by multiplex or ELISA. Graphs are represented as mean cytokine concentration (pg/ml) ± S.E.M, with each measurement performed 3 times on 6 replicates/group. Samples were collected from 3 experiments performed on separate days. * and ** represent a p value less than 0.05 compared to mock or RSV treated cells for each cytokine target, respectively.

    Techniques Used: Multiplex Assay, Enzyme-linked Immunosorbent Assay, Concentration Assay

    17) Product Images from "Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response"

    Article Title: Cationic nanocarriers induce cell necrosis through impairment of Na+/K+-ATPase and cause subsequent inflammatory response

    Journal: Cell Research

    doi: 10.1038/cr.2015.9

    Cell necrosis induced by cationic carriers involves impairment of Na + /K + -ATPase activity. (A) Representative images of A549 cells after the addition of DOTAP liposomes (50 μg/ml). Cells were loaded with fluorescence sodium indicator CoroNa Green and stained with PI. Scale bar, 20 μm. (B) The fluorescence intensities of CoroNa Green in one field were recorded and analyzed using ImageJ software. (C) A549 cells were cultured in medium with or without sodium for 1 h and then treated with cationic carriers for 10 min. DOTAP liposome (50 μg/ml); PEI (10 μg/ml); Chitosan (10 μg/ml). (D) A549 cells were pretreated with inhibitors including ouabain (2 μM), eosin (5 μM), Gd 3+ (20 μM), NiCl 2 (1 mM), LaCl 3 (0.1 mM) or 2-APB (50 μM) for 30 min and DOTAP liposomes (100 μg/ml) were added. Necrotic cells were detected by flow cytometry with PI staining in 10 min. (E , F) Na + /K + -ATPase activity in cultured cells and in tissues treated with cationic carriers. A549 cells were treated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (NeutralL, 50 μg/ml) or Anionic liposomes (AnionicL, 50 μg/ml) for 5 min and heavy-membrane fraction of the cells were used for Na + /K + -ATPase activity assay (E) . Mice were injected with DOTAP liposome (25 mg/kg), PEI (5 mg/kg), Chitosan (25 mg/kg), Neutral liposomes (25 mg/kg) or Anionic liposomes (25 mg/kg) and 20 min later homogenates of lungs were prepared for Na + /K + -ATPase activity assay (F) . (G) Uptake of 86 Rb + in A549 cells treated with cationic nanocarriers. A549 cells were incubated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (50 μg/ml) or Anionic liposomes (50 μg/ml) for 2 min and treatment of 5 μM ouabain was used as positive control. n = 3. (H) Mice were pretreated with or without ouabain (5 μg/mice) for 10 min and subsequently injected with DOTAP liposomes (100 mg/kg) through tail veins every 24 h for two days and mouse survival were recorded every 24 h, n = 10. (I) Complex structures were calculated. (a) for Na + /K + -ATPase-DOTAP and (b) for Na + /K + -ATPase-ouabain/DOTAP. (J) Control-shRNA, Na + /K + -ATPase-shRNA (ATP1A1-shRNA) and TRPM7-shRNA transfected A549 cells were treated with DOTAP liposomes (50 μg/ml) for 5 min before analysis by flow cytometry. Data are mean ± SEM; n = 3. ** P
    Figure Legend Snippet: Cell necrosis induced by cationic carriers involves impairment of Na + /K + -ATPase activity. (A) Representative images of A549 cells after the addition of DOTAP liposomes (50 μg/ml). Cells were loaded with fluorescence sodium indicator CoroNa Green and stained with PI. Scale bar, 20 μm. (B) The fluorescence intensities of CoroNa Green in one field were recorded and analyzed using ImageJ software. (C) A549 cells were cultured in medium with or without sodium for 1 h and then treated with cationic carriers for 10 min. DOTAP liposome (50 μg/ml); PEI (10 μg/ml); Chitosan (10 μg/ml). (D) A549 cells were pretreated with inhibitors including ouabain (2 μM), eosin (5 μM), Gd 3+ (20 μM), NiCl 2 (1 mM), LaCl 3 (0.1 mM) or 2-APB (50 μM) for 30 min and DOTAP liposomes (100 μg/ml) were added. Necrotic cells were detected by flow cytometry with PI staining in 10 min. (E , F) Na + /K + -ATPase activity in cultured cells and in tissues treated with cationic carriers. A549 cells were treated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (NeutralL, 50 μg/ml) or Anionic liposomes (AnionicL, 50 μg/ml) for 5 min and heavy-membrane fraction of the cells were used for Na + /K + -ATPase activity assay (E) . Mice were injected with DOTAP liposome (25 mg/kg), PEI (5 mg/kg), Chitosan (25 mg/kg), Neutral liposomes (25 mg/kg) or Anionic liposomes (25 mg/kg) and 20 min later homogenates of lungs were prepared for Na + /K + -ATPase activity assay (F) . (G) Uptake of 86 Rb + in A549 cells treated with cationic nanocarriers. A549 cells were incubated with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), Chitosan (50 μg/ml), Neutral liposomes (50 μg/ml) or Anionic liposomes (50 μg/ml) for 2 min and treatment of 5 μM ouabain was used as positive control. n = 3. (H) Mice were pretreated with or without ouabain (5 μg/mice) for 10 min and subsequently injected with DOTAP liposomes (100 mg/kg) through tail veins every 24 h for two days and mouse survival were recorded every 24 h, n = 10. (I) Complex structures were calculated. (a) for Na + /K + -ATPase-DOTAP and (b) for Na + /K + -ATPase-ouabain/DOTAP. (J) Control-shRNA, Na + /K + -ATPase-shRNA (ATP1A1-shRNA) and TRPM7-shRNA transfected A549 cells were treated with DOTAP liposomes (50 μg/ml) for 5 min before analysis by flow cytometry. Data are mean ± SEM; n = 3. ** P

    Techniques Used: Activity Assay, Fluorescence, Staining, Software, Cell Culture, Flow Cytometry, Cytometry, Mouse Assay, Injection, Incubation, Positive Control, shRNA, Transfection

    Cell depolarization and cell necrosis triggered by cationic carriers in a positive charge-dependent way. (A) A549 cell depolarization caused by cationic particles was determined by diBA-C4-(3) after 1 min of treatment. Heparin (400 μg/ml) was added to neutralize the cationic charge. (B) Percentages of necrotic cells after incubation of A549 cells with liposomes of various zeta-potentials. (C) Cationic charge was neutralized by BSA with concentrations of 40, 80, 160, 640 μg/ml. Data are mean ± SEM; n = 3. * P
    Figure Legend Snippet: Cell depolarization and cell necrosis triggered by cationic carriers in a positive charge-dependent way. (A) A549 cell depolarization caused by cationic particles was determined by diBA-C4-(3) after 1 min of treatment. Heparin (400 μg/ml) was added to neutralize the cationic charge. (B) Percentages of necrotic cells after incubation of A549 cells with liposomes of various zeta-potentials. (C) Cationic charge was neutralized by BSA with concentrations of 40, 80, 160, 640 μg/ml. Data are mean ± SEM; n = 3. * P

    Techniques Used: Dot Immunobinding, Incubation

    Acute cell necrosis induced by cationic carriers in vivo and in vitro . (A) The detection of propidium iodide (PI)-positive necrotic cells in mouse lungs. Various particles were injected through tail vein of mouse, 2 h later, PI and 4% formaldehyde were perfused through tail vein for the detection of the necrotic cells. (B) A representative experiment of the detection of the necrotic cells induced by the injection of cationic liposomes in vivo by flow cytometry with Annexin-V and PI staining. C57BL/6 mice were injected with DOTAP liposomes (25 mg/kg). Necrotic cells in BAL fluid were detected 4 h after injection by flow cytometry with Annexin-V and PI staining. n = 3/group. (C) The morphological change of the cells treated with various nanocarriers in vitro . Cells were treated in vitro with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), chitosan (50 μg/ml), anionic or neutral liposomes (abbreviated as AnionicL and NeutralL, 50 μg/ml) for 30 min. Cells were subjected to inverted microscope observation. (D) The detection of the necrotic cells induced in vitro by flow cytometry with Annexin-V and PI staining. Primary lung cells of C57BL/6 mice (left) and A549 cells (right) were treated with cationic carriers for 10 min. Percentages of necrotic cells in PI-positive region are shown. (E) A representative experiment of immunofluorescense of Cathepsin-B (green) and Caspase-3. A549 cells were treated with DOTAP liposomes (20 μg/ml) for 30 min. Diffused cytoplasmic cathepsin-B immunoreactivity was evident after the treatment of DOTAP liposome. In contrast, the activation of Caspase-3 was observed after 24 h of treatment. (F) A549 cells were treated with DOTAP liposomes, and intracellular Ca 2+ concentration and ROS levels were detected with Fluo-3/AM and H 2 DCF-DA by flow cytometry, respectively. Data are mean ± SEM; n = 3. ** P
    Figure Legend Snippet: Acute cell necrosis induced by cationic carriers in vivo and in vitro . (A) The detection of propidium iodide (PI)-positive necrotic cells in mouse lungs. Various particles were injected through tail vein of mouse, 2 h later, PI and 4% formaldehyde were perfused through tail vein for the detection of the necrotic cells. (B) A representative experiment of the detection of the necrotic cells induced by the injection of cationic liposomes in vivo by flow cytometry with Annexin-V and PI staining. C57BL/6 mice were injected with DOTAP liposomes (25 mg/kg). Necrotic cells in BAL fluid were detected 4 h after injection by flow cytometry with Annexin-V and PI staining. n = 3/group. (C) The morphological change of the cells treated with various nanocarriers in vitro . Cells were treated in vitro with DOTAP liposome (50 μg/ml), PEI (10 μg/ml), chitosan (50 μg/ml), anionic or neutral liposomes (abbreviated as AnionicL and NeutralL, 50 μg/ml) for 30 min. Cells were subjected to inverted microscope observation. (D) The detection of the necrotic cells induced in vitro by flow cytometry with Annexin-V and PI staining. Primary lung cells of C57BL/6 mice (left) and A549 cells (right) were treated with cationic carriers for 10 min. Percentages of necrotic cells in PI-positive region are shown. (E) A representative experiment of immunofluorescense of Cathepsin-B (green) and Caspase-3. A549 cells were treated with DOTAP liposomes (20 μg/ml) for 30 min. Diffused cytoplasmic cathepsin-B immunoreactivity was evident after the treatment of DOTAP liposome. In contrast, the activation of Caspase-3 was observed after 24 h of treatment. (F) A549 cells were treated with DOTAP liposomes, and intracellular Ca 2+ concentration and ROS levels were detected with Fluo-3/AM and H 2 DCF-DA by flow cytometry, respectively. Data are mean ± SEM; n = 3. ** P

    Techniques Used: In Vivo, In Vitro, Injection, Flow Cytometry, Cytometry, Staining, Mouse Assay, Inverted Microscopy, Activation Assay, Concentration Assay

    18) Product Images from "In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549"

    Article Title: In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549

    Journal: Radiology and Oncology

    doi: 10.2478/raon-2018-0014

    Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.
    Figure Legend Snippet: Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.

    Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Selection, Fluorescence, FACS, Isolation

    19) Product Images from "S100A6/miR193a regulates the proliferation, invasion, migration and angiogenesis of lung cancer cells through the P53 acetylation"

    Article Title: S100A6/miR193a regulates the proliferation, invasion, migration and angiogenesis of lung cancer cells through the P53 acetylation

    Journal: American Journal of Translational Research

    doi:

    MiR-193a overexpression inhibited the invasion, migration and angiogenesis of lung cancer cells. A. The cell invasion and migration was decreased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. ##P
    Figure Legend Snippet: MiR-193a overexpression inhibited the invasion, migration and angiogenesis of lung cancer cells. A. The cell invasion and migration was decreased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. ##P

    Techniques Used: Over Expression, Migration, Transfection

    MiR-193a overexpression inhibited the proliferation of lung cancer cells. A. The expression of miR-193a was increased in A549 cells after transfected with miR-193a mimic. ***P
    Figure Legend Snippet: MiR-193a overexpression inhibited the proliferation of lung cancer cells. A. The expression of miR-193a was increased in A549 cells after transfected with miR-193a mimic. ***P

    Techniques Used: Over Expression, Expressing, Transfection

    MiR-193a overexpression reduced the expression of S100A6. A. The expression of S100A6 was decreased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. ***P
    Figure Legend Snippet: MiR-193a overexpression reduced the expression of S100A6. A. The expression of S100A6 was decreased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. ***P

    Techniques Used: Over Expression, Expressing, Transfection

    MiR-193a overexpression increased the P53 acetylation. A. The expression of anti-acetylp53 (K373) and K-AC was increased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. **P
    Figure Legend Snippet: MiR-193a overexpression increased the P53 acetylation. A. The expression of anti-acetylp53 (K373) and K-AC was increased in A549 cells after transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. **P

    Techniques Used: Over Expression, Expressing, Transfection

    S100A6 expression was increased in lung cancer cells. The expression of S100A6 in HBE cells, A549 cells, H441 cells and H1975 cells was detected by Western blot. ***P
    Figure Legend Snippet: S100A6 expression was increased in lung cancer cells. The expression of S100A6 in HBE cells, A549 cells, H441 cells and H1975 cells was detected by Western blot. ***P

    Techniques Used: Expressing, Western Blot

    MiR-193a directly targets S100A6. A. The putative target sequence for miR-193a on the 3’UTR of S100A6. B. Luciferase reporter analysis revealed the target role of miR-193a on the 3’UTR of S100A6. C. The expression of miR-193a was decreased in A549 cells after transfected with pcDNA-S100A6. ***P
    Figure Legend Snippet: MiR-193a directly targets S100A6. A. The putative target sequence for miR-193a on the 3’UTR of S100A6. B. Luciferase reporter analysis revealed the target role of miR-193a on the 3’UTR of S100A6. C. The expression of miR-193a was decreased in A549 cells after transfected with pcDNA-S100A6. ***P

    Techniques Used: Sequencing, Luciferase, Expressing, Transfection

    Effect of miR-193a overexpression in vivo study. A. The tumor weight and mice body weight were decreased after mice transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. *P
    Figure Legend Snippet: Effect of miR-193a overexpression in vivo study. A. The tumor weight and mice body weight were decreased after mice transfected with miR-193a mimic and reversed in A549 cells after transfected with miR-193a mimic+pcDNA-S100A6. *P

    Techniques Used: Over Expression, In Vivo, Mouse Assay, Transfection

    S100A6 overexpression inhibited the P53 acetylation in lung cancer cells. A. The expression of anti-acetylp53 (K373) and K-AC was decreased in A549 cells after transfected with pcDNA-S100A6. ***P
    Figure Legend Snippet: S100A6 overexpression inhibited the P53 acetylation in lung cancer cells. A. The expression of anti-acetylp53 (K373) and K-AC was decreased in A549 cells after transfected with pcDNA-S100A6. ***P

    Techniques Used: Over Expression, Expressing, Transfection

    S100A6 overexpression promoted the invasion, migration and angiogenesis of lung cancer cells. A. The cell invasion and migration was increased in A549 cells after transfected with pcDNA-S100A6. **P
    Figure Legend Snippet: S100A6 overexpression promoted the invasion, migration and angiogenesis of lung cancer cells. A. The cell invasion and migration was increased in A549 cells after transfected with pcDNA-S100A6. **P

    Techniques Used: Over Expression, Migration, Transfection

    S100A6 overexpression promoted the proliferation of lung cancer cells. A. The expression of S100A6 was increased in A549 cells after transfected with pcDNA-S100A6. **P
    Figure Legend Snippet: S100A6 overexpression promoted the proliferation of lung cancer cells. A. The expression of S100A6 was increased in A549 cells after transfected with pcDNA-S100A6. **P

    Techniques Used: Over Expression, Expressing, Transfection

    20) Product Images from "Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin"

    Article Title: Long non-coding RNA CRYBG3 blocks cytokinesis by directly binding G-actin

    Journal: Cancer research

    doi: 10.1158/0008-5472.CAN-18-0988

    LNC CRYBG3 directly interacts with G-actin. (A) RNA pull-down analysis determined the Actin protein-LNC CRYBG3 interaction in A549 cells using 5’-biotin-linked RNAs. (B) Histogram of LNC CRYBG33 enrichment after RNA immunoprecipitation assays (RIP). Actin antibodies were used. (C) In vitro polymerisation of pyrene-labelled actin (0.1 μg/μL) was monitored using time-based fluorimetry. Additional components were: 20 μg/μL LNC CRYBG3 in vitro transcription product. Pyrene-labelled actin was monitored using time-based fluorimetry. (D) Diagram showing the truncated LNC CRYBG3. (E) In vitro polymerisation of pyrene-labelled actin treated with truncated LNC CRYBG3. (F G) Diagram showing the mutant of LNC CRYBG3. (H) In vitro polymerisation of pyrene-labelled actin treated with mutant LNC CRYBG3. (I) Immunofluorescence analysis determined the microfilaments morphology after A549 cells were transfected with actin mutant plasmid and then overexpress LNC CRYBG3 or negative control. Data represent the mean ± SE of three independent biological experiments.
    Figure Legend Snippet: LNC CRYBG3 directly interacts with G-actin. (A) RNA pull-down analysis determined the Actin protein-LNC CRYBG3 interaction in A549 cells using 5’-biotin-linked RNAs. (B) Histogram of LNC CRYBG33 enrichment after RNA immunoprecipitation assays (RIP). Actin antibodies were used. (C) In vitro polymerisation of pyrene-labelled actin (0.1 μg/μL) was monitored using time-based fluorimetry. Additional components were: 20 μg/μL LNC CRYBG3 in vitro transcription product. Pyrene-labelled actin was monitored using time-based fluorimetry. (D) Diagram showing the truncated LNC CRYBG3. (E) In vitro polymerisation of pyrene-labelled actin treated with truncated LNC CRYBG3. (F G) Diagram showing the mutant of LNC CRYBG3. (H) In vitro polymerisation of pyrene-labelled actin treated with mutant LNC CRYBG3. (I) Immunofluorescence analysis determined the microfilaments morphology after A549 cells were transfected with actin mutant plasmid and then overexpress LNC CRYBG3 or negative control. Data represent the mean ± SE of three independent biological experiments.

    Techniques Used: Immunoprecipitation, In Vitro, Mutagenesis, Immunofluorescence, Transfection, Plasmid Preparation, Negative Control

    LNC CRYBG3 regulates cytoskeleton formation. (A) Contractile ring dyeing in mitotic telophase. ( B C) Immunofluorescence analysis determined the microfilaments and microtubules morphology after cells overexpressed LNC CRYBG3. GFP shows the LNC CRYBG3 positive cells. (D E) F-actin and G-actin protein levels in the A549 cells overexpressed with LNC CRYBG3 or negative control. (F) Immunofluorescence analysis determined the microfilaments morphology. (G) Frozen sections of tumors were stained with Alexa Fluor® 647 phalloidine to indicate the microfilaments and nuclear were stained with DAPI. Data represent the mean ± SE of three independent biological experiments.
    Figure Legend Snippet: LNC CRYBG3 regulates cytoskeleton formation. (A) Contractile ring dyeing in mitotic telophase. ( B C) Immunofluorescence analysis determined the microfilaments and microtubules morphology after cells overexpressed LNC CRYBG3. GFP shows the LNC CRYBG3 positive cells. (D E) F-actin and G-actin protein levels in the A549 cells overexpressed with LNC CRYBG3 or negative control. (F) Immunofluorescence analysis determined the microfilaments morphology. (G) Frozen sections of tumors were stained with Alexa Fluor® 647 phalloidine to indicate the microfilaments and nuclear were stained with DAPI. Data represent the mean ± SE of three independent biological experiments.

    Techniques Used: Immunofluorescence, Negative Control, Staining

    LNC CRYBG3 prevents tumor growth and reduces tumor cell metastasis and invasion. (A) The tumor volume was detected for each group on 30 th day. (B) Calculate the theoretically time requirement for the tumor to grow to 100 mm 3 . The tumor formation frequency was calculated as described in methods (n = 10 for each group). (C) Xenografted tumor grows curve after overexpressed LNC CRYBG3 or negative control. The injection was begun on the time when xenografted tumor get 100 mm 3 twice a week for 1 month (n=10 for each group). (D) The xenografed tumors obtained after 8 times injection (n=10 for each group). (E) The weight of xenografed tumors from different groups was compared (n=10 for each group). (F) Representative H E staining of transplanted lung tumor tissues are shown. (G) In vivo cells metastasis experiment (n=5 for each group). (H I) Up-regulation of LNC CRYBG3 reduced the metastatic potential of A549 cells. Images showing trans-well invasion of the A549 cells. The three images in the upper panel show invasion of A549 cells in control or overexpressed with negative control or LNC CRYBG3. The images in the bottom two panels show the short hairpin of LNC CRYBG3 RNA expressed cells overexpressed with LNC NC or LNC CRYBG3. (J K) Up-regulation of LNC CRYBG3 influences the invasive property of A549 cells. Images showing matrigel invasion of the A549 cells. Data represent the mean ± SE of three independent biological experiments.
    Figure Legend Snippet: LNC CRYBG3 prevents tumor growth and reduces tumor cell metastasis and invasion. (A) The tumor volume was detected for each group on 30 th day. (B) Calculate the theoretically time requirement for the tumor to grow to 100 mm 3 . The tumor formation frequency was calculated as described in methods (n = 10 for each group). (C) Xenografted tumor grows curve after overexpressed LNC CRYBG3 or negative control. The injection was begun on the time when xenografted tumor get 100 mm 3 twice a week for 1 month (n=10 for each group). (D) The xenografed tumors obtained after 8 times injection (n=10 for each group). (E) The weight of xenografed tumors from different groups was compared (n=10 for each group). (F) Representative H E staining of transplanted lung tumor tissues are shown. (G) In vivo cells metastasis experiment (n=5 for each group). (H I) Up-regulation of LNC CRYBG3 reduced the metastatic potential of A549 cells. Images showing trans-well invasion of the A549 cells. The three images in the upper panel show invasion of A549 cells in control or overexpressed with negative control or LNC CRYBG3. The images in the bottom two panels show the short hairpin of LNC CRYBG3 RNA expressed cells overexpressed with LNC NC or LNC CRYBG3. (J K) Up-regulation of LNC CRYBG3 influences the invasive property of A549 cells. Images showing matrigel invasion of the A549 cells. Data represent the mean ± SE of three independent biological experiments.

    Techniques Used: Negative Control, Injection, Staining, In Vivo

    21) Product Images from "The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii"

    Article Title: The sensor kinase BfmS controls production of outer membrane vesicles in Acinetobacter baumannii

    Journal: BMC Microbiology

    doi: 10.1186/s12866-019-1679-0

    Pathogenic effect of OMVs derived from three A. baumannii strains. a Biofilm formation of A. baumannii strains cultured with OMVs from different A. baumannii strains. A. baumannii was inoculated in polystyrene tubes and then OMVs (5 μg/ml) obtained from different A. baumannii strains were added to bacterial culture media. Biofilms formed on polystyrene tubes were stained with crystal violet. The amount of crystal violet eluted from the biofilms with ethanol was quantified as the OD 570 normalized to total bacterial growth (OD 600 ). The data are presented as mean ± SD of three independent experiments. b Cytotoxicity of A549 cells treated with OMVs from A. baumannii strains. Cells were treated with various concentrations of A. baumannii OMVs for 24 h. Cell viability was determined using the MTT assay. Data are presented as mean ± SD of three independent experiments. + p
    Figure Legend Snippet: Pathogenic effect of OMVs derived from three A. baumannii strains. a Biofilm formation of A. baumannii strains cultured with OMVs from different A. baumannii strains. A. baumannii was inoculated in polystyrene tubes and then OMVs (5 μg/ml) obtained from different A. baumannii strains were added to bacterial culture media. Biofilms formed on polystyrene tubes were stained with crystal violet. The amount of crystal violet eluted from the biofilms with ethanol was quantified as the OD 570 normalized to total bacterial growth (OD 600 ). The data are presented as mean ± SD of three independent experiments. b Cytotoxicity of A549 cells treated with OMVs from A. baumannii strains. Cells were treated with various concentrations of A. baumannii OMVs for 24 h. Cell viability was determined using the MTT assay. Data are presented as mean ± SD of three independent experiments. + p

    Techniques Used: Derivative Assay, Cell Culture, Staining, MTT Assay

    22) Product Images from "Tumor suppressor function of miR-129-5p in lung cancer"

    Article Title: Tumor suppressor function of miR-129-5p in lung cancer

    Journal: Oncology Letters

    doi: 10.3892/ol.2019.10241

    Suppression of miR-129-5p levels promotes A549 and SPC-A-1 cell proliferation. A549 and SPC-A-1 cells were seeded and then transfected with miR-129-5p ASO. (A) At 24 h after transfection, miR-129-5p expression was determined using the reverse transcription-quantitative
    Figure Legend Snippet: Suppression of miR-129-5p levels promotes A549 and SPC-A-1 cell proliferation. A549 and SPC-A-1 cells were seeded and then transfected with miR-129-5p ASO. (A) At 24 h after transfection, miR-129-5p expression was determined using the reverse transcription-quantitative

    Techniques Used: Transfection, Allele-specific Oligonucleotide, Expressing

    HMGB1 is targeted by miR-129-5p in A549 cells. (A) The binding sites and its mutated version of HMGB1 and miR-129-5p. (B) miR-129-5p mimic and plasmid containing WT or mutated 3′-UTR of HMGB1 were transfected into A549 cells. After 48 h, the luciferase
    Figure Legend Snippet: HMGB1 is targeted by miR-129-5p in A549 cells. (A) The binding sites and its mutated version of HMGB1 and miR-129-5p. (B) miR-129-5p mimic and plasmid containing WT or mutated 3′-UTR of HMGB1 were transfected into A549 cells. After 48 h, the luciferase

    Techniques Used: Binding Assay, Plasmid Preparation, Transfection, Luciferase

    Overexpression of miR-129-5p inhibited the proliferation of A549 and SPC-A-1 cells and promoted cell apoptosis. (A) The miR-129-5p levels in normal lung tissues, A549 and SPC-A-1 cells were determined using RT-qPCR. The miR-129-5p levels in the tumor
    Figure Legend Snippet: Overexpression of miR-129-5p inhibited the proliferation of A549 and SPC-A-1 cells and promoted cell apoptosis. (A) The miR-129-5p levels in normal lung tissues, A549 and SPC-A-1 cells were determined using RT-qPCR. The miR-129-5p levels in the tumor

    Techniques Used: Over Expression, Quantitative RT-PCR

    23) Product Images from "Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment"

    Article Title: Impaired lysosomal activity mediated autophagic flux disruption by graphite carbon nanofibers induce apoptosis in human lung epithelial cells through oxidative stress and energetic impairment

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-017-0194-4

    Graphical diagram representing the mechanism of cell death in A549 cells by graphite carbon nanofibers. Exposure to GCNF causes the increased production of reactive oxygen species which activates the autophagosomes accumulation, apoptosis induction as well as damage to DNA. Further mechanistic studies revealed the interconnection between apoptosis and autophagy through the impairment of lysosomes and energy pool. Thus GCNF induced nanotoxicity through modulation of autophagy apoptosis axis via oxidative stress
    Figure Legend Snippet: Graphical diagram representing the mechanism of cell death in A549 cells by graphite carbon nanofibers. Exposure to GCNF causes the increased production of reactive oxygen species which activates the autophagosomes accumulation, apoptosis induction as well as damage to DNA. Further mechanistic studies revealed the interconnection between apoptosis and autophagy through the impairment of lysosomes and energy pool. Thus GCNF induced nanotoxicity through modulation of autophagy apoptosis axis via oxidative stress

    Techniques Used:

    ( a ) Western blot analysis of LC3, Beclin-1 and ( b ) Respective densitometry analysis and ( c ) Representative fluorescence photomicrographs of GFP – LC3 plasmid transfected A549 and their respective statistical analysis ( d ) in the presence and absence of chloroquine to determine the autophagic flux. Scale Bar - 20 μm. Imm unoblotting analysis ( e ), respective densitometry ( f ) and corresponding fluorescence photomicrographs ( g ) depicting the accumulation of SQSTM1/p62 after GCNF exposure in A549 cells. Scale Bar - 20 μm. Per view 10 cells and 4 views per group were analyzed. ( h ) Immunoblotting and ( i ) respective densitometry analysis of GCNF exposed A549 cells to assess the effect on mTOR signaling pathways. GAPDH was used as a loading control. Values are expressed as mean ± SE of three independent experiment. * p
    Figure Legend Snippet: ( a ) Western blot analysis of LC3, Beclin-1 and ( b ) Respective densitometry analysis and ( c ) Representative fluorescence photomicrographs of GFP – LC3 plasmid transfected A549 and their respective statistical analysis ( d ) in the presence and absence of chloroquine to determine the autophagic flux. Scale Bar - 20 μm. Imm unoblotting analysis ( e ), respective densitometry ( f ) and corresponding fluorescence photomicrographs ( g ) depicting the accumulation of SQSTM1/p62 after GCNF exposure in A549 cells. Scale Bar - 20 μm. Per view 10 cells and 4 views per group were analyzed. ( h ) Immunoblotting and ( i ) respective densitometry analysis of GCNF exposed A549 cells to assess the effect on mTOR signaling pathways. GAPDH was used as a loading control. Values are expressed as mean ± SE of three independent experiment. * p

    Techniques Used: Western Blot, Fluorescence, Plasmid Preparation, Transfection

    GCNF induced significant dose and time dependent increase in ROS production depicted by the increase in DCFDA fluorescence measured through plate reader ( a ) and respective fluorescence images ( b ). Scale Bar – 300 μm. Per view 100 cells and 4 views per group were analyzed. ( c ) Effect of GCNF on lipid peroxidation (LPO) was monitored compared to control cell. ( d , e ) NAC (ROS scavenger) have significant impact on viability of GCNF exposed A549 cells as well as ROS production in GCNF exposed A549 cells. Values are expressed as mean ± SE of three independent experiment. * p
    Figure Legend Snippet: GCNF induced significant dose and time dependent increase in ROS production depicted by the increase in DCFDA fluorescence measured through plate reader ( a ) and respective fluorescence images ( b ). Scale Bar – 300 μm. Per view 100 cells and 4 views per group were analyzed. ( c ) Effect of GCNF on lipid peroxidation (LPO) was monitored compared to control cell. ( d , e ) NAC (ROS scavenger) have significant impact on viability of GCNF exposed A549 cells as well as ROS production in GCNF exposed A549 cells. Values are expressed as mean ± SE of three independent experiment. * p

    Techniques Used: Fluorescence

    Representative flow cytograms ( a ) and corresponding TEM images ( b ) depicting cellular internalization of GCNF in A549 cells. In TEM images, red enclosure denotes presence of GCNF in membranous structure. ( c ) Phase contrast photomicrographs of A549 cells showing their cellular morphology. For flow cytometry, three independent experiments were performed and data represents mean ± SE value. * p
    Figure Legend Snippet: Representative flow cytograms ( a ) and corresponding TEM images ( b ) depicting cellular internalization of GCNF in A549 cells. In TEM images, red enclosure denotes presence of GCNF in membranous structure. ( c ) Phase contrast photomicrographs of A549 cells showing their cellular morphology. For flow cytometry, three independent experiments were performed and data represents mean ± SE value. * p

    Techniques Used: Flow Cytometry, Transmission Electron Microscopy, Cytometry

    Viability reduction and loss of membrane integrity in GCNF exposed A549 cells was assessed using MTT assay ( a ) and propidium iodide (PI) dye exclusion assay ( b ), respectively. Values represents mean ± SE of three independent experiment. * p
    Figure Legend Snippet: Viability reduction and loss of membrane integrity in GCNF exposed A549 cells was assessed using MTT assay ( a ) and propidium iodide (PI) dye exclusion assay ( b ), respectively. Values represents mean ± SE of three independent experiment. * p

    Techniques Used: MTT Assay, Exclusion Assay

    24) Product Images from "In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549"

    Article Title: In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549

    Journal: Radiology and Oncology

    doi: 10.2478/raon-2018-0014

    Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.
    Figure Legend Snippet: Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.

    Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Selection, Fluorescence, FACS, Isolation

    25) Product Images from "Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin"

    Article Title: Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25172

    Effect of p53 or p21 Waf1 siRNA depletion on RPA2 phosphorylation and stability of p53 complex with RPA ( A ) A549 cells were collected following one-hour 500 nM CPT treatment and the levels of p53 and p21 Waf1 as well as an overall RPA2 phosphorylation or phosphorylation at the residues Ser 29 , Tyr 21 and Ser 4/8 were analyzed by western blots; -b and -h indicate base non-phosphorylated and hyper-phosphorylated RPA2 forms. GAPDH and Ponceau Red staining were used as protein loading controls. ( B ) In CPT-treated cells, p21 Waf1 was silenced by siRNA as indicated. Stability of the RPA/p53 complex was analyzed by western blots following p53 or RPA1 immunoprecipitation. 15% of the total cell lysate was loaded in the last lane. ( C ) A scheme of cell treatment with CPT used in our experiments. Cells were pulse-treated with 500 nM CPT for one hour and later maintained in a drug-free medium. DNA synthesis in intact A549 cells or its p53- or p21 Waf1 siRNA depleted derivatives was analyzed by the [ 3 H]-thymidine incorporation assay at different time intervals following CPT pulse treatment. The results are normalized to the DNA synthesis rate of untreated cells.
    Figure Legend Snippet: Effect of p53 or p21 Waf1 siRNA depletion on RPA2 phosphorylation and stability of p53 complex with RPA ( A ) A549 cells were collected following one-hour 500 nM CPT treatment and the levels of p53 and p21 Waf1 as well as an overall RPA2 phosphorylation or phosphorylation at the residues Ser 29 , Tyr 21 and Ser 4/8 were analyzed by western blots; -b and -h indicate base non-phosphorylated and hyper-phosphorylated RPA2 forms. GAPDH and Ponceau Red staining were used as protein loading controls. ( B ) In CPT-treated cells, p21 Waf1 was silenced by siRNA as indicated. Stability of the RPA/p53 complex was analyzed by western blots following p53 or RPA1 immunoprecipitation. 15% of the total cell lysate was loaded in the last lane. ( C ) A scheme of cell treatment with CPT used in our experiments. Cells were pulse-treated with 500 nM CPT for one hour and later maintained in a drug-free medium. DNA synthesis in intact A549 cells or its p53- or p21 Waf1 siRNA depleted derivatives was analyzed by the [ 3 H]-thymidine incorporation assay at different time intervals following CPT pulse treatment. The results are normalized to the DNA synthesis rate of untreated cells.

    Techniques Used: Recombinase Polymerase Amplification, Cycling Probe Technology, Western Blot, Staining, Immunoprecipitation, DNA Synthesis, Thymidine Incorporation Assay

    Expression of conformational or DNA-binding p53 mutants, p53(His175) and p53 (His273), in A549 cells affects CPT-induced RPA2 phosphorylation and HR ( A ) Cells were harvested following one-hour CPT treatment at 500 nM. Expression of p53, p21 Waf1 and an overall RPA2 phosphorylation were analyzed by western blot with antibodies specific for p21 Waf1 , RPA1, mutant p53 (ab32049, Abcam) or for both wild-type and the mutant p53 (Pab240, Abcam). It was experimentally shown that ab32049 antibody (Abcam) does not react with wild-type p53 in A549 cells. Binding of p53 to RPA in the parental or the p21 Waf1 -depleted cells was analyzed by western blotting following p53 immunoprecipitation with anti-p53 antibody (Pab240, Abcam). ( B ) Parental A549 cells or cells expressing p53 (His175) or p53 (His273) were synchronized with nocodazole, pulse treated with CPT at the entry to S phase and later maintained in drug-free medium. Bars represent the relative cell frequencies at different stages of the cell cycle at the indicated times. ( C) HR frequency within pDR-GFP recombination substrate were measured 28 hours following CPT treatment.
    Figure Legend Snippet: Expression of conformational or DNA-binding p53 mutants, p53(His175) and p53 (His273), in A549 cells affects CPT-induced RPA2 phosphorylation and HR ( A ) Cells were harvested following one-hour CPT treatment at 500 nM. Expression of p53, p21 Waf1 and an overall RPA2 phosphorylation were analyzed by western blot with antibodies specific for p21 Waf1 , RPA1, mutant p53 (ab32049, Abcam) or for both wild-type and the mutant p53 (Pab240, Abcam). It was experimentally shown that ab32049 antibody (Abcam) does not react with wild-type p53 in A549 cells. Binding of p53 to RPA in the parental or the p21 Waf1 -depleted cells was analyzed by western blotting following p53 immunoprecipitation with anti-p53 antibody (Pab240, Abcam). ( B ) Parental A549 cells or cells expressing p53 (His175) or p53 (His273) were synchronized with nocodazole, pulse treated with CPT at the entry to S phase and later maintained in drug-free medium. Bars represent the relative cell frequencies at different stages of the cell cycle at the indicated times. ( C) HR frequency within pDR-GFP recombination substrate were measured 28 hours following CPT treatment.

    Techniques Used: Expressing, Binding Assay, Cycling Probe Technology, Western Blot, Mutagenesis, Recombinase Polymerase Amplification, Immunoprecipitation

    Phosphorylation of RPA in response to replication arrest induced by CPT contributes to the dissociation of the RPA/p53 complex ( A ) Cells were collected for analysis following one-hour treatment with 500 nM CPT. p53-negative H1299 cells were transfected with the transactivation-deficient p53(22.23) mutant. p53 binding was assessed following RPA1 immunoprecipitation. The expression levels of p53(22.23), RPA1 and phosphorylation of RPA2 in untreated cells were used as loading controls. ( B ) The residues within the N-terminal RPA2 domain reported to be phosphorylated by CDKs, ATR/ATM and DNA-PK were replaced with alanine or glutamic acid, thus producing RPA2A or RPA2D4 mutants that imitate non-phosphorylated or phosphorylated forms of RPA2, respectively. The consensus sites for the kinases are indicated. Expression levels of the recombinant wild type RPA2 and the mutants in A549 cells prior to or after siRNA silencing of endogenous RPA2. The cells with the silenced endogenous RPA2 were treated with 500 nM CPT for one hour. After p53 immunoprecipitation, RPA binding was analyzed on western blots with anti-RPA1 antibody.
    Figure Legend Snippet: Phosphorylation of RPA in response to replication arrest induced by CPT contributes to the dissociation of the RPA/p53 complex ( A ) Cells were collected for analysis following one-hour treatment with 500 nM CPT. p53-negative H1299 cells were transfected with the transactivation-deficient p53(22.23) mutant. p53 binding was assessed following RPA1 immunoprecipitation. The expression levels of p53(22.23), RPA1 and phosphorylation of RPA2 in untreated cells were used as loading controls. ( B ) The residues within the N-terminal RPA2 domain reported to be phosphorylated by CDKs, ATR/ATM and DNA-PK were replaced with alanine or glutamic acid, thus producing RPA2A or RPA2D4 mutants that imitate non-phosphorylated or phosphorylated forms of RPA2, respectively. The consensus sites for the kinases are indicated. Expression levels of the recombinant wild type RPA2 and the mutants in A549 cells prior to or after siRNA silencing of endogenous RPA2. The cells with the silenced endogenous RPA2 were treated with 500 nM CPT for one hour. After p53 immunoprecipitation, RPA binding was analyzed on western blots with anti-RPA1 antibody.

    Techniques Used: Recombinase Polymerase Amplification, Cycling Probe Technology, Transfection, Mutagenesis, Binding Assay, Immunoprecipitation, Expressing, Recombinant, Western Blot

    Involvement of the p53/p21 Waf1 axis in regulation of HR in response to replication arrest by CPT ( A ) Control A549 cells or cells with p53 or p21 Waf1 silenced by siRNA were synchronized in mitosis by nocodazole. After release from nocodazole arrest, cells were allowed to progress through the cell cycle and collected at different time-points to evaluate cell cycle progression by flow cytometry. Upon entrance into S phase, the cells were pulse-treated with CPT and later maintained in a drug-free medium. Cell cycle profiles and derivative bar graphs show the relative frequencies of cells in each stage of cell cycle at the indicated time points. ( B ) DNA synthesis was assessed at different time intervals following CPT treatment by [ 3 H]-thymidine incorporation assay and normalized to the DNA synthesis rate of untreated cells. ( C ) The percentages of apoptotic cells at the indicated times were assessed by flow cytometry following staining with FITC-conjugated Annexin V. ( D ) The cell line A549 carries a stably-transfected recombinant reporter construct, pDR-GFP [ 49 ]. The cells were harvested 27 hours following CPT addition and the HR frequencies were analyzed. Chi-square tests detected significant differences between p21siRNA and p53siRNA depleted cells (χ 2 = 26.3; p
    Figure Legend Snippet: Involvement of the p53/p21 Waf1 axis in regulation of HR in response to replication arrest by CPT ( A ) Control A549 cells or cells with p53 or p21 Waf1 silenced by siRNA were synchronized in mitosis by nocodazole. After release from nocodazole arrest, cells were allowed to progress through the cell cycle and collected at different time-points to evaluate cell cycle progression by flow cytometry. Upon entrance into S phase, the cells were pulse-treated with CPT and later maintained in a drug-free medium. Cell cycle profiles and derivative bar graphs show the relative frequencies of cells in each stage of cell cycle at the indicated time points. ( B ) DNA synthesis was assessed at different time intervals following CPT treatment by [ 3 H]-thymidine incorporation assay and normalized to the DNA synthesis rate of untreated cells. ( C ) The percentages of apoptotic cells at the indicated times were assessed by flow cytometry following staining with FITC-conjugated Annexin V. ( D ) The cell line A549 carries a stably-transfected recombinant reporter construct, pDR-GFP [ 49 ]. The cells were harvested 27 hours following CPT addition and the HR frequencies were analyzed. Chi-square tests detected significant differences between p21siRNA and p53siRNA depleted cells (χ 2 = 26.3; p

    Techniques Used: Cycling Probe Technology, Flow Cytometry, Cytometry, DNA Synthesis, Thymidine Incorporation Assay, Staining, Stable Transfection, Transfection, Recombinant, Construct

    26) Product Images from "Interferon-β Stimulation Elicited by the Influenza Virus Is Regulated by the Histone Methylase Dot1L through the RIG-I-TRIM25 Signaling Axis"

    Article Title: Interferon-β Stimulation Elicited by the Influenza Virus Is Regulated by the Histone Methylase Dot1L through the RIG-I-TRIM25 Signaling Axis

    Journal: Cells

    doi: 10.3390/cells9030732

    The overexpression of TRIM25 counteracts the effect of Dot1L inhibition on interferon signaling. ( A ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with a TRIM25 expressing plasmid (TRIM25) or a control plasmid. After 24 h, the cells were infected with influenza virus PR8 (PR8 and PR8 TRIM25) at MOI 1 and the luciferase activity was evaluated at 16 hpi. ( B ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with plasmids expressing RIG-I (RIG-I), or RIG-I and TRIM25 (RIG-I TRIM25), or RIG-I 2CARDs (RIG-I CARD), or RIG-I 2CARDs and TRIM25 (RIG-I CARD TRIM25). The luciferase activity was evaluated 16 h later. The luciferase activity was normalized by Renilla luciferase and it was expressed relative to that of the untreated MOCK condition. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: The overexpression of TRIM25 counteracts the effect of Dot1L inhibition on interferon signaling. ( A ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with a TRIM25 expressing plasmid (TRIM25) or a control plasmid. After 24 h, the cells were infected with influenza virus PR8 (PR8 and PR8 TRIM25) at MOI 1 and the luciferase activity was evaluated at 16 hpi. ( B ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with plasmids expressing RIG-I (RIG-I), or RIG-I and TRIM25 (RIG-I TRIM25), or RIG-I 2CARDs (RIG-I CARD), or RIG-I 2CARDs and TRIM25 (RIG-I CARD TRIM25). The luciferase activity was evaluated 16 h later. The luciferase activity was normalized by Renilla luciferase and it was expressed relative to that of the untreated MOCK condition. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Over Expression, Inhibition, Transfection, Expressing, Plasmid Preparation, Infection, Luciferase, Activity Assay

    INF-β driven expression triggered by a mutated RIG-I at Ser 8 is not regulated by Dot1L. A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with plasmids expressing the wild type RIG-I (RIG-I), or a mutated form of RIG-I with a Ser to Asp substitution at position 8 of the CARDS domains (RIG-I S8D). The luciferase activity was evaluated at 16 hpt. The cells were left uninfected or infected with PR8 at MOI 3 for 8 h. Luciferase activity was normalized by Renilla luciferase and it was expressed relative to that of the untreated MOCK condition. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: INF-β driven expression triggered by a mutated RIG-I at Ser 8 is not regulated by Dot1L. A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+) for 48 h. Then the cells were transfected with pIF-LukTer alone (MOCK), or together with plasmids expressing the wild type RIG-I (RIG-I), or a mutated form of RIG-I with a Ser to Asp substitution at position 8 of the CARDS domains (RIG-I S8D). The luciferase activity was evaluated at 16 hpt. The cells were left uninfected or infected with PR8 at MOI 3 for 8 h. Luciferase activity was normalized by Renilla luciferase and it was expressed relative to that of the untreated MOCK condition. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Expressing, Transfection, Luciferase, Activity Assay, Infection

    Dot1L inhibition or downregulation decreases IFN-β promoter stimulation in infected cells. ( A ) Upper part: A549 cells were left untreated (control), treated with a Dot1L inhibitor (EPZ, 1 μM, 48 h) or infected with lentiviruses expressing an irrelevant shRNA (shTM) or specific Dot1L shRNAs (shDOT1L 1 and shDOT1L 2) for 5 days. The cells were then transfected with plasmid pIF-LukTer and the luciferase activity was evaluated 16 h later. The luciferase activity was normalized by Renilla luciferase and the value obtained in the control cells was set as 100%. Lower part: Total extracts were used for Western blot analysis against the indicated proteins. ( B ) A549 cells were left untreated (control), treated with Dot1L inhibitor (EPZ, 1 μM, 48 h), or infected with lentiviruses expressing the indicated shRNAs for 5 days. The cells were then transfected with plasmid pIF-LuKter and 24 h later they were infected with influenza (FLU-PR8), or Sendai (SeV), or vesicular stomatitis virus (VSV) at MOI of 1. The luciferase activity was measured at 16 hpi and it is shown as the fold change relative to the levels in each control condition after normalization with the Renilla luciferase levels in each sample. The relative luciferase units obtained in each condition are represented in the lower part. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: Dot1L inhibition or downregulation decreases IFN-β promoter stimulation in infected cells. ( A ) Upper part: A549 cells were left untreated (control), treated with a Dot1L inhibitor (EPZ, 1 μM, 48 h) or infected with lentiviruses expressing an irrelevant shRNA (shTM) or specific Dot1L shRNAs (shDOT1L 1 and shDOT1L 2) for 5 days. The cells were then transfected with plasmid pIF-LukTer and the luciferase activity was evaluated 16 h later. The luciferase activity was normalized by Renilla luciferase and the value obtained in the control cells was set as 100%. Lower part: Total extracts were used for Western blot analysis against the indicated proteins. ( B ) A549 cells were left untreated (control), treated with Dot1L inhibitor (EPZ, 1 μM, 48 h), or infected with lentiviruses expressing the indicated shRNAs for 5 days. The cells were then transfected with plasmid pIF-LuKter and 24 h later they were infected with influenza (FLU-PR8), or Sendai (SeV), or vesicular stomatitis virus (VSV) at MOI of 1. The luciferase activity was measured at 16 hpi and it is shown as the fold change relative to the levels in each control condition after normalization with the Renilla luciferase levels in each sample. The relative luciferase units obtained in each condition are represented in the lower part. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Inhibition, Infection, Expressing, shRNA, Transfection, Plasmid Preparation, Luciferase, Activity Assay, Western Blot

    Dot1L modulates MAVS activation, RIG-I-MAVS association, and the antiviral response mediated by the RIG-I sensor. ( A ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+), 48 h later the cells were transfected with pIF-LukTer (MOCK), or cotransfected with pIF-LukTer together with plasmids expressing MAVS (MAVS), or mutant IRF3 (IRF3 5D) and the luciferase activity was evaluated at 16 hpt. ( B ) Scheme of the mitochondria purification. ( C ) Control or EPZ-treated cells were left uninfected (MOCK), or infected with PR8 virus at MOI 3 for 8 h. Input, P1 (P1), and purified mitochondria (Mito) fractions were used for Western blot analysis against the indicated proteins. ( D ) Control or EPZ-treated cells (1 μM (+), 48 h) were left uninfected (MOCK), or infected with PR8 virus at MOI 3 (PR8). At 16 hpi, the total extract was used for immunoprecipitation analysis using antibodies against MAVS. The immunoprecipitate was used for Western blot analysis against the indicated proteins. ( E ) Control or EPZ-treated cells were transfected with pIF-LukTer (MOCK), or cotransfected with pIF-LukTer, together with plasmids expressing RIG-I (RIG-I), or RIG-I 2CARDs (RIG-I CARDs), or mutant IRF3 (IRF35D) and the luciferase activity was evaluated at 16 hpt. In panels A and E, the luciferase activity was normalized by Renilla luciferase. The MOCK condition without EPZ treatment was taken as 100%. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: Dot1L modulates MAVS activation, RIG-I-MAVS association, and the antiviral response mediated by the RIG-I sensor. ( A ) A549 cells were left untreated (MOCK) or treated with 1 μM EPZ (+), 48 h later the cells were transfected with pIF-LukTer (MOCK), or cotransfected with pIF-LukTer together with plasmids expressing MAVS (MAVS), or mutant IRF3 (IRF3 5D) and the luciferase activity was evaluated at 16 hpt. ( B ) Scheme of the mitochondria purification. ( C ) Control or EPZ-treated cells were left uninfected (MOCK), or infected with PR8 virus at MOI 3 for 8 h. Input, P1 (P1), and purified mitochondria (Mito) fractions were used for Western blot analysis against the indicated proteins. ( D ) Control or EPZ-treated cells (1 μM (+), 48 h) were left uninfected (MOCK), or infected with PR8 virus at MOI 3 (PR8). At 16 hpi, the total extract was used for immunoprecipitation analysis using antibodies against MAVS. The immunoprecipitate was used for Western blot analysis against the indicated proteins. ( E ) Control or EPZ-treated cells were transfected with pIF-LukTer (MOCK), or cotransfected with pIF-LukTer, together with plasmids expressing RIG-I (RIG-I), or RIG-I 2CARDs (RIG-I CARDs), or mutant IRF3 (IRF35D) and the luciferase activity was evaluated at 16 hpt. In panels A and E, the luciferase activity was normalized by Renilla luciferase. The MOCK condition without EPZ treatment was taken as 100%. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Activation Assay, Transfection, Expressing, Mutagenesis, Luciferase, Activity Assay, Purification, Infection, Western Blot, Immunoprecipitation

    Dot1L does not affect viral replication or IFN-β promoter stimulation in delNS1 virus infected cells. ( A ) A549 cells were plated in the presence or absence of EPZ 1 µM and 48 h later they were infected at MOI 10 −3 with PR8 or delNS1 (PR8ΔNS1). The virus titer was determined with a plaque assay on MDCK cells. ( B ) A549 cells were left untreated or treated with 1 μM Dot1L inhibitor (+) and 48 h later they were transfected with the plasmid pIF-LukTer. At 24 h post-transfection, the cells were left uninfected (MOCK), or infected with influenza virus (PR8) or delNS1 virus (PR8ΔNS1) at MOI 1 and the luciferase activity was evaluated 16 h later. The luciferase activity normalized by Renilla luciferase in the MOCK condition without EPZ treatment was taken as 100%. ( C ) MDCK or MDCK-NS1 cells were plated in the presence or absence of EPZ 1 µM and 48 h later they were infected at MOI 10 −3 with delNS1. The virus titer was determined by plaque assay on MDCK cells. EPZ was present all throughout the experiment in the EPZ-treated cells. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: Dot1L does not affect viral replication or IFN-β promoter stimulation in delNS1 virus infected cells. ( A ) A549 cells were plated in the presence or absence of EPZ 1 µM and 48 h later they were infected at MOI 10 −3 with PR8 or delNS1 (PR8ΔNS1). The virus titer was determined with a plaque assay on MDCK cells. ( B ) A549 cells were left untreated or treated with 1 μM Dot1L inhibitor (+) and 48 h later they were transfected with the plasmid pIF-LukTer. At 24 h post-transfection, the cells were left uninfected (MOCK), or infected with influenza virus (PR8) or delNS1 virus (PR8ΔNS1) at MOI 1 and the luciferase activity was evaluated 16 h later. The luciferase activity normalized by Renilla luciferase in the MOCK condition without EPZ treatment was taken as 100%. ( C ) MDCK or MDCK-NS1 cells were plated in the presence or absence of EPZ 1 µM and 48 h later they were infected at MOI 10 −3 with delNS1. The virus titer was determined by plaque assay on MDCK cells. EPZ was present all throughout the experiment in the EPZ-treated cells. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Infection, Plaque Assay, Transfection, Plasmid Preparation, Luciferase, Activity Assay

    Dot1L inhibition does not modify RIG-I-MAVS interaction and nuclear translocation of NF-κB in delNS1 infection. ( A ) Control or EPZ-treated cells were left uninfected (MOCK), or infected with PR8 or with delNS1 virus at MOI 3. Input, P1 (P1) and purified mitochondria (Mito) fractions were used for Western blot analysis against the indicated proteins. ( B ) A549 cells were plated in the presence or absence of EPZ 1 µM and 48 h later, the cells were processed for immunofluorescence using anti-p65. ( C , D ) Cells were processed as in ( B ) and then infected at MOI 3 with PR8 at 8 h ( C ), or delNS1 at 16 h ( D ). EPZ was present all throughout the experiment in the EPZ-treated cells. The experiment was repeated twice, and representative images are shown. ( E ) Nuclear NF-κB translocation was measured by orthogonal projection image analysis and quantified in at least 200 cells/condition. The ratio was calculated by quantitation of the relative p65 intensity in the nucleus and the cytoplasm of each cell in all different conditions. Ratios are shown in dispersion graphs. ns p > 0.05; * p
    Figure Legend Snippet: Dot1L inhibition does not modify RIG-I-MAVS interaction and nuclear translocation of NF-κB in delNS1 infection. ( A ) Control or EPZ-treated cells were left uninfected (MOCK), or infected with PR8 or with delNS1 virus at MOI 3. Input, P1 (P1) and purified mitochondria (Mito) fractions were used for Western blot analysis against the indicated proteins. ( B ) A549 cells were plated in the presence or absence of EPZ 1 µM and 48 h later, the cells were processed for immunofluorescence using anti-p65. ( C , D ) Cells were processed as in ( B ) and then infected at MOI 3 with PR8 at 8 h ( C ), or delNS1 at 16 h ( D ). EPZ was present all throughout the experiment in the EPZ-treated cells. The experiment was repeated twice, and representative images are shown. ( E ) Nuclear NF-κB translocation was measured by orthogonal projection image analysis and quantified in at least 200 cells/condition. The ratio was calculated by quantitation of the relative p65 intensity in the nucleus and the cytoplasm of each cell in all different conditions. Ratios are shown in dispersion graphs. ns p > 0.05; * p

    Techniques Used: Inhibition, Translocation Assay, Infection, Purification, Western Blot, Immunofluorescence, Quantitation Assay

    Changes of TRIM25 in control and Dot1L inhibited cells. ( A ) Comparison of the TRIM25 expression from the RNA-seq and qRT-PCR data of A549 cells that were untreated or treated with EPZ (1 μM, 48 h) and then infected with PR8 at MOI 3 for 8 h. The differences between the untreated and treated cells were analyzed and are represented as the log2 fold change. Three technical replicates of three independent experiments for qRT-PCR detection were performed.. ns p > 0.05; * p
    Figure Legend Snippet: Changes of TRIM25 in control and Dot1L inhibited cells. ( A ) Comparison of the TRIM25 expression from the RNA-seq and qRT-PCR data of A549 cells that were untreated or treated with EPZ (1 μM, 48 h) and then infected with PR8 at MOI 3 for 8 h. The differences between the untreated and treated cells were analyzed and are represented as the log2 fold change. Three technical replicates of three independent experiments for qRT-PCR detection were performed.. ns p > 0.05; * p

    Techniques Used: Expressing, RNA Sequencing Assay, Quantitative RT-PCR, Infection

    Dot1L controls NF-κB and IRF3 activation. A549 cells were left untreated or treated with 1 μM EPZ (+), 48 h later the cells were ( A ) transfected with a plasmid expressing luciferase under the NF-κB promoter (NF-κB) and at 24 hpt they were stimulated with TNF-α β20 ng/mL, NF-κB-TNFα), or infected with influenza virus (PR8, MOI 1, NF-κB-PR8), and the luciferase activity was evaluated at 16 h later; ( B ) transfected with reporter plasmid p55-IRF3 expressing luciferase under the IRF3 promoter, and the luciferase activity was evaluated 16 h later. In all cases, the luciferase activity values were normalized by Renilla luciferase and they were expressed relative to their corresponding EPZ untreated control conditions. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p
    Figure Legend Snippet: Dot1L controls NF-κB and IRF3 activation. A549 cells were left untreated or treated with 1 μM EPZ (+), 48 h later the cells were ( A ) transfected with a plasmid expressing luciferase under the NF-κB promoter (NF-κB) and at 24 hpt they were stimulated with TNF-α β20 ng/mL, NF-κB-TNFα), or infected with influenza virus (PR8, MOI 1, NF-κB-PR8), and the luciferase activity was evaluated at 16 h later; ( B ) transfected with reporter plasmid p55-IRF3 expressing luciferase under the IRF3 promoter, and the luciferase activity was evaluated 16 h later. In all cases, the luciferase activity values were normalized by Renilla luciferase and they were expressed relative to their corresponding EPZ untreated control conditions. Three technical replicates of three independent experiments were analyzed. ns p > 0.05; * p

    Techniques Used: Activation Assay, Transfection, Plasmid Preparation, Expressing, Luciferase, Infection, Activity Assay

    27) Product Images from "A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation"

    Article Title: A Serpin Shapes the Extracellular Environment to Prevent Influenza A Virus Maturation

    Journal: Cell

    doi: 10.1016/j.cell.2015.01.040

    SERPINE1 Gene Expression Profiles and the Role of Extracellular PAI-1 Protein for IAV Inhibition (A–C) A549 cells were infected with IAV WSN/33, treated with IFN-β, or mock-treated. (A) SERPINE1 mRNA expression was normalized relative to housekeeping gene RPS-11 . Fold increase over pre-treatment control levels is shown. Accumulated total (B) intracellular and (C) extracellular protein levels of PAI-1 were determined by ELISA. Data are represented as mean ± SEM from n = 3 experiments. (D) HAEC infected with IAV WSN/33 or mock treated. Accumulated PAI-1 protein in repeated apical washes was determined by ELISA. Data are shown as mean ± SEM from n = 3 replicates. (E) A549 cells were infected with IAV WSN/33 in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM at 48 hpi. The number of infected cells was normalized to buffer (1) or IgG (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests. (F) HAECs were infected with IAV WSN/33 in the presence of either rPAI (1) or α-PAI-1 (2) and buffer (1) or IgG (2). Progeny virus was collected from apical washes, and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by plaque assay. Data are shown as mean ± SEM from n = 3 replicates. See also Figure S2 .
    Figure Legend Snippet: SERPINE1 Gene Expression Profiles and the Role of Extracellular PAI-1 Protein for IAV Inhibition (A–C) A549 cells were infected with IAV WSN/33, treated with IFN-β, or mock-treated. (A) SERPINE1 mRNA expression was normalized relative to housekeeping gene RPS-11 . Fold increase over pre-treatment control levels is shown. Accumulated total (B) intracellular and (C) extracellular protein levels of PAI-1 were determined by ELISA. Data are represented as mean ± SEM from n = 3 experiments. (D) HAEC infected with IAV WSN/33 or mock treated. Accumulated PAI-1 protein in repeated apical washes was determined by ELISA. Data are shown as mean ± SEM from n = 3 replicates. (E) A549 cells were infected with IAV WSN/33 in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM at 48 hpi. The number of infected cells was normalized to buffer (1) or IgG (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests. (F) HAECs were infected with IAV WSN/33 in the presence of either rPAI (1) or α-PAI-1 (2) and buffer (1) or IgG (2). Progeny virus was collected from apical washes, and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by plaque assay. Data are shown as mean ± SEM from n = 3 replicates. See also Figure S2 .

    Techniques Used: Expressing, Inhibition, Infection, Enzyme-linked Immunosorbent Assay, Plaque Assay

    SERPINE1 Gene Expression Profiles and the Role of Extracellular PAI-1 Protein in IAV Inhibition, Related to Figure 2 (A–C) Extended Figure 2 A. ISG15 , IFITM3 , and BST2 mRNA expression was normalized to housekeeping gene RPS-11 , fold increase over pre-treatment control levels is shown. (D–F) Extended Figure 2 A-C, showing the effect of TGF-β on SERPINE1 mRNA expression (A), or on intracellular (B) and extracellular (C) PAI-1 protein levels. (G). Extended Figure 2 D, showing the amount of extracellular PAI-1 protein levels from HAEC after induction with TGF-β. (H). A549 cells were infected with HPIV3-GFP for 48 hpi in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM. The number of infected cells was normalized to buffer (1) or IgG control (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests. (I). HAEC were infected with HPIV3-GFP in the presence of either recombinant stable human PAI-1 (rPAI-1, 1) or α-PAI-1 antibody (2), and buffer (1) or IgG (2) as controls. Progeny virus was collected from apical washes at the indicated times and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by TCID 50 assay. Data are represented as mean ± SEM from n = 3 replicates.
    Figure Legend Snippet: SERPINE1 Gene Expression Profiles and the Role of Extracellular PAI-1 Protein in IAV Inhibition, Related to Figure 2 (A–C) Extended Figure 2 A. ISG15 , IFITM3 , and BST2 mRNA expression was normalized to housekeeping gene RPS-11 , fold increase over pre-treatment control levels is shown. (D–F) Extended Figure 2 A-C, showing the effect of TGF-β on SERPINE1 mRNA expression (A), or on intracellular (B) and extracellular (C) PAI-1 protein levels. (G). Extended Figure 2 D, showing the amount of extracellular PAI-1 protein levels from HAEC after induction with TGF-β. (H). A549 cells were infected with HPIV3-GFP for 48 hpi in the presence of either rPAI-1 (1) or α-PAI-1 antibody (2), and virus spread was assayed by HTM. The number of infected cells was normalized to buffer (1) or IgG control (2). Data are represented as mean ± SEM from n = 4 independent experiments. Statistical significance relative to empty control was determined by t tests. (I). HAEC were infected with HPIV3-GFP in the presence of either recombinant stable human PAI-1 (rPAI-1, 1) or α-PAI-1 antibody (2), and buffer (1) or IgG (2) as controls. Progeny virus was collected from apical washes at the indicated times and rPAI-1 or α-PAI-1 were replenished after each wash. Virus titers were determined by TCID 50 assay. Data are represented as mean ± SEM from n = 3 replicates.

    Techniques Used: Expressing, Inhibition, Infection, Recombinant

    High-Throughput Microscopy Screens for Inhibitors of IAV Spread, Related to Figure 1 (A) Example of automated cell scoring from the HTM screen. Images show one representative out of 48 views per 96-well; original images from individual channels on the left (blue, DAPI-stained nuclei; red, transduced cells; green, NP-positive cells), and segmented images on the right (gray, nuclei; red, transduced cells; green, NP-positive cells). (B) Establishing the spread ratio as a stable measure of spread over a large range of transduction efficiencies. A549 cells were transduced with a dilution series of BST2- expressing lentivirus, or a high dose of empty vector, the latter yielding 90% of transduced cells. A/WSN/33 spread was determined as described in Figure 1 A. (C) Establishing α-HA antibody to block IAV spread. A549 cells were infected with IAV WSN/33 for 24h, in the presence or absence of an α-HA antibody serial dilution, then fixed and stained for IAV NP. Shown are results from automated quantification, and representative images of two wells with the minimum inhibitory concentration, 0.125 μg/ml, or without α-HA are shown. (D) Cytotoxicity assays of selected hits from the screen. A549 cells in 96-well plates were transduced with high-titer lentiviral stocks, yielding 90% transduced cells. Cells were then assayed by HTM for (from top to bottom): cell survival/proliferation, by DAPI stain and cell count; apoptosis, by Annexin V stain; DNA damage, by staining for phosphorylated H2AX; and cell membrane integrity, by Sytox stain. Tamoxifen and staurosporine were used as positive controls, untreated and untransduced cells as negative controls. Representative images from the respective assays are shown on the right.
    Figure Legend Snippet: High-Throughput Microscopy Screens for Inhibitors of IAV Spread, Related to Figure 1 (A) Example of automated cell scoring from the HTM screen. Images show one representative out of 48 views per 96-well; original images from individual channels on the left (blue, DAPI-stained nuclei; red, transduced cells; green, NP-positive cells), and segmented images on the right (gray, nuclei; red, transduced cells; green, NP-positive cells). (B) Establishing the spread ratio as a stable measure of spread over a large range of transduction efficiencies. A549 cells were transduced with a dilution series of BST2- expressing lentivirus, or a high dose of empty vector, the latter yielding 90% of transduced cells. A/WSN/33 spread was determined as described in Figure 1 A. (C) Establishing α-HA antibody to block IAV spread. A549 cells were infected with IAV WSN/33 for 24h, in the presence or absence of an α-HA antibody serial dilution, then fixed and stained for IAV NP. Shown are results from automated quantification, and representative images of two wells with the minimum inhibitory concentration, 0.125 μg/ml, or without α-HA are shown. (D) Cytotoxicity assays of selected hits from the screen. A549 cells in 96-well plates were transduced with high-titer lentiviral stocks, yielding 90% transduced cells. Cells were then assayed by HTM for (from top to bottom): cell survival/proliferation, by DAPI stain and cell count; apoptosis, by Annexin V stain; DNA damage, by staining for phosphorylated H2AX; and cell membrane integrity, by Sytox stain. Tamoxifen and staurosporine were used as positive controls, untreated and untransduced cells as negative controls. Representative images from the respective assays are shown on the right.

    Techniques Used: High Throughput Screening Assay, Microscopy, Staining, Transduction, Expressing, Plasmid Preparation, Blocking Assay, Infection, Serial Dilution, Concentration Assay, Cell Counting

    High-Throughput Microscopy Screens for Inhibitors of IAV Spread (A) Screening workflow. Shown are hypothetical effects of expressing inhibitory (antiviral) or non-inhibitory ISGs on single or multiple rounds of virus replication. Red, transduced cells; green, infected cells; blue, DAPI-stained nuclei. (B) Effect of 401 single ISGs on IAV spread. ISGs inhibiting more than 2-fold SD in two independent screens are shown in red. Spread ratio, the ratio of infected cells at 24/8 hpi. A positive control for inhibition is α-HA antibody. (C) Confirmation assays for selected ISGs on A549 cells or primary NHBE cells. Data are represented as mean ± SEM from n = 6 values in two independent experiments for A549, and n = 3 for NHBE cells. (D) SERPINE1 (PAI-1)-mediated inhibition of A/Vietnam/1203/2004(HALo), A/California/04/2009, and A/sw/Texas/4199-2/1998 in A549 cells. Empty vector, negative control; IFITM3 and BST2 (tetherin), positive controls. Data are represented as mean ± SEM from n = 4 independent experiments. One-way ANOVA and Dunn’s multiple comparison test versus “empty.” (E) ISG-expressing A549 cells were infected with IAV WSN/33 at MOI 0.01, and virus titers were measured by plaque assay on MDCK cells. Data are represented as mean ± SEM from n = 4 independent experiments. See also Figure S1 .
    Figure Legend Snippet: High-Throughput Microscopy Screens for Inhibitors of IAV Spread (A) Screening workflow. Shown are hypothetical effects of expressing inhibitory (antiviral) or non-inhibitory ISGs on single or multiple rounds of virus replication. Red, transduced cells; green, infected cells; blue, DAPI-stained nuclei. (B) Effect of 401 single ISGs on IAV spread. ISGs inhibiting more than 2-fold SD in two independent screens are shown in red. Spread ratio, the ratio of infected cells at 24/8 hpi. A positive control for inhibition is α-HA antibody. (C) Confirmation assays for selected ISGs on A549 cells or primary NHBE cells. Data are represented as mean ± SEM from n = 6 values in two independent experiments for A549, and n = 3 for NHBE cells. (D) SERPINE1 (PAI-1)-mediated inhibition of A/Vietnam/1203/2004(HALo), A/California/04/2009, and A/sw/Texas/4199-2/1998 in A549 cells. Empty vector, negative control; IFITM3 and BST2 (tetherin), positive controls. Data are represented as mean ± SEM from n = 4 independent experiments. One-way ANOVA and Dunn’s multiple comparison test versus “empty.” (E) ISG-expressing A549 cells were infected with IAV WSN/33 at MOI 0.01, and virus titers were measured by plaque assay on MDCK cells. Data are represented as mean ± SEM from n = 4 independent experiments. See also Figure S1 .

    Techniques Used: High Throughput Screening Assay, Microscopy, Expressing, Infection, Staining, Positive Control, Inhibition, Plasmid Preparation, Negative Control, Plaque Assay

    Effect of PAI-1 on Various Steps of the IAV Life Cycle (A–E) A549 cells were transduced to express the indicated ISGs. Empty vector served as negative control, and the following positive controls were used: Diphyllin for IAV entry, Ribavirin for IAV replication. Data are represented as mean ± SEM from at least n = 4 independent experiments for all panels. (A) Cells were challenged with IAV WSN/33 at MOI 1 and the number of NP-positive nuclei was determined at 6 hpi. Statistics by 1-way ANOVA and Dunn’s multiple comparison test. (B) IAV replication efficiency was assayed by a luciferase-based IAV minigenome assay. Expression constructs for components of the IAV replication machinery (PB1, PB2, PA and NP, of A/WSN/33 origin) were co-transfected with a reporter construct mimicking the viral genome, leading to expression of firefly luciferase (Fluc) when the genome mimic is replicated. Individual t tests compared to empty control. (C) Influenza A/Puerto Rico/8/34-NS1-GFP virus replication during infection was assayed by FACS assay, determining the percentage of infected cells at 10 hpi (GFP-positive) in the ISG-overexpressing (RFP-positive) population. Statistical significance relative to empty control was determined by t tests. (D–F) Cells were infected with IAV WSN/33 at MOI = 0.01. At 24 hpi, vRNA was extracted from cells (D) or supernatants (E), and vRNA copy number was determined by qRT-PCR. Infectious virus titers in supernatants at 48 hpi were determined by plaque assay on MDCK cells (F).
    Figure Legend Snippet: Effect of PAI-1 on Various Steps of the IAV Life Cycle (A–E) A549 cells were transduced to express the indicated ISGs. Empty vector served as negative control, and the following positive controls were used: Diphyllin for IAV entry, Ribavirin for IAV replication. Data are represented as mean ± SEM from at least n = 4 independent experiments for all panels. (A) Cells were challenged with IAV WSN/33 at MOI 1 and the number of NP-positive nuclei was determined at 6 hpi. Statistics by 1-way ANOVA and Dunn’s multiple comparison test. (B) IAV replication efficiency was assayed by a luciferase-based IAV minigenome assay. Expression constructs for components of the IAV replication machinery (PB1, PB2, PA and NP, of A/WSN/33 origin) were co-transfected with a reporter construct mimicking the viral genome, leading to expression of firefly luciferase (Fluc) when the genome mimic is replicated. Individual t tests compared to empty control. (C) Influenza A/Puerto Rico/8/34-NS1-GFP virus replication during infection was assayed by FACS assay, determining the percentage of infected cells at 10 hpi (GFP-positive) in the ISG-overexpressing (RFP-positive) population. Statistical significance relative to empty control was determined by t tests. (D–F) Cells were infected with IAV WSN/33 at MOI = 0.01. At 24 hpi, vRNA was extracted from cells (D) or supernatants (E), and vRNA copy number was determined by qRT-PCR. Infectious virus titers in supernatants at 48 hpi were determined by plaque assay on MDCK cells (F).

    Techniques Used: Plasmid Preparation, Negative Control, Luciferase, Expressing, Construct, Transfection, Infection, FACS, Quantitative RT-PCR, Plaque Assay

    28) Product Images from "Osteopontin production by TM4SF4 signaling drives a positive feedback autocrine loop with the STAT3 pathway to maintain cancer stem cell-like properties in lung cancer cells"

    Article Title: Osteopontin production by TM4SF4 signaling drives a positive feedback autocrine loop with the STAT3 pathway to maintain cancer stem cell-like properties in lung cancer cells

    Journal: Oncotarget

    doi: 10.18632/oncotarget.21021

    Effect of TM4SF4 on EMT and CSC-like properties through osteopontin secretion in lung cancer cells ( A ) Changes of transcriptional and protein levels of osteopontin in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Cytokine array analysis. Changes of osteopontin in cell lysate and cell cultured media of TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( C ) Suppression of migration/invasion capacity (left panel) and EMT markers such as N-cadherin and vimentin (right panel) that were increased in TM4SF4 -overexpressing A 549 cells. ( D ) Suppression of sphere-forming capacity (left panel) and CSC markers such as Sox2 and Oct3/4 (right panel) that were increased in TM4SF4-overexpressing A549 cells. Data represent the mean of three independent experiments. Scale bar = 20 mm. The quantified results are presented as mean ± s.d. using two-tailed t -test. * p
    Figure Legend Snippet: Effect of TM4SF4 on EMT and CSC-like properties through osteopontin secretion in lung cancer cells ( A ) Changes of transcriptional and protein levels of osteopontin in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Cytokine array analysis. Changes of osteopontin in cell lysate and cell cultured media of TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( C ) Suppression of migration/invasion capacity (left panel) and EMT markers such as N-cadherin and vimentin (right panel) that were increased in TM4SF4 -overexpressing A 549 cells. ( D ) Suppression of sphere-forming capacity (left panel) and CSC markers such as Sox2 and Oct3/4 (right panel) that were increased in TM4SF4-overexpressing A549 cells. Data represent the mean of three independent experiments. Scale bar = 20 mm. The quantified results are presented as mean ± s.d. using two-tailed t -test. * p

    Techniques Used: Cell Culture, Migration, Two Tailed Test

    TM4SF4 is involved in transcriptional regulation of osteopontin through the GSK3β/β-catenin signaling pathway ( A ) Western blot analysis of cellular GSK3β (phosphorylation) and β-catenin levels(upper panel) and nucleus fraction β-catenin(lower panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Changes of cellular β-catenin, osteopontin, and EMT markers (N-cadherin and vimentin) levels after treatment with CHIR99021 (10μM, 24hr), a GSK3β inhibitor, in TM4SF4 -overexpressing A549 cells. ( C ) Changes of sphere-forming (upper panel) and migration/invasion capacity (lower panel) in GSK3β inhibitor–treated TM4SF4 -overexpressing A549 cells. Scale bar = 20 mm. ( D ) RT-PCR and Western blot analysis of transcriptional and protein levels of osteopontin in β-catenin and TCF4- knockdown A549 cells with siRNA. ( E ) Identification of β-catenin in complexes captured by TCF4 antibody (left panel) and chromatin immunoprecipitation assay between osteopontin promoter region and TCF4 in TM4SF4 -overexpressing A549 cells (right panel). Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. ns = not significant. ** p
    Figure Legend Snippet: TM4SF4 is involved in transcriptional regulation of osteopontin through the GSK3β/β-catenin signaling pathway ( A ) Western blot analysis of cellular GSK3β (phosphorylation) and β-catenin levels(upper panel) and nucleus fraction β-catenin(lower panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Changes of cellular β-catenin, osteopontin, and EMT markers (N-cadherin and vimentin) levels after treatment with CHIR99021 (10μM, 24hr), a GSK3β inhibitor, in TM4SF4 -overexpressing A549 cells. ( C ) Changes of sphere-forming (upper panel) and migration/invasion capacity (lower panel) in GSK3β inhibitor–treated TM4SF4 -overexpressing A549 cells. Scale bar = 20 mm. ( D ) RT-PCR and Western blot analysis of transcriptional and protein levels of osteopontin in β-catenin and TCF4- knockdown A549 cells with siRNA. ( E ) Identification of β-catenin in complexes captured by TCF4 antibody (left panel) and chromatin immunoprecipitation assay between osteopontin promoter region and TCF4 in TM4SF4 -overexpressing A549 cells (right panel). Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. ns = not significant. ** p

    Techniques Used: Western Blot, Migration, Reverse Transcription Polymerase Chain Reaction, Chromatin Immunoprecipitation, Two Tailed Test

    TM4SF4 is involved in the transcriptional regulation of osteopontin via the JAK2/STAT3 or FAK/STAT3 pathway ( A ) Western blot analysis of cellular, p-JAK2, p-FAK, p-SRC and p-STAT3 levels in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Changes in metastatic capacity (left panel) and cellular EMT markers (N-cadherin and vimentin) and osteopontin levels (right panel) after treatment of STAT3 inhibitor VII (10 μM, 24 hr), JAK2/STAT3 inhibtior. ( C ) Changes of metastatic capacity (left panel) and cellular EMT markers (N-cadherin and vimentin), p-STAT, p-SRC, and osteopontin levels (right panel) after treatment with FAK inhibitor 14 (10 μM, 24 hr) in TM4SF4 -overexpressing A549 cells. ( D ) Changes of sphere-forming cellular capacity after treatment of FAK inhibitor 14 and STAT3 inhibitor VII in TM4SF4 -overexpressing A549 cells. ( E ) Changes of transcriptional and protein levels of osteopontin after treatment with FAK inhibitor 14 and STAT3 inhibitor VII. Scale bar = 20 mm. Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. ** p
    Figure Legend Snippet: TM4SF4 is involved in the transcriptional regulation of osteopontin via the JAK2/STAT3 or FAK/STAT3 pathway ( A ) Western blot analysis of cellular, p-JAK2, p-FAK, p-SRC and p-STAT3 levels in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( B ) Changes in metastatic capacity (left panel) and cellular EMT markers (N-cadherin and vimentin) and osteopontin levels (right panel) after treatment of STAT3 inhibitor VII (10 μM, 24 hr), JAK2/STAT3 inhibtior. ( C ) Changes of metastatic capacity (left panel) and cellular EMT markers (N-cadherin and vimentin), p-STAT, p-SRC, and osteopontin levels (right panel) after treatment with FAK inhibitor 14 (10 μM, 24 hr) in TM4SF4 -overexpressing A549 cells. ( D ) Changes of sphere-forming cellular capacity after treatment of FAK inhibitor 14 and STAT3 inhibitor VII in TM4SF4 -overexpressing A549 cells. ( E ) Changes of transcriptional and protein levels of osteopontin after treatment with FAK inhibitor 14 and STAT3 inhibitor VII. Scale bar = 20 mm. Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. ** p

    Techniques Used: Western Blot, Two Tailed Test

    Changes of cellular TM4SF4/osteopontin levels and their related down-stream targets in ALDH1 high or fractionated γ-irradiation-exposed cells and control of EMT and CSC properties by TM4SF4 in lung cancer cells ( A ) Western blot analysis of TM4SF4 and osteopontin levels in ALDH1 high [AL(+)] and ALDH1 low [AL(–)] cells sorted from A549 cell lines(left panel) and fractionated γ-radiation-exposed cells (2 Gy × 3 times, 2 Gy × 9 times: right panel). ( B ) Changes of migration/ invasion capacity (left panel) and EMT markers including N-cadherin, Vimentin, Snail, and Twist (right panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( C ) Changes of sphere-forming capacity (left panel) and CSC markers such as Oct3/4, Sox2, CD133, CD44, β-catenin, and ALDH1 (left panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( D ) Changes of sphere-forming capacity and cellular osteopontin level after treatment with neutralizing antibody against TM4SF4. Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. * p
    Figure Legend Snippet: Changes of cellular TM4SF4/osteopontin levels and their related down-stream targets in ALDH1 high or fractionated γ-irradiation-exposed cells and control of EMT and CSC properties by TM4SF4 in lung cancer cells ( A ) Western blot analysis of TM4SF4 and osteopontin levels in ALDH1 high [AL(+)] and ALDH1 low [AL(–)] cells sorted from A549 cell lines(left panel) and fractionated γ-radiation-exposed cells (2 Gy × 3 times, 2 Gy × 9 times: right panel). ( B ) Changes of migration/ invasion capacity (left panel) and EMT markers including N-cadherin, Vimentin, Snail, and Twist (right panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( C ) Changes of sphere-forming capacity (left panel) and CSC markers such as Oct3/4, Sox2, CD133, CD44, β-catenin, and ALDH1 (left panel) in TM4SF4 -overexpressing or TM4SF4 -suppressing A549 cells. ( D ) Changes of sphere-forming capacity and cellular osteopontin level after treatment with neutralizing antibody against TM4SF4. Data represent the mean of three independent experiments. The quantified results are presented as mean ± s.d. using two-tailed t -test. * p

    Techniques Used: Irradiation, Western Blot, Migration, Two Tailed Test

    29) Product Images from "Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy"

    Article Title: Heparin-binding Hemagglutinin of Mycobacterium tuberculosis Is an Inhibitor of Autophagy

    Journal: Frontiers in Cellular and Infection Microbiology

    doi: 10.3389/fcimb.2017.00033

    HBHA protein and rMS-HBHA infection could induce cell death on A549. (A) A549 cells were treated with HBHA of different concentrations for 24 h, and LDH release was detected. ( *** P
    Figure Legend Snippet: HBHA protein and rMS-HBHA infection could induce cell death on A549. (A) A549 cells were treated with HBHA of different concentrations for 24 h, and LDH release was detected. ( *** P

    Techniques Used: Infection

    Schematic model of the role of HBHA during mycobacterial infection . HBHA inhibited autophagy in mycobacterial infected A549 cells, thereby promoted intracellular bacterial survival and replication. Subsequently, HBHA induced apoptosis on A549 cells through activation of caspase-3 which may facilitate bacterial escape from lung epithelial cells and dissemination to the adjacent cells.
    Figure Legend Snippet: Schematic model of the role of HBHA during mycobacterial infection . HBHA inhibited autophagy in mycobacterial infected A549 cells, thereby promoted intracellular bacterial survival and replication. Subsequently, HBHA induced apoptosis on A549 cells through activation of caspase-3 which may facilitate bacterial escape from lung epithelial cells and dissemination to the adjacent cells.

    Techniques Used: Infection, Activation Assay

    HBHA inhibited the maturation of autophagosome in A549 cells. (A) A549 cells were starved for 1 h and subsequently treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml) for 4 h. The cells were then stained with monodansylcadaverine (MDC), and MDC-labeled autophagic vacuoles were detected by confocal microscopy. Scale bars: 20 μm. (B) MDC-positive autophagic vacuoles were quantified in each group. ( *** P
    Figure Legend Snippet: HBHA inhibited the maturation of autophagosome in A549 cells. (A) A549 cells were starved for 1 h and subsequently treated with HBHA (8 μg/ml) for 90 min or 3-MA (100 μg/ml) for 4 h. The cells were then stained with monodansylcadaverine (MDC), and MDC-labeled autophagic vacuoles were detected by confocal microscopy. Scale bars: 20 μm. (B) MDC-positive autophagic vacuoles were quantified in each group. ( *** P

    Techniques Used: Staining, Labeling, Confocal Microscopy

    Recombinant Mycobacterium smegmatis ( MS ) expressing HBHA ( rMS-HBHA ) inhibited autophagy among starved A549 cells. (A) Total proteins of MC 2 155 and rMS-HBHA were extracted, and Western blot was carried out to detect HBHA using anti-HBHA antibody. (B) A549 cells were infected with the wild-type strain MC 2 155 or rMS-HBHA at the multiplicity of infection (MOI) of 10:1 for indicated time or treated with HBHA (8 μg/ml) for 90 min. The expression levels of LC3 were detected by Western blot. (C) The intensities of LC3II bands were normalized to the intensity of β-actin. ( *** P
    Figure Legend Snippet: Recombinant Mycobacterium smegmatis ( MS ) expressing HBHA ( rMS-HBHA ) inhibited autophagy among starved A549 cells. (A) Total proteins of MC 2 155 and rMS-HBHA were extracted, and Western blot was carried out to detect HBHA using anti-HBHA antibody. (B) A549 cells were infected with the wild-type strain MC 2 155 or rMS-HBHA at the multiplicity of infection (MOI) of 10:1 for indicated time or treated with HBHA (8 μg/ml) for 90 min. The expression levels of LC3 were detected by Western blot. (C) The intensities of LC3II bands were normalized to the intensity of β-actin. ( *** P

    Techniques Used: Recombinant, Mass Spectrometry, Expressing, Western Blot, Infection

    Inhibition of autophagy could promote survival of rMS-HBHA within A549 cells. (A) Growth curves of MS and rMS-HBHA were measured by OD 600 absorbance. (B) A549 cells were infected with MS or rMS-HBHA at the MOI of 10:1. The cells were lysed at 1, 10, or 18 h post-infection, and lysates were diluted and plated on agar plates to determine the number of viable intracellular bacteria. ( *** P
    Figure Legend Snippet: Inhibition of autophagy could promote survival of rMS-HBHA within A549 cells. (A) Growth curves of MS and rMS-HBHA were measured by OD 600 absorbance. (B) A549 cells were infected with MS or rMS-HBHA at the MOI of 10:1. The cells were lysed at 1, 10, or 18 h post-infection, and lysates were diluted and plated on agar plates to determine the number of viable intracellular bacteria. ( *** P

    Techniques Used: Inhibition, Mass Spectrometry, Infection

    HBHA treatment induced apoptosis on A549 cells through activation of caspase-3. (A) A549 cells were treated with HBHA (8 μg/ml) for 24 h in the presence or absence of RIPK1 inhibitor Nec-1 (30 μM), and LDH release was detected. ( *** P
    Figure Legend Snippet: HBHA treatment induced apoptosis on A549 cells through activation of caspase-3. (A) A549 cells were treated with HBHA (8 μg/ml) for 24 h in the presence or absence of RIPK1 inhibitor Nec-1 (30 μM), and LDH release was detected. ( *** P

    Techniques Used: Activation Assay

    Heparin-binding hemagglutinin (HBHA) inhibited the expression of LC3 and Beclin-1 in A549 cells. (A–D) A549 cells were starved for 1 h, and HBHA proteins of different concentrations were subsequently added to the cells for 90 min. LC3 (A) and ATG5/Beclin-1 (C) expression was detected by Western blot. The intensities of LC3II (B) and Beclin-1 (D) bands were normalized to the intensity of β-actin. ( * P
    Figure Legend Snippet: Heparin-binding hemagglutinin (HBHA) inhibited the expression of LC3 and Beclin-1 in A549 cells. (A–D) A549 cells were starved for 1 h, and HBHA proteins of different concentrations were subsequently added to the cells for 90 min. LC3 (A) and ATG5/Beclin-1 (C) expression was detected by Western blot. The intensities of LC3II (B) and Beclin-1 (D) bands were normalized to the intensity of β-actin. ( * P

    Techniques Used: Binding Assay, Expressing, Western Blot

    30) Product Images from "ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells"

    Article Title: ADAM23 is downregulated in side population and suppresses lung metastasis of lung carcinoma cells

    Journal: Cancer Science

    doi: 10.1111/cas.12895

    ADAM 23 expression in A549‐derived side population ( SP ) and main population ( MP ) cells and other lung adenocarcinoma cell lines. (a) mRNA expression of ADAM 23 in A549 parent, SP (1), MP (1), SP (9) and MP (9) cells by RT ‐ PCR (upper panel) and qPCR (lower panel) ( n = 4). (b) mRNA expression of ADAM 23 in parent, SP and MP cells derived from lung adenocarcinoma cell lines measured by qPCR ( n = 3). Bars, mean ± SD . ** P
    Figure Legend Snippet: ADAM 23 expression in A549‐derived side population ( SP ) and main population ( MP ) cells and other lung adenocarcinoma cell lines. (a) mRNA expression of ADAM 23 in A549 parent, SP (1), MP (1), SP (9) and MP (9) cells by RT ‐ PCR (upper panel) and qPCR (lower panel) ( n = 4). (b) mRNA expression of ADAM 23 in parent, SP and MP cells derived from lung adenocarcinoma cell lines measured by qPCR ( n = 3). Bars, mean ± SD . ** P

    Techniques Used: Expressing, Derivative Assay, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    Involvement of ADAM 23 and αvβ3 integrin in cell adhesion and migration. (a) Cell adhesion of control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was assayed ( n = 4). (b) Cell adhesion of A549 cells transfected with empty vectors or ADAM 23 expression vectors, ADAM 23 transfectants treated with non‐immune IgG ( NI , ADAM 23) or anti‐ ADAM 23 antibody (α‐ ADAM 23), ADAM 23 transfectants treated with scrambled peptide or disintegrin peptide, ADAM 23 transfectants treated with disintegrin peptide and non‐immune IgG ( NI , disintegrin peptide) or disintegrin peptide and anti‐αvβ3 antibody (α‐αvβ3, disintegrin peptide), and empty vector transfectants treated with non‐immune IgG ( NI , empty vector) or anti‐αvβ3 integrin antibody (α‐αvβ3, empty vector) was determined ( n = 4). (c) Migration of control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was measured by migration assay ( n = 4). (d) Migration of A549 cells treated with non‐immune IgG ( NI ) or anti‐αvβ3 integrin antibody (α‐αvβ3) was measured using the xCELL igence system ( n = 4). Bars, mean ± SD . * P
    Figure Legend Snippet: Involvement of ADAM 23 and αvβ3 integrin in cell adhesion and migration. (a) Cell adhesion of control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was assayed ( n = 4). (b) Cell adhesion of A549 cells transfected with empty vectors or ADAM 23 expression vectors, ADAM 23 transfectants treated with non‐immune IgG ( NI , ADAM 23) or anti‐ ADAM 23 antibody (α‐ ADAM 23), ADAM 23 transfectants treated with scrambled peptide or disintegrin peptide, ADAM 23 transfectants treated with disintegrin peptide and non‐immune IgG ( NI , disintegrin peptide) or disintegrin peptide and anti‐αvβ3 antibody (α‐αvβ3, disintegrin peptide), and empty vector transfectants treated with non‐immune IgG ( NI , empty vector) or anti‐αvβ3 integrin antibody (α‐αvβ3, empty vector) was determined ( n = 4). (c) Migration of control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was measured by migration assay ( n = 4). (d) Migration of A549 cells treated with non‐immune IgG ( NI ) or anti‐αvβ3 integrin antibody (α‐αvβ3) was measured using the xCELL igence system ( n = 4). Bars, mean ± SD . * P

    Techniques Used: Migration, Transfection, Expressing, Plasmid Preparation

    Lung metastasis in NOD / SCID mice after i.v. injection of A549 ffLuc‐cp156 transfectants. (a) Lung metastasis of the transfectants with empty vectors, ADAM 23 expression vectors, mock vectors or sh RNA vectors (sh5) was monitored at 1 h, 1 day and 2 weeks by bioluminescence imaging ( n = 6 per group). Representative mice of each group are shown. (b) Photon counts were analyzed ( n = 6). (c) Representative microscopic views of the lung sections. Hematoxylin‐positive blue‐colored nodules indicate metastatic foci. Bar, 1 mm. (d) Evaluation of lung metastasis by calculating the numbers/mm 2 ( n = 3). Bars, mean ± SD . * P
    Figure Legend Snippet: Lung metastasis in NOD / SCID mice after i.v. injection of A549 ffLuc‐cp156 transfectants. (a) Lung metastasis of the transfectants with empty vectors, ADAM 23 expression vectors, mock vectors or sh RNA vectors (sh5) was monitored at 1 h, 1 day and 2 weeks by bioluminescence imaging ( n = 6 per group). Representative mice of each group are shown. (b) Photon counts were analyzed ( n = 6). (c) Representative microscopic views of the lung sections. Hematoxylin‐positive blue‐colored nodules indicate metastatic foci. Bar, 1 mm. (d) Evaluation of lung metastasis by calculating the numbers/mm 2 ( n = 3). Bars, mean ± SD . * P

    Techniques Used: Mouse Assay, Injection, Expressing, Imaging

    Propagation of A549‐derived side population ( SP ) cells by successive rounds of FACS and relative gene expression of the ADAM , ADAMTS and MMP family members in main population ( MP ) and SP . (a) SP cell fraction was sequentially sorted up to nine times. Percentage of SP cells re‐sorted from SP (2), MP (2), SP (3), MP (3), SP (9) and MP (9) is shown ( n = 3). (b) The ADAM , ADAMTS and MMP family members with significantly increased expression in MP (9) compared to SP (9) by the PCR array ( n = 3). Bars, mean ± SD . * P
    Figure Legend Snippet: Propagation of A549‐derived side population ( SP ) cells by successive rounds of FACS and relative gene expression of the ADAM , ADAMTS and MMP family members in main population ( MP ) and SP . (a) SP cell fraction was sequentially sorted up to nine times. Percentage of SP cells re‐sorted from SP (2), MP (2), SP (3), MP (3), SP (9) and MP (9) is shown ( n = 3). (b) The ADAM , ADAMTS and MMP family members with significantly increased expression in MP (9) compared to SP (9) by the PCR array ( n = 3). Bars, mean ± SD . * P

    Techniques Used: Derivative Assay, FACS, Expressing, Polymerase Chain Reaction

    Effects of ADAM 23 expression, anti‐ ADAM 23 antibody and anti‐αvβ3 integrin antibody on colony formation. (a) Expression of ADAM 23 in A549 cells (control) or A549 transfectants with empty vectors, ADAM 23 expression vectors, non‐targeting mock vectors or ADAM 23‐targeting lentiviral vectors (sh1 and sh5) by RT ‐ PCR and by immunoblotting of the immunoprecipitates ( IP ‐ IMB ). Arrow indicates ADAM 23 and the lower band is a non‐specific protein band. (b) Colony formation by control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was assayed ( n = 6). (c) Colony formation by A549 cells transfected with empty vectors or ADAM 23 expression vectors, ADAM 23 transfectants treated with non‐immune IgG ( NI , ADAM 23) or anti‐ ADAM 23 antibody (α‐ ADAM 23), and empty vector transfectants treated with non‐immune IgG ( NI , empty vector) or anti‐αvβ3 integrin antibody (α‐αvβ3) was determined ( n = 6). Bars, mean ± SD . * P
    Figure Legend Snippet: Effects of ADAM 23 expression, anti‐ ADAM 23 antibody and anti‐αvβ3 integrin antibody on colony formation. (a) Expression of ADAM 23 in A549 cells (control) or A549 transfectants with empty vectors, ADAM 23 expression vectors, non‐targeting mock vectors or ADAM 23‐targeting lentiviral vectors (sh1 and sh5) by RT ‐ PCR and by immunoblotting of the immunoprecipitates ( IP ‐ IMB ). Arrow indicates ADAM 23 and the lower band is a non‐specific protein band. (b) Colony formation by control A549 cells (control) and A549 cells transfected with empty vectors, ADAM 23 expression vectors, non‐targeting vectors (mock) or ADAM 23‐targeting vectors (sh1 and sh5) was assayed ( n = 6). (c) Colony formation by A549 cells transfected with empty vectors or ADAM 23 expression vectors, ADAM 23 transfectants treated with non‐immune IgG ( NI , ADAM 23) or anti‐ ADAM 23 antibody (α‐ ADAM 23), and empty vector transfectants treated with non‐immune IgG ( NI , empty vector) or anti‐αvβ3 integrin antibody (α‐αvβ3) was determined ( n = 6). Bars, mean ± SD . * P

    Techniques Used: Expressing, Reverse Transcription Polymerase Chain Reaction, Transfection, Plasmid Preparation

    Characteristics of A549‐derived side population ( SP ) and main population ( MP ) cells. (a) SP and MP in the absence (left) or presence of verapamil (right) are outlined as a percentage of the total cell population. (b) Proliferation of parent, SP and MP cells was measured by cell counting (left) and BrdU labeling methods (right) at 3 days ( n = 6). (c) Photos of colonies formed by parent, SP and MP cells (upper panel) and numbers of colonies/cm 2 ( n = 6). Scale bar = 5 mm. (d) Adherent cells were counted and results are expressed as number of cells/mm 2 ( n = 4). (e) Migration activity at 24 h was determined and results are expressed as percentage of wound closure ( n = 4). Bars, mean ± SD . * P
    Figure Legend Snippet: Characteristics of A549‐derived side population ( SP ) and main population ( MP ) cells. (a) SP and MP in the absence (left) or presence of verapamil (right) are outlined as a percentage of the total cell population. (b) Proliferation of parent, SP and MP cells was measured by cell counting (left) and BrdU labeling methods (right) at 3 days ( n = 6). (c) Photos of colonies formed by parent, SP and MP cells (upper panel) and numbers of colonies/cm 2 ( n = 6). Scale bar = 5 mm. (d) Adherent cells were counted and results are expressed as number of cells/mm 2 ( n = 4). (e) Migration activity at 24 h was determined and results are expressed as percentage of wound closure ( n = 4). Bars, mean ± SD . * P

    Techniques Used: Derivative Assay, Cell Counting, Labeling, Migration, Activity Assay

    Relationship between ADAM 23 expression and stem cell functions. (a) Relative expression levels of ABCG 2 and ABCC 2 to GAPDH in SP (9) and MP (9) were measured by qPCR ( n = 3). (b) Relative expression levels of AKRC 1/2, TM 4 SF 1 and NR 0B1 in ADAM 23‐knockdown (sh5) cells compared to A549 (mock) cells were measured by qPCR ( n = 3). (c,d) Effects of ADAM 23 overexpression in SP (9) cells or treatment of MP (9) cells with anti‐ ADAM 23 antibody on cell adhesion ( n = 4) and colony formation ( n = 6) were examined. (e) Tumorigenicity of A549 ffLuc‐cp156 (mock) and ADAM 23‐knockdown (sh5) cells was monitored at 2 weeks by bioluminescence imaging ( n = 6 per group; bilateral sites of 3 mice), and photon counts were analyzed by LIVING IMAGE 3.0 software. Bars, mean ± SD . * P
    Figure Legend Snippet: Relationship between ADAM 23 expression and stem cell functions. (a) Relative expression levels of ABCG 2 and ABCC 2 to GAPDH in SP (9) and MP (9) were measured by qPCR ( n = 3). (b) Relative expression levels of AKRC 1/2, TM 4 SF 1 and NR 0B1 in ADAM 23‐knockdown (sh5) cells compared to A549 (mock) cells were measured by qPCR ( n = 3). (c,d) Effects of ADAM 23 overexpression in SP (9) cells or treatment of MP (9) cells with anti‐ ADAM 23 antibody on cell adhesion ( n = 4) and colony formation ( n = 6) were examined. (e) Tumorigenicity of A549 ffLuc‐cp156 (mock) and ADAM 23‐knockdown (sh5) cells was monitored at 2 weeks by bioluminescence imaging ( n = 6 per group; bilateral sites of 3 mice), and photon counts were analyzed by LIVING IMAGE 3.0 software. Bars, mean ± SD . * P

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Over Expression, Imaging, Mouse Assay, Software

    31) Product Images from "Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity"

    Article Title: Role of the B Allele of Influenza A Virus Segment 8 in Setting Mammalian Host Range and Pathogenicity

    Journal: Journal of Virology

    doi: 10.1128/JVI.01205-16

    A- and B-allele reassortant viruses replicate efficiently in mammalian cell culture. (A, B) MDCK cells were infected at an MOI of 0.001 with PR8-based viruses, and the supernatants were titrated by plaque assay after 48 h (A) or at the plotted time points (B). Data in panel A are the mean ± SD ( n = 5), while panel B presents the results of a single experiment. (C) A549 cells were infected with the indicated viruses at an MOI of 0.001, and endpoint titers were determined after 48 h. Data are the mean ± range ( n = 2). (D) The replication kinetics of PR8 NS segment reassortants in A549 cells were determined as described in the legend to panel B. Data are the mean ± SD ( n = 3). (E) Primary human CD14 + MDM cells were infected with PR8-based viruses at an MOI of 3, and the titers in the supernatant were determined after 24 h. Data are the mean ± range ( n = 2). A duplicate sample of PR8-infected cells was taken immediately after the virus adsorption period (postwash), and titers were determined to confirm that the virus in samples collected at later times reflected true virus replication and not carryover of the virus inoculum. (F) Results of assays performed as described in the legends to panels A and B. Data represent the mean ± range ( n = 2). (G) MDCK-SIAT cells were infected with Cal7-based viruses at an MOI of 0.01, and the supernatant was titrated at the plotted time points. Data are the mean ± range ( n = 2). (H) MDCK cells were infected with Udorn72-based viruses as described in the legend to panel B. Data represent the mean ± range ( n = 2). Dotted lines indicate the limit of detection.
    Figure Legend Snippet: A- and B-allele reassortant viruses replicate efficiently in mammalian cell culture. (A, B) MDCK cells were infected at an MOI of 0.001 with PR8-based viruses, and the supernatants were titrated by plaque assay after 48 h (A) or at the plotted time points (B). Data in panel A are the mean ± SD ( n = 5), while panel B presents the results of a single experiment. (C) A549 cells were infected with the indicated viruses at an MOI of 0.001, and endpoint titers were determined after 48 h. Data are the mean ± range ( n = 2). (D) The replication kinetics of PR8 NS segment reassortants in A549 cells were determined as described in the legend to panel B. Data are the mean ± SD ( n = 3). (E) Primary human CD14 + MDM cells were infected with PR8-based viruses at an MOI of 3, and the titers in the supernatant were determined after 24 h. Data are the mean ± range ( n = 2). A duplicate sample of PR8-infected cells was taken immediately after the virus adsorption period (postwash), and titers were determined to confirm that the virus in samples collected at later times reflected true virus replication and not carryover of the virus inoculum. (F) Results of assays performed as described in the legends to panels A and B. Data represent the mean ± range ( n = 2). (G) MDCK-SIAT cells were infected with Cal7-based viruses at an MOI of 0.01, and the supernatant was titrated at the plotted time points. Data are the mean ± range ( n = 2). (H) MDCK cells were infected with Udorn72-based viruses as described in the legend to panel B. Data represent the mean ± range ( n = 2). Dotted lines indicate the limit of detection.

    Techniques Used: Cell Culture, Infection, Plaque Assay, Adsorption

    A B-allele reassortant is not deficient in controlling the host IFN response in mammalian cell culture. (A, B) Ability of viruses to replicate despite established antiviral conditions. A549 cells were pretreated with various concentrations of human recombinant IFN-β for 24 h prior to infection with the indicated PR8 viruses at an MOI of 0.01. (A) Virus in the supernatant was titrated by plaque assay at 24 to 48 h p.i. Data are the mean titers from 24-h and 48-h multicycle infections. (B) Cell lysates were prepared at 48 h p.i., subjected to SDS-PAGE, and immunoblotted for cellular IFN-inducible Mx-1, viral NP, and tubulin. (C to E) Induction of host cell type I IFN response during infection with reassortant viruses. (C) Human lung A549 cells were infected for 24 h at various multiplicities, and active type I IFN in the supernatant was quantified using the HEK-Blue reporter cell line. (D, E) Results of assay performed as described in the legend to panel C but at an MOI of 3.
    Figure Legend Snippet: A B-allele reassortant is not deficient in controlling the host IFN response in mammalian cell culture. (A, B) Ability of viruses to replicate despite established antiviral conditions. A549 cells were pretreated with various concentrations of human recombinant IFN-β for 24 h prior to infection with the indicated PR8 viruses at an MOI of 0.01. (A) Virus in the supernatant was titrated by plaque assay at 24 to 48 h p.i. Data are the mean titers from 24-h and 48-h multicycle infections. (B) Cell lysates were prepared at 48 h p.i., subjected to SDS-PAGE, and immunoblotted for cellular IFN-inducible Mx-1, viral NP, and tubulin. (C to E) Induction of host cell type I IFN response during infection with reassortant viruses. (C) Human lung A549 cells were infected for 24 h at various multiplicities, and active type I IFN in the supernatant was quantified using the HEK-Blue reporter cell line. (D, E) Results of assay performed as described in the legend to panel C but at an MOI of 3.

    Techniques Used: Cell Culture, Recombinant, Infection, Plaque Assay, SDS Page

    Induction of host innate immune response during infection with reassortant viruses. (A) Human CD14 + MDMs were infected at an MOI of 1 for 24 h, and the levels of various cytokines and chemokines in the supernatant were determined using an immunospot blot array. Values represent those from a heat map of the mean fold change in the level of expression with respect to the level in a mock-infected sample (Mock). sTREM-1, serum soluble triggering receptor expressed on myeloid cells-1; sICAM-1, soluble intercellular adhesion molecule-1; CSF2 and CSF3, colony-stimulating factors 2 and 3, respectively. (B, C) A549 cells were infected at an MOI of 5, cell lysates were generated at the indicated times, and the polypeptide composition was determined by TMT-based quantitative mass spectrometry. (B) Values represent those from a heat map of the mean fold change with respect to the value for mock-infected samples. The zoomed portion shows a subcluster of heavily upregulated antiviral proteins. (C) The quantitative temporal expression of specific antiviral restriction factors Mx-1, IFIT2, OASL, IFIT3, IFIT1, and IFIH1 is plotted.
    Figure Legend Snippet: Induction of host innate immune response during infection with reassortant viruses. (A) Human CD14 + MDMs were infected at an MOI of 1 for 24 h, and the levels of various cytokines and chemokines in the supernatant were determined using an immunospot blot array. Values represent those from a heat map of the mean fold change in the level of expression with respect to the level in a mock-infected sample (Mock). sTREM-1, serum soluble triggering receptor expressed on myeloid cells-1; sICAM-1, soluble intercellular adhesion molecule-1; CSF2 and CSF3, colony-stimulating factors 2 and 3, respectively. (B, C) A549 cells were infected at an MOI of 5, cell lysates were generated at the indicated times, and the polypeptide composition was determined by TMT-based quantitative mass spectrometry. (B) Values represent those from a heat map of the mean fold change with respect to the value for mock-infected samples. The zoomed portion shows a subcluster of heavily upregulated antiviral proteins. (C) The quantitative temporal expression of specific antiviral restriction factors Mx-1, IFIT2, OASL, IFIT3, IFIT1, and IFIH1 is plotted.

    Techniques Used: Infection, Expressing, Generated, Mass Spectrometry

    32) Product Images from "Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases"

    Article Title: Nonproteolytic K29-Linked Ubiquitination of the PB2 Replication Protein of Influenza A Viruses by Proviral Cullin 4-Based E3 Ligases

    Journal: mBio

    doi: 10.1128/mBio.00305-20

    CRL4 E3 ligase complexes mediate PB2 ubiquitination. (A) HEK293T cells were transiently transfected with the indicated expression plasmids. At 36 h posttransfection, cells were treated with MG132 (10 μM) for 4 h. Anti-Flag immunoprecipitation (IP) was performed from cell lysates prepared under strong denaturing conditions (2% SDS), and the ubiquitinated forms of PB2 were detected using antiubiquitin antibody, followed by an anti-Flag immunoblot assay to detect PB2. Expression of Flag-PB2 and the Strep fusion proteins was monitored in cell lysate (Input). A dashed line marks that a lane from the initial membrane has been removed (not shown here). (B) HEK293 cells stably expressing Strep-DDB1, Strep-DCAF12L1 (D12L1), or Strep-DCAF11 (D11) were transfected with the indicated siRNA and 24 h later transfected with 3×Flag-PB2 for 36 h and treated with MG132 (10 μM) for 4 h. Cell lysate was processed for anti-Flag immunoprecipitation and immunoblotting as shown in panel A. (C) HEK293T cells were infected with H1N1 WSN -Strep at an MOI of 3 for different times and treated with MG132 (10 μM) for 4 h before cell lysis (left). Similarly, A549 cells were infected with H1N1 WSN -Strep at an MOI of 3 for 8 h and treated or not with 10 μM MG132 4 h before cell lysis (right). Strep-PB2 protein was pulled using Strep-Tactin Sepharose beads, and antiubiquitin and anti-PB2 immunoblotting assays were performed. (D) HEK293T and A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 WSN -Strep at an MOI of 3 for 6 and 8 h, respectively, and treated with MG132 (10 μM) for 4 h before cell lysis. PB2-Strep pulldown and immunoblot assays were performed as described for panel C.
    Figure Legend Snippet: CRL4 E3 ligase complexes mediate PB2 ubiquitination. (A) HEK293T cells were transiently transfected with the indicated expression plasmids. At 36 h posttransfection, cells were treated with MG132 (10 μM) for 4 h. Anti-Flag immunoprecipitation (IP) was performed from cell lysates prepared under strong denaturing conditions (2% SDS), and the ubiquitinated forms of PB2 were detected using antiubiquitin antibody, followed by an anti-Flag immunoblot assay to detect PB2. Expression of Flag-PB2 and the Strep fusion proteins was monitored in cell lysate (Input). A dashed line marks that a lane from the initial membrane has been removed (not shown here). (B) HEK293 cells stably expressing Strep-DDB1, Strep-DCAF12L1 (D12L1), or Strep-DCAF11 (D11) were transfected with the indicated siRNA and 24 h later transfected with 3×Flag-PB2 for 36 h and treated with MG132 (10 μM) for 4 h. Cell lysate was processed for anti-Flag immunoprecipitation and immunoblotting as shown in panel A. (C) HEK293T cells were infected with H1N1 WSN -Strep at an MOI of 3 for different times and treated with MG132 (10 μM) for 4 h before cell lysis (left). Similarly, A549 cells were infected with H1N1 WSN -Strep at an MOI of 3 for 8 h and treated or not with 10 μM MG132 4 h before cell lysis (right). Strep-PB2 protein was pulled using Strep-Tactin Sepharose beads, and antiubiquitin and anti-PB2 immunoblotting assays were performed. (D) HEK293T and A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 WSN -Strep at an MOI of 3 for 6 and 8 h, respectively, and treated with MG132 (10 μM) for 4 h before cell lysis. PB2-Strep pulldown and immunoblot assays were performed as described for panel C.

    Techniques Used: Transfection, Expressing, Immunoprecipitation, Stable Transfection, Infection, Lysis

    Involvement of CRL4 factors in IAV infection. (A) A549 cells were transfected with siRNA nontarget (NT) or siRNA targeting CRL4 factors (si target) for 48 h and then infected at an MOI of 0.0001 PFU/cell (H1N1 WSN ) or 0.001 PFU/cell (H1N1 pdm09 and H3N2). Viral titers were determined by plaque-forming assay at the indicated time points. Statistical significances are given in Table S1 . (B) A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 pdm09 at an MOI of 3 PFU/cell. Total cell lysates were prepared at the indicated time postinfection and analyzed by Western blotting using the indicated antibodies. The relative amounts of viral proteins in siRNA-treated samples compared to the corresponding siRNA nontarget (NT) samples are indicated. (C) A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 WSN at an MOI of 5 PFU/cell. At 6 h postinfection, cells were fixed, permeabilized, and stained with an anti-NP antibody (green) and with Hoechst 33342 (blue). Representative images of NP localization are shown. Quantification of the NP labeling is provided in Fig. S2C . (D) HEK293T cells were noninfected (NI) or infected with H1N1 WSN (left) or H1N1 WSN -PB2-Strep (right) virus at an MOI of 3 for 6 h. Cell lysate was subjected to anti-PB2 antibody and IgG (left) or to Strep-Tactin pulldown (right). Proteins in the pulled fractions and in 1/10 of whole-cell extract (inputs) were assessed in an immunoblot assay as indicated. (E) HEK293 cells stably expressing Strep-DDB1, Strep-DCAFs (D12L1 for DCAF12L1 or D11 for DCAF11), or Strep-mCherry (control) were infected with H1N1 WSN at an MOI of 3 for 6 h and subjected to Strep-Tactin pulldown. The PB2 protein copulled with the Strep-CRL4 factors was detected using anti-PB2 antibody. One-fourth of whole-cell extract was used to detect the Strep-CRL4 factors in the input since they were undetected in a 1/10 fraction, indicating a low level of Strep fusion expression. The asterisk indicates an aspecific band.
    Figure Legend Snippet: Involvement of CRL4 factors in IAV infection. (A) A549 cells were transfected with siRNA nontarget (NT) or siRNA targeting CRL4 factors (si target) for 48 h and then infected at an MOI of 0.0001 PFU/cell (H1N1 WSN ) or 0.001 PFU/cell (H1N1 pdm09 and H3N2). Viral titers were determined by plaque-forming assay at the indicated time points. Statistical significances are given in Table S1 . (B) A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 pdm09 at an MOI of 3 PFU/cell. Total cell lysates were prepared at the indicated time postinfection and analyzed by Western blotting using the indicated antibodies. The relative amounts of viral proteins in siRNA-treated samples compared to the corresponding siRNA nontarget (NT) samples are indicated. (C) A549 cells transfected with the indicated siRNA for 48 h were infected with H1N1 WSN at an MOI of 5 PFU/cell. At 6 h postinfection, cells were fixed, permeabilized, and stained with an anti-NP antibody (green) and with Hoechst 33342 (blue). Representative images of NP localization are shown. Quantification of the NP labeling is provided in Fig. S2C . (D) HEK293T cells were noninfected (NI) or infected with H1N1 WSN (left) or H1N1 WSN -PB2-Strep (right) virus at an MOI of 3 for 6 h. Cell lysate was subjected to anti-PB2 antibody and IgG (left) or to Strep-Tactin pulldown (right). Proteins in the pulled fractions and in 1/10 of whole-cell extract (inputs) were assessed in an immunoblot assay as indicated. (E) HEK293 cells stably expressing Strep-DDB1, Strep-DCAFs (D12L1 for DCAF12L1 or D11 for DCAF11), or Strep-mCherry (control) were infected with H1N1 WSN at an MOI of 3 for 6 h and subjected to Strep-Tactin pulldown. The PB2 protein copulled with the Strep-CRL4 factors was detected using anti-PB2 antibody. One-fourth of whole-cell extract was used to detect the Strep-CRL4 factors in the input since they were undetected in a 1/10 fraction, indicating a low level of Strep fusion expression. The asterisk indicates an aspecific band.

    Techniques Used: Infection, Transfection, Western Blot, Staining, Labeling, Stable Transfection, Expressing

    CRL4s mediate different patterns of PB2 ubiquitination that are involved in infection. (A) HEK293 cells stably expressing Strep-DCAF12L1 or Strep-DCAF11 were cotransfected with the indicated PB2 mutants fused with 3×Flag tag together with HA-ubiquitin. At 48 h posttransfection, cell lysates were subjected to Flag pulldown and analyzed by immunoblotting with the indicated antibodies. Expression of Flag-PB2 and Strep fusions was monitored in cell lysate (Input). (B) A549 cells infected at an MOI of 0.001 with recombinant H1N1 pdm09 viruses, either wild type or mutated in the PB2 lysines targeted by each of the CRL4 E3 ligases as indicated. Viral titers were determined in triplicates by plaque-forming assay at the indicated time points, and significance was measured using one-way ANOVA (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).
    Figure Legend Snippet: CRL4s mediate different patterns of PB2 ubiquitination that are involved in infection. (A) HEK293 cells stably expressing Strep-DCAF12L1 or Strep-DCAF11 were cotransfected with the indicated PB2 mutants fused with 3×Flag tag together with HA-ubiquitin. At 48 h posttransfection, cell lysates were subjected to Flag pulldown and analyzed by immunoblotting with the indicated antibodies. Expression of Flag-PB2 and Strep fusions was monitored in cell lysate (Input). (B) A549 cells infected at an MOI of 0.001 with recombinant H1N1 pdm09 viruses, either wild type or mutated in the PB2 lysines targeted by each of the CRL4 E3 ligases as indicated. Viral titers were determined in triplicates by plaque-forming assay at the indicated time points, and significance was measured using one-way ANOVA (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001).

    Techniques Used: Infection, Stable Transfection, Expressing, Recombinant

    33) Product Images from "Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing"

    Article Title: Bacterial Outer Membrane Vesicles Induce Vitronectin Release Into the Bronchoalveolar Space Conferring Protection From Complement-Mediated Killing

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2018.01559

    Human type II alveolar cells (A549) produce vitronectin after stimulation with bacterial OMV. Surface-bound vitronectin was detected on A549 epithelial cells by flow cytometry (A) . Cells were starved for 18 h prior to the experiment. Following the addition of OMV (5 μg) from P. aeruginosa (solid squares) or nontypeable H. influenzae (solid triangles), the cells were harvested at indicated time points. Untreated cells were used as controls (open circles). Mean values of two biological replicates are shown, each comprising three technical repetitions. ANOVA was used to calculate the p -values. A corresponding increase in vitronectin ( VTN ) mRNA levels was seen 1–3 h after challenge with 1 μg OMV from either bacterial species (B) . Symbols represent mean values and error bars represent standard error of the mean of three biological replicates. The difference in mean values between respective bacterial species and controls were assessed using a t -test.
    Figure Legend Snippet: Human type II alveolar cells (A549) produce vitronectin after stimulation with bacterial OMV. Surface-bound vitronectin was detected on A549 epithelial cells by flow cytometry (A) . Cells were starved for 18 h prior to the experiment. Following the addition of OMV (5 μg) from P. aeruginosa (solid squares) or nontypeable H. influenzae (solid triangles), the cells were harvested at indicated time points. Untreated cells were used as controls (open circles). Mean values of two biological replicates are shown, each comprising three technical repetitions. ANOVA was used to calculate the p -values. A corresponding increase in vitronectin ( VTN ) mRNA levels was seen 1–3 h after challenge with 1 μg OMV from either bacterial species (B) . Symbols represent mean values and error bars represent standard error of the mean of three biological replicates. The difference in mean values between respective bacterial species and controls were assessed using a t -test.

    Techniques Used: Flow Cytometry, Cytometry

    34) Product Images from "Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin"

    Article Title: Transcriptional activation of p21Waf1 contributes to suppression of HR by p53 in response to replication arrest induced by camptothecin

    Journal: Oncotarget

    doi: 10.18632/oncotarget.25172

    Effect of p53 or p21 Waf1 siRNA depletion on RPA2 phosphorylation and stability of p53 complex with RPA ( A ) A549 cells were collected following one-hour 500 nM CPT treatment and the levels of p53 and p21 Waf1 as well as an overall RPA2 phosphorylation or phosphorylation at the residues Ser 29 , Tyr 21 and Ser 4/8 were analyzed by western blots; -b and -h indicate base non-phosphorylated and hyper-phosphorylated RPA2 forms. GAPDH and Ponceau Red staining were used as protein loading controls. ( B ) In CPT-treated cells, p21 Waf1 was silenced by siRNA as indicated. Stability of the RPA/p53 complex was analyzed by western blots following p53 or RPA1 immunoprecipitation. 15% of the total cell lysate was loaded in the last lane. ( C ) A scheme of cell treatment with CPT used in our experiments. Cells were pulse-treated with 500 nM CPT for one hour and later maintained in a drug-free medium. DNA synthesis in intact A549 cells or its p53- or p21 Waf1 siRNA depleted derivatives was analyzed by the [ 3 H]-thymidine incorporation assay at different time intervals following CPT pulse treatment. The results are normalized to the DNA synthesis rate of untreated cells.
    Figure Legend Snippet: Effect of p53 or p21 Waf1 siRNA depletion on RPA2 phosphorylation and stability of p53 complex with RPA ( A ) A549 cells were collected following one-hour 500 nM CPT treatment and the levels of p53 and p21 Waf1 as well as an overall RPA2 phosphorylation or phosphorylation at the residues Ser 29 , Tyr 21 and Ser 4/8 were analyzed by western blots; -b and -h indicate base non-phosphorylated and hyper-phosphorylated RPA2 forms. GAPDH and Ponceau Red staining were used as protein loading controls. ( B ) In CPT-treated cells, p21 Waf1 was silenced by siRNA as indicated. Stability of the RPA/p53 complex was analyzed by western blots following p53 or RPA1 immunoprecipitation. 15% of the total cell lysate was loaded in the last lane. ( C ) A scheme of cell treatment with CPT used in our experiments. Cells were pulse-treated with 500 nM CPT for one hour and later maintained in a drug-free medium. DNA synthesis in intact A549 cells or its p53- or p21 Waf1 siRNA depleted derivatives was analyzed by the [ 3 H]-thymidine incorporation assay at different time intervals following CPT pulse treatment. The results are normalized to the DNA synthesis rate of untreated cells.

    Techniques Used: Recombinase Polymerase Amplification, Cycling Probe Technology, Western Blot, Staining, Immunoprecipitation, DNA Synthesis, Thymidine Incorporation Assay

    Expression of conformational or DNA-binding p53 mutants, p53(His175) and p53 (His273), in A549 cells affects CPT-induced RPA2 phosphorylation and HR ( A ) Cells were harvested following one-hour CPT treatment at 500 nM. Expression of p53, p21 Waf1 and an overall RPA2 phosphorylation were analyzed by western blot with antibodies specific for p21 Waf1 , RPA1, mutant p53 (ab32049, Abcam) or for both wild-type and the mutant p53 (Pab240, Abcam). It was experimentally shown that ab32049 antibody (Abcam) does not react with wild-type p53 in A549 cells. Binding of p53 to RPA in the parental or the p21 Waf1 -depleted cells was analyzed by western blotting following p53 immunoprecipitation with anti-p53 antibody (Pab240, Abcam). ( B ) Parental A549 cells or cells expressing p53 (His175) or p53 (His273) were synchronized with nocodazole, pulse treated with CPT at the entry to S phase and later maintained in drug-free medium. Bars represent the relative cell frequencies at different stages of the cell cycle at the indicated times. ( C) HR frequency within pDR-GFP recombination substrate were measured 28 hours following CPT treatment.
    Figure Legend Snippet: Expression of conformational or DNA-binding p53 mutants, p53(His175) and p53 (His273), in A549 cells affects CPT-induced RPA2 phosphorylation and HR ( A ) Cells were harvested following one-hour CPT treatment at 500 nM. Expression of p53, p21 Waf1 and an overall RPA2 phosphorylation were analyzed by western blot with antibodies specific for p21 Waf1 , RPA1, mutant p53 (ab32049, Abcam) or for both wild-type and the mutant p53 (Pab240, Abcam). It was experimentally shown that ab32049 antibody (Abcam) does not react with wild-type p53 in A549 cells. Binding of p53 to RPA in the parental or the p21 Waf1 -depleted cells was analyzed by western blotting following p53 immunoprecipitation with anti-p53 antibody (Pab240, Abcam). ( B ) Parental A549 cells or cells expressing p53 (His175) or p53 (His273) were synchronized with nocodazole, pulse treated with CPT at the entry to S phase and later maintained in drug-free medium. Bars represent the relative cell frequencies at different stages of the cell cycle at the indicated times. ( C) HR frequency within pDR-GFP recombination substrate were measured 28 hours following CPT treatment.

    Techniques Used: Expressing, Binding Assay, Cycling Probe Technology, Western Blot, Mutagenesis, Recombinase Polymerase Amplification, Immunoprecipitation

    Phosphorylation of RPA in response to replication arrest induced by CPT contributes to the dissociation of the RPA/p53 complex ( A ) Cells were collected for analysis following one-hour treatment with 500 nM CPT. p53-negative H1299 cells were transfected with the transactivation-deficient p53(22.23) mutant. p53 binding was assessed following RPA1 immunoprecipitation. The expression levels of p53(22.23), RPA1 and phosphorylation of RPA2 in untreated cells were used as loading controls. ( B ) The residues within the N-terminal RPA2 domain reported to be phosphorylated by CDKs, ATR/ATM and DNA-PK were replaced with alanine or glutamic acid, thus producing RPA2A or RPA2D4 mutants that imitate non-phosphorylated or phosphorylated forms of RPA2, respectively. The consensus sites for the kinases are indicated. Expression levels of the recombinant wild type RPA2 and the mutants in A549 cells prior to or after siRNA silencing of endogenous RPA2. The cells with the silenced endogenous RPA2 were treated with 500 nM CPT for one hour. After p53 immunoprecipitation, RPA binding was analyzed on western blots with anti-RPA1 antibody.
    Figure Legend Snippet: Phosphorylation of RPA in response to replication arrest induced by CPT contributes to the dissociation of the RPA/p53 complex ( A ) Cells were collected for analysis following one-hour treatment with 500 nM CPT. p53-negative H1299 cells were transfected with the transactivation-deficient p53(22.23) mutant. p53 binding was assessed following RPA1 immunoprecipitation. The expression levels of p53(22.23), RPA1 and phosphorylation of RPA2 in untreated cells were used as loading controls. ( B ) The residues within the N-terminal RPA2 domain reported to be phosphorylated by CDKs, ATR/ATM and DNA-PK were replaced with alanine or glutamic acid, thus producing RPA2A or RPA2D4 mutants that imitate non-phosphorylated or phosphorylated forms of RPA2, respectively. The consensus sites for the kinases are indicated. Expression levels of the recombinant wild type RPA2 and the mutants in A549 cells prior to or after siRNA silencing of endogenous RPA2. The cells with the silenced endogenous RPA2 were treated with 500 nM CPT for one hour. After p53 immunoprecipitation, RPA binding was analyzed on western blots with anti-RPA1 antibody.

    Techniques Used: Recombinase Polymerase Amplification, Cycling Probe Technology, Transfection, Mutagenesis, Binding Assay, Immunoprecipitation, Expressing, Recombinant, Western Blot

    Involvement of the p53/p21 Waf1 axis in regulation of HR in response to replication arrest by CPT ( A ) Control A549 cells or cells with p53 or p21 Waf1 silenced by siRNA were synchronized in mitosis by nocodazole. After release from nocodazole arrest, cells were allowed to progress through the cell cycle and collected at different time-points to evaluate cell cycle progression by flow cytometry. Upon entrance into S phase, the cells were pulse-treated with CPT and later maintained in a drug-free medium. Cell cycle profiles and derivative bar graphs show the relative frequencies of cells in each stage of cell cycle at the indicated time points. ( B ) DNA synthesis was assessed at different time intervals following CPT treatment by [ 3 H]-thymidine incorporation assay and normalized to the DNA synthesis rate of untreated cells. ( C ) The percentages of apoptotic cells at the indicated times were assessed by flow cytometry following staining with FITC-conjugated Annexin V. ( D ]. The cells were harvested 27 hours following CPT addition and the HR frequencies were analyzed. Chi-square tests detected significant differences between p21siRNA and p53siRNA depleted cells (χ 2 = 26.3; p
    Figure Legend Snippet: Involvement of the p53/p21 Waf1 axis in regulation of HR in response to replication arrest by CPT ( A ) Control A549 cells or cells with p53 or p21 Waf1 silenced by siRNA were synchronized in mitosis by nocodazole. After release from nocodazole arrest, cells were allowed to progress through the cell cycle and collected at different time-points to evaluate cell cycle progression by flow cytometry. Upon entrance into S phase, the cells were pulse-treated with CPT and later maintained in a drug-free medium. Cell cycle profiles and derivative bar graphs show the relative frequencies of cells in each stage of cell cycle at the indicated time points. ( B ) DNA synthesis was assessed at different time intervals following CPT treatment by [ 3 H]-thymidine incorporation assay and normalized to the DNA synthesis rate of untreated cells. ( C ) The percentages of apoptotic cells at the indicated times were assessed by flow cytometry following staining with FITC-conjugated Annexin V. ( D ]. The cells were harvested 27 hours following CPT addition and the HR frequencies were analyzed. Chi-square tests detected significant differences between p21siRNA and p53siRNA depleted cells (χ 2 = 26.3; p

    Techniques Used: Cycling Probe Technology, Flow Cytometry, Cytometry, DNA Synthesis, Thymidine Incorporation Assay, Staining

    35) Product Images from "Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions"

    Article Title: Nitrative DNA damage in lung epithelial cells exposed to indium nanoparticles and indium ions

    Journal: Scientific Reports

    doi: 10.1038/s41598-020-67488-3

    Effects of iNOS and endocytosis inhibitors on indium-induced 8-nitroG formation. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. The cells were co-treated with 1400 W, Bay, MBCD, MDC and CytoD and 8-nitroG formation was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitive image analysis for the effects of iNOS and endocytosis inhibitors on indium-exposed A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of iNOS and endocytosis inhibitors on indium-induced 8-nitroG formation. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. The cells were co-treated with 1400 W, Bay, MBCD, MDC and CytoD and 8-nitroG formation was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitive image analysis for the effects of iNOS and endocytosis inhibitors on indium-exposed A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Immunocytochemistry, Staining, Software

    8-NitroG formation in indium-treated cells. A549 cells were incubated with the indicated concentrations of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. Positive control was prepared by incubating A549 cells with culture supernatant of MNCNT-exposed cells as described in “ Methods ” section. 8-NitroG formation was detected by immunocytochemistry as described in “ Methods ” section. ( A ) Fluorescent images of indium-induced 8-nitroG formation in A549 cells. The red fluorescence shows 8-nitroG formation and the blue fluorescence shows the nucleus stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis for indium-induced 8-nitroG formation in A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 4–8 independent experiments. * p
    Figure Legend Snippet: 8-NitroG formation in indium-treated cells. A549 cells were incubated with the indicated concentrations of In 2 O 3 , ITO and InCl 3 for 4 h at 37 °C. Positive control was prepared by incubating A549 cells with culture supernatant of MNCNT-exposed cells as described in “ Methods ” section. 8-NitroG formation was detected by immunocytochemistry as described in “ Methods ” section. ( A ) Fluorescent images of indium-induced 8-nitroG formation in A549 cells. The red fluorescence shows 8-nitroG formation and the blue fluorescence shows the nucleus stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis for indium-induced 8-nitroG formation in A549 cells. The staining intensity per area was quantified with an ImageJ software, and the relative intensity of the control was set at 1. The data were expressed as means ± SD of 4–8 independent experiments. * p

    Techniques Used: Incubation, Positive Control, Immunocytochemistry, Fluorescence, Staining, Software

    Proposed mechanism of indium-induced DNA damage in A549 cells.
    Figure Legend Snippet: Proposed mechanism of indium-induced DNA damage in A549 cells.

    Techniques Used:

    Time course of 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of indium-treated A549 cells at different incubation times. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 at 37 °C for indicated durations. 8-NitroG was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis of 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control at 2 h was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. * p
    Figure Legend Snippet: Time course of 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of indium-treated A549 cells at different incubation times. A549 cells were treated with 200 ng/ml of In 2 O 3 , ITO and InCl 3 at 37 °C for indicated durations. 8-NitroG was detected by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( B ) Quantitative image analysis of 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control at 2 h was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. * p

    Techniques Used: Incubation, Immunocytochemistry, Staining, Software

    Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of siRNA on 8-nitroG formation in indium-treated A549 cells. ( A ) Reduction in HMGB1, RAGE and TLR9 expression by siRNA transfection into A549 cells. Effects of siRNA on protein expression were evaluated by Western blotting. These blots were cropped from different parts in the same gel, and each blot was divided with white lines. Full-length blots are shown in Supplementary Figure S2 online. ( B ) Image analysis for HMGB1, RAGE and TLR9 expression in siRNA-transfected A549 cells. These values were expressed as fold changes compared with control. ( C ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of siRNA. Cells were transfected with 10 nM siRNA for HMGB1 , AGER and TLR9 or negative control siRNA for 2 days and then treated with 200 ng/ml indium compounds for 4 h as described in “ Methods ” section. 8-NitroG formation was evaluated by immunocytochemistry as described in “ Methods ” section. The nucleus was stained with Hoechst 33258. Magnification, × 200. ( D ) Quantitative image analysis for the effects of siRNA on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an image J software. The relative intensity of the control was set at 1. ( B , D ) The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Expressing, Transfection, Western Blot, Negative Control, Immunocytochemistry, Staining, Software

    Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p
    Figure Legend Snippet: Effects of HMGB1 and RAGE antibodies on 8-nitroG formation in indium-treated A549 cells. ( A ) Fluorescent images of 8-nitroG formation in indium-treated A549 cells and effects of antibodies. A549 cells were pretreated with 10 µg/ml of anti-HMGB1 and anti-RAGE antbodies and their isotype control IgGs for 30 min, followed by the treatment with 200 ng/ml of In 2 O 3 , ITO and InCl 3 as described in “ Methods ” section. 8-NitroG was detected by immunocytochemistry. The nucleus was stained with Hoechst 33258. Magnification × 200. ( B ) Quantitative image analysis for the effects of antbodies on 8-nitroG formation in indium-treated A549 cells. Staining intensities of 8-nitroG per area were analyzed with an ImageJ software. The relative intensity of the control was set at 1. The data were expressed as means ± SD of 3–4 independent experiments. ** p

    Techniques Used: Immunocytochemistry, Staining, Software

    36) Product Images from "Organ-restricted vascular delivery of nanoparticles for lung cancer therapy"

    Article Title: Organ-restricted vascular delivery of nanoparticles for lung cancer therapy

    Journal: bioRxiv

    doi: 10.1101/2020.03.05.969212

    Increased uptake of EGFR targeted MSNs in human lung cancer cells in vitro . a) Untargeted versus EGFR-targeted uptake of ATTO 633-labeled MSN AVI and MSN tEGFR (red in the upper panel, gray in the lower panel) by human lung cancer cells (A549) in 1 h, co-stained for EGFR (green) and cell nuclei (blue) by immunofluorescence, measured by confocal microscopy. b) Increased MSN tEGFR uptake after 1 h in A549 cells normalized to MSN AVI as measured by flow cytometry analysis. ** p = 0.0079; Values given are an average of four independent experiments ± standard error of the mean. Mann-Whitney test. c) Different modes of nanoparticle uptake ( i.e., receptor-mediated, blue arrow at (1) and unspecific endocytosis, red arrow at (2)) and endosomal escape, orange arrow at (3) observed in TEM micrographs of A549 cells exposed to MSN tEGFR for 3 h. Cell membrane is shown with the black arrow. Scale bars = 2 μm (upper left), 500 nm for insets 1-3.
    Figure Legend Snippet: Increased uptake of EGFR targeted MSNs in human lung cancer cells in vitro . a) Untargeted versus EGFR-targeted uptake of ATTO 633-labeled MSN AVI and MSN tEGFR (red in the upper panel, gray in the lower panel) by human lung cancer cells (A549) in 1 h, co-stained for EGFR (green) and cell nuclei (blue) by immunofluorescence, measured by confocal microscopy. b) Increased MSN tEGFR uptake after 1 h in A549 cells normalized to MSN AVI as measured by flow cytometry analysis. ** p = 0.0079; Values given are an average of four independent experiments ± standard error of the mean. Mann-Whitney test. c) Different modes of nanoparticle uptake ( i.e., receptor-mediated, blue arrow at (1) and unspecific endocytosis, red arrow at (2)) and endosomal escape, orange arrow at (3) observed in TEM micrographs of A549 cells exposed to MSN tEGFR for 3 h. Cell membrane is shown with the black arrow. Scale bars = 2 μm (upper left), 500 nm for insets 1-3.

    Techniques Used: In Vitro, Labeling, Staining, Immunofluorescence, Confocal Microscopy, Flow Cytometry, MANN-WHITNEY, Transmission Electron Microscopy

    Increased uptake of CCR2 targeted nanoparticles in murine alveolar macrophages in vitro . a) EGFR expression in A549 cells in comparison to another NSCLC cell line, H520 cells, b) Western blot analysis of CCR2 expression in murine alveolar macrophage (MH-S) cells in comparison to A549 cells. c) Untargeted versus CCR2-targeted uptake of ATTO 633-labeled MSN AVI and MSN tCCR2 (red in the upper panel, gray in the lower panel) by MH-S cells after 1 h; CCR2 (green) and cell nuclei (blue) visualized by confocal microscopy. Scale bar = 25 μm. d) Increased uptake of ATTO 488-labeled MSN tCCR2 versus MSN AVI after 1 h by MH-S cells measured by flow cytometry analysis. * p = 0.0286, Mann-Whitney test; values given are an average of four independent experiments ± standard error of the mean.
    Figure Legend Snippet: Increased uptake of CCR2 targeted nanoparticles in murine alveolar macrophages in vitro . a) EGFR expression in A549 cells in comparison to another NSCLC cell line, H520 cells, b) Western blot analysis of CCR2 expression in murine alveolar macrophage (MH-S) cells in comparison to A549 cells. c) Untargeted versus CCR2-targeted uptake of ATTO 633-labeled MSN AVI and MSN tCCR2 (red in the upper panel, gray in the lower panel) by MH-S cells after 1 h; CCR2 (green) and cell nuclei (blue) visualized by confocal microscopy. Scale bar = 25 μm. d) Increased uptake of ATTO 488-labeled MSN tCCR2 versus MSN AVI after 1 h by MH-S cells measured by flow cytometry analysis. * p = 0.0286, Mann-Whitney test; values given are an average of four independent experiments ± standard error of the mean.

    Techniques Used: In Vitro, Expressing, Western Blot, Labeling, Confocal Microscopy, Flow Cytometry, MANN-WHITNEY

    37) Product Images from "Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection"

    Article Title: Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection

    Journal: Emerging Microbes & Infections

    doi: 10.1080/22221751.2020.1754135

    Gene mutation and virus replication capability. A: A circos diagram showing the gene mutations. The genome of A/Zhejiang /DTID-ZJU01/2013(H7N9) was used as a reference genome. The circos diagram was constructed to show the mutations in the different genes (HA, NA, PA, PB1, PB2, NP, MA, and NS). In the circos diagram, the innermost circle represents the virus isolated from the plasma. The other circles represent the viruses isolated from the sputum collected at different times. The marked lines show the mutation sites. B-C: The virus replication capability in embryonated chicken eggs. Replication capability was measured by the quantity of RNA and TCID50 titre. D-E: The virus replication capability in embryonated A549 cells. Replication capability was measured by the quantity of RNA and TCID50 titre. F: Cell fluorescence map. The virus replication capability was indicated by fluorescence intensity.
    Figure Legend Snippet: Gene mutation and virus replication capability. A: A circos diagram showing the gene mutations. The genome of A/Zhejiang /DTID-ZJU01/2013(H7N9) was used as a reference genome. The circos diagram was constructed to show the mutations in the different genes (HA, NA, PA, PB1, PB2, NP, MA, and NS). In the circos diagram, the innermost circle represents the virus isolated from the plasma. The other circles represent the viruses isolated from the sputum collected at different times. The marked lines show the mutation sites. B-C: The virus replication capability in embryonated chicken eggs. Replication capability was measured by the quantity of RNA and TCID50 titre. D-E: The virus replication capability in embryonated A549 cells. Replication capability was measured by the quantity of RNA and TCID50 titre. F: Cell fluorescence map. The virus replication capability was indicated by fluorescence intensity.

    Techniques Used: Mutagenesis, Construct, Isolation, Fluorescence

    Isolation of exosomes and identification of viral genes. A: The culture supernatant from A549 cell model without virus infection was observed using transmission electronic microscope. The exosomes were showed by blue arrow. B: The culture supernatant from A549 cell model with ZJU01 strain infection was observed using transmission electronic microscope. The exosome was showed by blue arrow. The viral particles were viewed and showed by red arrow. C: The culture supernatant from A549 cell model with GZ8H002 strain infection was observed using transmission electronic microscope. The exosome was showed by blue arrow. The viral particles were viewed and showed by red arrow. D: The viral content in A549 cell model infected with ZJU01 strain. E: The viral content in A549 cell model infected with GZ8H002 strain. F: The H7 gene content in exosomes. G: The isolation of H7N9 virus from exosomes. H: The TCID50 titre of the H7N9 virus isolated from the exosomes.
    Figure Legend Snippet: Isolation of exosomes and identification of viral genes. A: The culture supernatant from A549 cell model without virus infection was observed using transmission electronic microscope. The exosomes were showed by blue arrow. B: The culture supernatant from A549 cell model with ZJU01 strain infection was observed using transmission electronic microscope. The exosome was showed by blue arrow. The viral particles were viewed and showed by red arrow. C: The culture supernatant from A549 cell model with GZ8H002 strain infection was observed using transmission electronic microscope. The exosome was showed by blue arrow. The viral particles were viewed and showed by red arrow. D: The viral content in A549 cell model infected with ZJU01 strain. E: The viral content in A549 cell model infected with GZ8H002 strain. F: The H7 gene content in exosomes. G: The isolation of H7N9 virus from exosomes. H: The TCID50 titre of the H7N9 virus isolated from the exosomes.

    Techniques Used: Isolation, Infection, Transmission Assay, Microscopy

    38) Product Images from "The Use of PET Imaging for Prognostic Integrin α2β1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses"

    Article Title: The Use of PET Imaging for Prognostic Integrin α2β1 Phenotyping to Detect Non-Small Cell Lung Cancer and Monitor Drug Resistance Responses

    Journal: Theranostics

    doi: 10.7150/thno.19304

    Non-invasive PET imaging of 68 Ga-DOTA-A2B1 with/without a blocking dose of c(DGEAyK) peptide and 18 F-FDG in integrin α2β1-positive A549 and CL1-5 xenograft mouse models. (A) Decay-corrected whole-body planar coronal PET images of the A549 (or CL1-5) tumor-bearing animal model at 10 min post-injection of 7-8 MBq of 68 Ga-DOTA-A2B1 or 11.1 MBq 18 F-FDG tracer (p.i. 60 min). The blocking group was injected with a high dose of the non-radiolabeled c(DGEAyK) peptide (10 mg/kg), and all images are coronal views. The tumors are indicated with arrows. (B) Region of interest (ROI) analysis of the PET images showing 68 Ga-DOTA-A2B1 and 18 F-FDG uptake values in the tumor and major organs, such as the heart, lung, kidneys, liver and muscle; values are presented as the % ID/g ± SD (n = 5). The quantified PET imaging data (Figure 5 B) indicated the binding specificity and favorable biodistribution pattern of integrin tracers. The tumor/muscle ratios of the 68 Ga-DOTA-A2B1 A549 (n = 5) and CL1-5 (n = 5) groups were significantly better (* p ) than that of the 18 F-FDG group (n = 5), in which the A549 tumor was barely visible ( p
    Figure Legend Snippet: Non-invasive PET imaging of 68 Ga-DOTA-A2B1 with/without a blocking dose of c(DGEAyK) peptide and 18 F-FDG in integrin α2β1-positive A549 and CL1-5 xenograft mouse models. (A) Decay-corrected whole-body planar coronal PET images of the A549 (or CL1-5) tumor-bearing animal model at 10 min post-injection of 7-8 MBq of 68 Ga-DOTA-A2B1 or 11.1 MBq 18 F-FDG tracer (p.i. 60 min). The blocking group was injected with a high dose of the non-radiolabeled c(DGEAyK) peptide (10 mg/kg), and all images are coronal views. The tumors are indicated with arrows. (B) Region of interest (ROI) analysis of the PET images showing 68 Ga-DOTA-A2B1 and 18 F-FDG uptake values in the tumor and major organs, such as the heart, lung, kidneys, liver and muscle; values are presented as the % ID/g ± SD (n = 5). The quantified PET imaging data (Figure 5 B) indicated the binding specificity and favorable biodistribution pattern of integrin tracers. The tumor/muscle ratios of the 68 Ga-DOTA-A2B1 A549 (n = 5) and CL1-5 (n = 5) groups were significantly better (* p ) than that of the 18 F-FDG group (n = 5), in which the A549 tumor was barely visible ( p

    Techniques Used: Positron Emission Tomography, Imaging, Blocking Assay, Animal Model, Injection, Binding Assay

    Bone metastasis targeting and treatment response monitoring using 68 Ga-DOTA-A2B1 PET imaging in vivo . (A) Optical bioluminescence imaging results demonstrated the A549 tumor lesion location, and the PET image clearly shows that 68 Ga-DOTA-A2B1 specifically accumulated in osseous tumor grafts located in the left tibia of the mouse, not in the contralateral limb tibia. (B) Schematic representation of the treatment response workflow. Immediately after the first image scan, male mice received a single dose of doxorubicin (5 mg/kg), and at 6 h post-treatment, changes in the drug resistance phenotype were monitored with the 68 Ga-DOTA-A2B1 tracer. PET identified a clear region of contrast in the tumor-implanted tibia, where the tracer uptake was significantly higher than in the pretreatment image. (C) Quantitative analysis of 68 Ga-DOTA-A2B1 PET tracer uptake. The results indicated significantly elevated (19 %) tumor uptake and an increased T/N ratio, as observed by PET imaging, within 6 h of doxorubicin treatment. * p
    Figure Legend Snippet: Bone metastasis targeting and treatment response monitoring using 68 Ga-DOTA-A2B1 PET imaging in vivo . (A) Optical bioluminescence imaging results demonstrated the A549 tumor lesion location, and the PET image clearly shows that 68 Ga-DOTA-A2B1 specifically accumulated in osseous tumor grafts located in the left tibia of the mouse, not in the contralateral limb tibia. (B) Schematic representation of the treatment response workflow. Immediately after the first image scan, male mice received a single dose of doxorubicin (5 mg/kg), and at 6 h post-treatment, changes in the drug resistance phenotype were monitored with the 68 Ga-DOTA-A2B1 tracer. PET identified a clear region of contrast in the tumor-implanted tibia, where the tracer uptake was significantly higher than in the pretreatment image. (C) Quantitative analysis of 68 Ga-DOTA-A2B1 PET tracer uptake. The results indicated significantly elevated (19 %) tumor uptake and an increased T/N ratio, as observed by PET imaging, within 6 h of doxorubicin treatment. * p

    Techniques Used: Positron Emission Tomography, Imaging, In Vivo, Mouse Assay

    Integrin α2β1 is up-regulated after doxorubicin treatment. (A) In vitro FACS analysis of integrin α2β1 expression in wild-type A549 cells (WT) and after doxorubicin treatment (4 μg/mL; 48 h) in A549 cells (DXN). A clear shift in the abundance of integrin α2β1 was observed in the DXN group. (B) Doxorubicin treatment affected the protein levels of integrin α2 and β1 in A549 cells. Semi-quantitative analysis of the Western blot results revealed a markedly higher level of both integrin proteins in DXN cells (** p
    Figure Legend Snippet: Integrin α2β1 is up-regulated after doxorubicin treatment. (A) In vitro FACS analysis of integrin α2β1 expression in wild-type A549 cells (WT) and after doxorubicin treatment (4 μg/mL; 48 h) in A549 cells (DXN). A clear shift in the abundance of integrin α2β1 was observed in the DXN group. (B) Doxorubicin treatment affected the protein levels of integrin α2 and β1 in A549 cells. Semi-quantitative analysis of the Western blot results revealed a markedly higher level of both integrin proteins in DXN cells (** p

    Techniques Used: In Vitro, FACS, Expressing, Western Blot

    Over-expression of integrin α2β1 is associated with the malignant phenotype of human non-small cell lung cancer. (A) Western blot analysis of integrin α2 or β1 expression in the lung cancer cell lines CL1-0, CL1-5 and A549. (B) Flow cytometry was performed to evaluate the distribution level of integrin α2β1 on the tumor surface. (C) Using cell sorting, A549 cells were separated into two populations, non-sorted A549 and integrin α2β1-high A549 ++ cells, and the correlations between the integrin α2β1 level and the association with migration and invasion capabilities were determined. The A549 ++ population displayed a marked expression signature corresponding to a more aggressive NSCLC phenotype. Error bars represent the standard deviation of the mean. * p
    Figure Legend Snippet: Over-expression of integrin α2β1 is associated with the malignant phenotype of human non-small cell lung cancer. (A) Western blot analysis of integrin α2 or β1 expression in the lung cancer cell lines CL1-0, CL1-5 and A549. (B) Flow cytometry was performed to evaluate the distribution level of integrin α2β1 on the tumor surface. (C) Using cell sorting, A549 cells were separated into two populations, non-sorted A549 and integrin α2β1-high A549 ++ cells, and the correlations between the integrin α2β1 level and the association with migration and invasion capabilities were determined. The A549 ++ population displayed a marked expression signature corresponding to a more aggressive NSCLC phenotype. Error bars represent the standard deviation of the mean. * p

    Techniques Used: Over Expression, Western Blot, Expressing, Flow Cytometry, Cytometry, FACS, Migration, Standard Deviation

    In vitro inhibition of 68 Ga-DOTA-A2B1 binding to integrin α2β1 on CL1-5 and A549 cells by varying amounts of A2B1 peptides (n = 3, mean ± SD).
    Figure Legend Snippet: In vitro inhibition of 68 Ga-DOTA-A2B1 binding to integrin α2β1 on CL1-5 and A549 cells by varying amounts of A2B1 peptides (n = 3, mean ± SD).

    Techniques Used: In Vitro, Inhibition, Binding Assay

    Cell uptake assay (left) and cell efflux assay (right) of 68 Ga-DOTA-A2B1 on CL1-5 and A549 cells (n = 3, means ± SD).
    Figure Legend Snippet: Cell uptake assay (left) and cell efflux assay (right) of 68 Ga-DOTA-A2B1 on CL1-5 and A549 cells (n = 3, means ± SD).

    Techniques Used:

    39) Product Images from "Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation"

    Article Title: Multi-walled carbon nanotube induces nitrative DNA damage in human lung epithelial cells via HMGB1-RAGE interaction and Toll-like receptor 9 activation

    Journal: Particle and Fibre Toxicology

    doi: 10.1186/s12989-016-0127-7

    Colocalization of RAGE with HMGB1 and LAMP1 in MWCNT-treated cells. A549 cells were treated with 1 μg/mL MWCNT for 8 h, and then double immunofluorescent analysis was performed to examine the colocalization of RAGE with HMGB1 ( a ) and LAMP1, a lysosomal marker ( b ), as described in Methods. RAGE was stained with the mouse monoclonal antibody and Alexa 488-labeled goat anti-mouse IgG antibody. HMGB1 and LAMP were stained with the corresponding rabbit polyclonal antibodies and Alexa 594-labeled goat anti-rabbit IgG antibody. Hoechst, Hoechst 33258. Magnification, X400. Arrows indicate the colocalization of RAGE with HMGB1 or LAMP in the cytosol
    Figure Legend Snippet: Colocalization of RAGE with HMGB1 and LAMP1 in MWCNT-treated cells. A549 cells were treated with 1 μg/mL MWCNT for 8 h, and then double immunofluorescent analysis was performed to examine the colocalization of RAGE with HMGB1 ( a ) and LAMP1, a lysosomal marker ( b ), as described in Methods. RAGE was stained with the mouse monoclonal antibody and Alexa 488-labeled goat anti-mouse IgG antibody. HMGB1 and LAMP were stained with the corresponding rabbit polyclonal antibodies and Alexa 594-labeled goat anti-rabbit IgG antibody. Hoechst, Hoechst 33258. Magnification, X400. Arrows indicate the colocalization of RAGE with HMGB1 or LAMP in the cytosol

    Techniques Used: Marker, Staining, Labeling

    Association of TLR9 with HMGB1 and RAGE in MWCNT-treated cells. A549 cells were treated with 1 μg/mL MWCNT for 8 h. Double immunofluorescent analysis was performed to examine the colocalization of TLR9 with HMGB1 ( a ) and RAGE ( b ) as described in Methods. HMGB1 and RAGE were stained with the corresponding mouse monoclonal antibodies and Alexa 488-labeled goat anti-mouse IgG antibody. TLR9 was stained with the rabbit polyclonal antibody and Alexa 594-labeled goat anti-rabbit IgG antibody. Hoechst, Hoechst 33258. Magnification, X400. Arrows indicate the colocalization of TLR9 with HMGB1 or RAGE in the cytosol. c Interaction of TLR9 with RAGE and HMGB1. Immunoprecipitation (IP) was performed using anti-TLR9 rabbit polyclonal antibody and TLR9, RAGE and HMGB1 were detected by Western immunoblotting (IB). The membrane was treated with corresponding mouse monoclonal antibodies. Data are representative of 3 independent experiments
    Figure Legend Snippet: Association of TLR9 with HMGB1 and RAGE in MWCNT-treated cells. A549 cells were treated with 1 μg/mL MWCNT for 8 h. Double immunofluorescent analysis was performed to examine the colocalization of TLR9 with HMGB1 ( a ) and RAGE ( b ) as described in Methods. HMGB1 and RAGE were stained with the corresponding mouse monoclonal antibodies and Alexa 488-labeled goat anti-mouse IgG antibody. TLR9 was stained with the rabbit polyclonal antibody and Alexa 594-labeled goat anti-rabbit IgG antibody. Hoechst, Hoechst 33258. Magnification, X400. Arrows indicate the colocalization of TLR9 with HMGB1 or RAGE in the cytosol. c Interaction of TLR9 with RAGE and HMGB1. Immunoprecipitation (IP) was performed using anti-TLR9 rabbit polyclonal antibody and TLR9, RAGE and HMGB1 were detected by Western immunoblotting (IB). The membrane was treated with corresponding mouse monoclonal antibodies. Data are representative of 3 independent experiments

    Techniques Used: Staining, Labeling, Immunoprecipitation, Western Blot

    NO release and GSH decrease in MWCNT-treated cells. A549 cells were treated with 1 μg/ml MWCNT for indicated durations at 37 °C. a NO release from MWCNT-treated cells. The concentrations of NO 2 - and NO 3 - in culture supernatant were measured by the Griess method as described in Methods. b Decrease of GSH contents in MWCNT-treated cells. Intracellular GSH contents were measured with HPLC coupled with an ECD as described in Methods. Data represent means ± SD of 3 independent experiments. * p
    Figure Legend Snippet: NO release and GSH decrease in MWCNT-treated cells. A549 cells were treated with 1 μg/ml MWCNT for indicated durations at 37 °C. a NO release from MWCNT-treated cells. The concentrations of NO 2 - and NO 3 - in culture supernatant were measured by the Griess method as described in Methods. b Decrease of GSH contents in MWCNT-treated cells. Intracellular GSH contents were measured with HPLC coupled with an ECD as described in Methods. Data represent means ± SD of 3 independent experiments. * p

    Techniques Used: High Performance Liquid Chromatography

    Release of HMGB1 and dsDNA from MWCNT-treated cells and inhibitory effects of antibodies on 8-nitroG formation. a Release of HMGB1 from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT for indicated durations at 37 °C. Then the concentration of HMGB1 in the culture supernatant was measured by a commercial ELISA kit. b Release of dsDNA from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT at 37 °C, and the concentration of dsDNA in the culture supernatant was measured with a Quantus fluorometer. a , b Data represent means ± SD of 3 independent experiments. * p
    Figure Legend Snippet: Release of HMGB1 and dsDNA from MWCNT-treated cells and inhibitory effects of antibodies on 8-nitroG formation. a Release of HMGB1 from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT for indicated durations at 37 °C. Then the concentration of HMGB1 in the culture supernatant was measured by a commercial ELISA kit. b Release of dsDNA from MWCNT-treated cells. A549 cells were treated with 1 μg/ml of MWCNT at 37 °C, and the concentration of dsDNA in the culture supernatant was measured with a Quantus fluorometer. a , b Data represent means ± SD of 3 independent experiments. * p

    Techniques Used: Concentration Assay, Enzyme-linked Immunosorbent Assay

    Intracellular distribution of MWCNT and effects of endocytosis inhibitors on inflammatory responses. a Intracellular distribution of CNT-S and effects of endocytosis inhibitors. A549 cells were incubated with 1 μg/ml of CNT-S for 4 h at 37 °C. In certain experiments, the cells were pretreated with 2 mM MBCD or 50 μM MDC for 30 min. Then the cells were observed by TEM as described in Methods. Numerous fibers can be seen in vesicular structures in the cytosol (arrows and inset), but no or few fibers are observed in MBCD- and MDC-pretreated cells. b Intracellular distribution of CNT-L. A549 cells were incubated with 1 μg/ml of CNT-L for 4 h at 37 °C. Only a few fibers are observed in vesicular structures in the cytosol (arrow and inset), and long fibers penetrated the plasma and nuclear membranes (black arrowheads). a , b Bars = 1 μm. N = nucleus. Nuclear membrane is indicated by white arrowheads. Effects of various inhibitors on CNT-L-induced 8-nitroG formation ( c ) and iNOS expression ( d ). A549 cells were treated with 1 μg/ml of CNT-L for 8 h at 37 °C in the presence of an inhibitor (1 μM 1400 W, 10 μM Bay, 1 μM CytoD, 2 mM MBCD or 50 μM MDC). Then, fluorescent immunocytochemistry was performed as described in Methods. Hoechst, Hoechst 33258. Magnification, X200
    Figure Legend Snippet: Intracellular distribution of MWCNT and effects of endocytosis inhibitors on inflammatory responses. a Intracellular distribution of CNT-S and effects of endocytosis inhibitors. A549 cells were incubated with 1 μg/ml of CNT-S for 4 h at 37 °C. In certain experiments, the cells were pretreated with 2 mM MBCD or 50 μM MDC for 30 min. Then the cells were observed by TEM as described in Methods. Numerous fibers can be seen in vesicular structures in the cytosol (arrows and inset), but no or few fibers are observed in MBCD- and MDC-pretreated cells. b Intracellular distribution of CNT-L. A549 cells were incubated with 1 μg/ml of CNT-L for 4 h at 37 °C. Only a few fibers are observed in vesicular structures in the cytosol (arrow and inset), and long fibers penetrated the plasma and nuclear membranes (black arrowheads). a , b Bars = 1 μm. N = nucleus. Nuclear membrane is indicated by white arrowheads. Effects of various inhibitors on CNT-L-induced 8-nitroG formation ( c ) and iNOS expression ( d ). A549 cells were treated with 1 μg/ml of CNT-L for 8 h at 37 °C in the presence of an inhibitor (1 μM 1400 W, 10 μM Bay, 1 μM CytoD, 2 mM MBCD or 50 μM MDC). Then, fluorescent immunocytochemistry was performed as described in Methods. Hoechst, Hoechst 33258. Magnification, X200

    Techniques Used: Incubation, Transmission Electron Microscopy, Expressing, Immunocytochemistry

    Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. a Immunofluorescent images of reduced TLR9 expression and the absence of 8-nitroG formation in siRNA-transfected cells. A549 cells were transfected with negative control or TLR9 siRNA (siRNA-1 and siRNA-2). TLR9 expression and 8-nitroG formation were examined by fluorescent immunocytochemistry. Hoechst, Hoechst 33258. Magnification, X400. b Relative staining intensity of TLR9 in siRNA-transfected A549 cells. The staining intensity of TLR9 per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 4 independent experiments. *** p
    Figure Legend Snippet: Inhibitory effect of TLR9 siRNA on MWCNT-induced 8-nitroG formation. a Immunofluorescent images of reduced TLR9 expression and the absence of 8-nitroG formation in siRNA-transfected cells. A549 cells were transfected with negative control or TLR9 siRNA (siRNA-1 and siRNA-2). TLR9 expression and 8-nitroG formation were examined by fluorescent immunocytochemistry. Hoechst, Hoechst 33258. Magnification, X400. b Relative staining intensity of TLR9 in siRNA-transfected A549 cells. The staining intensity of TLR9 per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 4 independent experiments. *** p

    Techniques Used: Expressing, Transfection, Negative Control, Immunocytochemistry, Staining, Software

    Cytotoxic effect of MWCNT. A549 cells were treated with 1 μg/ml MWCNT for 24 h at 37 °C, and the cell viability was examined by MTT assay as described in Methods. Viability of the control cells was set at 100 %. Data represent means ± SD of 4-6 independent experiments. *** p
    Figure Legend Snippet: Cytotoxic effect of MWCNT. A549 cells were treated with 1 μg/ml MWCNT for 24 h at 37 °C, and the cell viability was examined by MTT assay as described in Methods. Viability of the control cells was set at 100 %. Data represent means ± SD of 4-6 independent experiments. *** p

    Techniques Used: MTT Assay

    Time course of 8-nitroG formation and iNOS expression in MWCNT-treated cells. Immunofluorescent images of 8-nitroG formation ( a ) and iNOS expression ( b ) in MWCNT-treated A549 cells. A549 cells were treated with 1 μg/ml MWCNT for indicated durations at 37 °C, and 8-nitroG formation and iNOS expression were examined by fluorescent immunocytochemistry as described in Methods. Hoechst, Hoechst 33258. Magnification, X200. ( c , d ) Relative staining intensity of 8-nitroG and iNOS in A549 cells. Staining intensities of 8-nitroG ( c ) and iNOS ( d ) per cell were analyzed by an ImageJ software. Relative staining intensity of the control at 4 h was set at 1. Data represent means ± SD of 4 (control) and 6 (CNT-S and CNT-L) independent experiments. ** p
    Figure Legend Snippet: Time course of 8-nitroG formation and iNOS expression in MWCNT-treated cells. Immunofluorescent images of 8-nitroG formation ( a ) and iNOS expression ( b ) in MWCNT-treated A549 cells. A549 cells were treated with 1 μg/ml MWCNT for indicated durations at 37 °C, and 8-nitroG formation and iNOS expression were examined by fluorescent immunocytochemistry as described in Methods. Hoechst, Hoechst 33258. Magnification, X200. ( c , d ) Relative staining intensity of 8-nitroG and iNOS in A549 cells. Staining intensities of 8-nitroG ( c ) and iNOS ( d ) per cell were analyzed by an ImageJ software. Relative staining intensity of the control at 4 h was set at 1. Data represent means ± SD of 4 (control) and 6 (CNT-S and CNT-L) independent experiments. ** p

    Techniques Used: Expressing, Immunocytochemistry, Staining, Software

    DNA damage in CNT-L-treated cells. a Immunofluorescent images of CNT-L-induced 8-oxodG and 8-nitroG formation in A549 cells. A549 cells were treated with CNT-L at indicated concentrations for 8 h at 37 °C, and 8-oxodG and 8-nitroG formation was examined by double immunofluorescent technique as described in Methods. Hoechst, Hoechst 33258. Magnification, X200. b Relative staining intensity of 8-nitroG formed in CNT-L-treated A549 cells. Staining intensity per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 8 (control) or 4 (CNT-L) independent experiments. * p
    Figure Legend Snippet: DNA damage in CNT-L-treated cells. a Immunofluorescent images of CNT-L-induced 8-oxodG and 8-nitroG formation in A549 cells. A549 cells were treated with CNT-L at indicated concentrations for 8 h at 37 °C, and 8-oxodG and 8-nitroG formation was examined by double immunofluorescent technique as described in Methods. Hoechst, Hoechst 33258. Magnification, X200. b Relative staining intensity of 8-nitroG formed in CNT-L-treated A549 cells. Staining intensity per cell was analyzed by an ImageJ software. Relative staining intensity of the control was set at 1. Data represent means ± SD of 8 (control) or 4 (CNT-L) independent experiments. * p

    Techniques Used: Staining, Software

    40) Product Images from "In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549"

    Article Title: In Silico Selection Approach to Develop DNA Aptamers for a Stem-like Cell Subpopulation of Non-small Lung Cancer Adenocarcinoma Cell Line A549

    Journal: Radiology and Oncology

    doi: 10.2478/raon-2018-0014

    Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.
    Figure Legend Snippet: Flow cytometry of cells double labelled with CD90 and aptamers. (A) Flow cytometry was performed for FITC-labelled aptamer pools comparing binding to A549 cell binding of the pool of the library), the sixth cycle and the pool following negative selection cycle against blood cells. The density plots are showing that there was no difference in the percentage of labelled cells for each of the pools. Displacement of the population in the density plot, and shift of position on the histograms for the cells labelled with the pool, which followed the negative selection against blood cells, was observed, showing a cell population with increased fluorescence intensity compared to the cells labelled with the initial library or pool of 6 th cycle. (B) CD90 binding and cell sorting: After adjustment of cellular gates for un-labelled cells, CD90+, the library and the pool following negative selection, the double-labelled cells labelled for CD90/library and CD90/negative selection pool were compared. Here, the gate was set to collect the most intensely labelled cells with the aptamer pool following negative selection. To confirm the profile of the isolated population, the sorted cells were analysed again by flow cytometry (post sort). The density plots show the presence of a well-defined secondary population that was double labelled, suggesting that aptamers were recognizing a sub-population of CD90, which was positive for stem cells within the A549 cell population.

    Techniques Used: Flow Cytometry, Cytometry, Binding Assay, Selection, Fluorescence, FACS, Isolation

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    Flow Cytometry:

    Article Title: Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog
    Article Snippet: .. Flow cytometry screening and cell sorting A549, 293T or 293T (+ pCMV Shh) cells were labeled with Shh antibody (Abcam 53281, 1:100) or therapeutic test antibodies (1:100) for 1 hour at room temperature after serum blocking. .. Following two PBS washes, a FITC-conjugated secondary antibody (Abcam 97029, 1:100) or an Alexa-Fluor 647 (Invitrogen, 1:1000) was used to label cells.

    Cytometry:

    Article Title: Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog
    Article Snippet: .. Flow cytometry screening and cell sorting A549, 293T or 293T (+ pCMV Shh) cells were labeled with Shh antibody (Abcam 53281, 1:100) or therapeutic test antibodies (1:100) for 1 hour at room temperature after serum blocking. .. Following two PBS washes, a FITC-conjugated secondary antibody (Abcam 97029, 1:100) or an Alexa-Fluor 647 (Invitrogen, 1:1000) was used to label cells.

    Cell Culture:

    Article Title: Novel pathogenic characteristics of highly pathogenic avian influenza virus H7N9: viraemia and extrapulmonary infection
    Article Snippet: .. A549 cells were cultured in DMEM supplemented with 10% FBS at 37°C and 5% CO2. ..

    Incubation:

    Article Title: Circular RNA screening from EIF3a in lung cancer, et al. Circular RNA screening from EIF3a in lung cancer
    Article Snippet: .. A549 cell lysates were incubated with anti‐rabbit AGO2 antibodies (Abcam, Cambridge, MA) or anti‐rabbit IgG antibodies (Millipore, Billerica, MA). .. 2.5 Western blot analysis After transfection for 48 hours, western blot assay was carried out using the standard method, and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA).

    Article Title: Hsa_circ_0001946 Inhibits Lung Cancer Progression and Mediates Cisplatin Sensitivity in Non-small Cell Lung Cancer via the Nucleotide Excision Repair Signaling Pathway
    Article Snippet: .. A549 cell lysates were incubated with anti-rabbit IgG or anti-rabbit AGO2 antibodies (Abcam, Cambridge, MA) with rotation at 4°C overnight. .. Next, the expression of circRNAs was determined by qRT-PCR.

    Labeling:

    Article Title: Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog
    Article Snippet: .. Flow cytometry screening and cell sorting A549, 293T or 293T (+ pCMV Shh) cells were labeled with Shh antibody (Abcam 53281, 1:100) or therapeutic test antibodies (1:100) for 1 hour at room temperature after serum blocking. .. Following two PBS washes, a FITC-conjugated secondary antibody (Abcam 97029, 1:100) or an Alexa-Fluor 647 (Invitrogen, 1:1000) was used to label cells.

    FACS:

    Article Title: Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog
    Article Snippet: .. Flow cytometry screening and cell sorting A549, 293T or 293T (+ pCMV Shh) cells were labeled with Shh antibody (Abcam 53281, 1:100) or therapeutic test antibodies (1:100) for 1 hour at room temperature after serum blocking. .. Following two PBS washes, a FITC-conjugated secondary antibody (Abcam 97029, 1:100) or an Alexa-Fluor 647 (Invitrogen, 1:1000) was used to label cells.

    Western Blot:

    Article Title: MicroRNA-422a functions as a tumor suppressor in non-small cell lung cancer through SULF2-mediated TGF-β/SMAD signaling pathway
    Article Snippet: .. Western blot analysisThe H522 and A549 cells were rinsed 3 times with cold PBS, and lysed on ice with the total protein lysis buffer for 10 min. ..

    Article Title: Investigation of Cytotoxicity Apoptotic and Inflammatory Responses of Biosynthesized Zinc Oxide Nanoparticles from Ocimum sanctum Linn in Human Skin Keratinocyte (Hacat) and Human Lung Epithelial (A549) Cells
    Article Snippet: .. Western Blot Assay HaCaT and A549 cells were treated with gZnNPs (0, 25, and 35 μ g/ml) for 24 h, and after exposure, the cell lysate was prepared in RIPA buffer (ab156034). ..

    Lysis:

    Article Title: MicroRNA-422a functions as a tumor suppressor in non-small cell lung cancer through SULF2-mediated TGF-β/SMAD signaling pathway
    Article Snippet: .. Western blot analysisThe H522 and A549 cells were rinsed 3 times with cold PBS, and lysed on ice with the total protein lysis buffer for 10 min. ..

    Blocking Assay:

    Article Title: Preclinical characterization of therapeutic antibodies targeted at the carboxy-terminus of Sonic hedgehog
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    Abcam a549 taxolr cells
    ERK1/2 activation in drug-resistant cancer cells was involved in decreased FBXW7 expression. a Cells of <t>A549,</t> <t>A549-taxolR,</t> T47D, T47D-doxR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with indicated concentrations of paclitaxel for 24 h, and western blotting was performed. b Cells of A549, A549-taxolR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with U0126, an ERK1/2 inhibitor (10 μM), for 12 h, and western blotting was performed. c Cell lysates of A549 and A549-taxolR with or without U0126 treatment (10 μM) for 12 h were immunoprecipitated with a ubiquitin construct (Ub) and immunoblotted with HSF1. Western blotting was also performed. d A549 and A549-taxolR cells were pretreated with or without U0126 treatment (10 μM) for 1 h and were treated with indicated doses of paclitaxel. Cell death was analyzed by western blot analysis, and cell viability was determined by the MTT assay. Values are presented as percentages of cell survival in paclitaxel-treated cells relative to untreated cells and as the mean ± SD of at least three independent experiments. Statistics calculated based on a Student’s t -test, * p
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    Abcam a549 mdr cells
    NOLC1 knockdown significantly promoted cell apoptosis in <t>A549/MDR</t> cells. a Flow cytometry analysis of A549/MDR cells stained with Annexin V and PI. The effect of siRNA-mediated NOLC1 knockdown on apoptosis was analyzed. Data were expressed as means ± SD of at least three experiments. b Hoechst staining assay was used to analyze the percentage of cells with fragmented nuclei in A549/MDR cells. ** p
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    Abcam a549 cell culture supernatants
    Increased expression of TNC following IAV infection of <t>A549</t> cells. ( a ) The abundance of tnc transcripts was measured with qRT-PCR (open bars) 24 hours after IAV infection (IAV+) of A549 cells and compared to uninfected A549 cells (IAV−). The closed bar shows the results obtained with DNA microarrays. The y-axis shows the fold-increase in transcripts associated with IAV-infected cells compared to uninfected controls. ( b ) The abundance of TNC was measured in A549 cells 24 hours after IAV infection (IAV+) and compared to uninfected A549 cells (IAV−). Additionally, TNC was similarly measured in BALF and plasma collected from mice 8 days after being inoculated with IAV (IAV+; n = 4 mice) or allantoic fluid (IAV−; n = 4 mice). The mean and sem are indicated. A student’s t-test was used to measure statistical significance.
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    Abcam a549 cells
    Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in <t>A549</t> cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P
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    ERK1/2 activation in drug-resistant cancer cells was involved in decreased FBXW7 expression. a Cells of A549, A549-taxolR, T47D, T47D-doxR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with indicated concentrations of paclitaxel for 24 h, and western blotting was performed. b Cells of A549, A549-taxolR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with U0126, an ERK1/2 inhibitor (10 μM), for 12 h, and western blotting was performed. c Cell lysates of A549 and A549-taxolR with or without U0126 treatment (10 μM) for 12 h were immunoprecipitated with a ubiquitin construct (Ub) and immunoblotted with HSF1. Western blotting was also performed. d A549 and A549-taxolR cells were pretreated with or without U0126 treatment (10 μM) for 1 h and were treated with indicated doses of paclitaxel. Cell death was analyzed by western blot analysis, and cell viability was determined by the MTT assay. Values are presented as percentages of cell survival in paclitaxel-treated cells relative to untreated cells and as the mean ± SD of at least three independent experiments. Statistics calculated based on a Student’s t -test, * p

    Journal: Cell Death & Disease

    Article Title: Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

    doi: 10.1038/s41419-020-2600-3

    Figure Lengend Snippet: ERK1/2 activation in drug-resistant cancer cells was involved in decreased FBXW7 expression. a Cells of A549, A549-taxolR, T47D, T47D-doxR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with indicated concentrations of paclitaxel for 24 h, and western blotting was performed. b Cells of A549, A549-taxolR, HSF1−/− mouse embryonic fibroblasts, and HSF1+/+ mouse embryonic fibroblasts were treated with U0126, an ERK1/2 inhibitor (10 μM), for 12 h, and western blotting was performed. c Cell lysates of A549 and A549-taxolR with or without U0126 treatment (10 μM) for 12 h were immunoprecipitated with a ubiquitin construct (Ub) and immunoblotted with HSF1. Western blotting was also performed. d A549 and A549-taxolR cells were pretreated with or without U0126 treatment (10 μM) for 1 h and were treated with indicated doses of paclitaxel. Cell death was analyzed by western blot analysis, and cell viability was determined by the MTT assay. Values are presented as percentages of cell survival in paclitaxel-treated cells relative to untreated cells and as the mean ± SD of at least three independent experiments. Statistics calculated based on a Student’s t -test, * p

    Article Snippet: ChIP assay A549 and A549-taxolR cells were fixed, nuclei were isolated, and chromatin was sheared by sonication using the ChIP Kit according to the manufacturer’s instructions (ab500; Abcam).

    Techniques: Activation Assay, Expressing, Western Blot, Immunoprecipitation, Construct, MTT Assay

    Increased phosphorylation of HSF1 at Ser303/307 in drug-resistant cancer cells was responsible for paclitaxel resistance. A549, A549-taxolR, T47D, and T47D-doxR cells after transient transfection of a WT-HSF1 and S303/307E, or b siRNA-HSF1 and S303/307A treated with indicated concentrations of paclitaxel for 24 h. Western blotting was performed. Cell viability was determined by the MTT assay. Data are expressed as the mean ± SD of at least three independent experiments. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control. Statistics calculated based on a Student’s t -test, * p

    Journal: Cell Death & Disease

    Article Title: Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

    doi: 10.1038/s41419-020-2600-3

    Figure Lengend Snippet: Increased phosphorylation of HSF1 at Ser303/307 in drug-resistant cancer cells was responsible for paclitaxel resistance. A549, A549-taxolR, T47D, and T47D-doxR cells after transient transfection of a WT-HSF1 and S303/307E, or b siRNA-HSF1 and S303/307A treated with indicated concentrations of paclitaxel for 24 h. Western blotting was performed. Cell viability was determined by the MTT assay. Data are expressed as the mean ± SD of at least three independent experiments. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the negative control. Statistics calculated based on a Student’s t -test, * p

    Article Snippet: ChIP assay A549 and A549-taxolR cells were fixed, nuclei were isolated, and chromatin was sheared by sonication using the ChIP Kit according to the manufacturer’s instructions (ab500; Abcam).

    Techniques: Transfection, Western Blot, MTT Assay, Software, Negative Control

    HSF1 depletion down-regulated the transcriptional level of mdr1 in drug-resistant cancer cells. a, b After A549 lung cancer cells, paclitaxel-resistant A549 cells (A549-taxolR), T47D breast cancer cells, and doxorubicin-resistant T47D cells (T47D-doxR) were transfected with a control siRNA (siCont) or siHSF1, western blotting or RT-PCR was performed. c After A549 and A549-taxolR were transfected with shRNA (shCont), shHSF1, CRISPR/Cas9-Control (Cont), or HSF1 CRISPR/Cas9 knockout (KO) plasmid, western blotting (top) or RT-PCR (middle) was performed. c , bottom Luciferase assays in A549 and A549-taxolR with CRISPR/Cas9-Control or HSF1 CRISPR/Cas9 KO plasmid were performed after transfection with a luciferase reporter construct with the mdr1 promoter. Values are presented as the mean ± SD of at least three independent experiments. Statistics calculated based on one-way ANOVA, * p

    Journal: Cell Death & Disease

    Article Title: Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

    doi: 10.1038/s41419-020-2600-3

    Figure Lengend Snippet: HSF1 depletion down-regulated the transcriptional level of mdr1 in drug-resistant cancer cells. a, b After A549 lung cancer cells, paclitaxel-resistant A549 cells (A549-taxolR), T47D breast cancer cells, and doxorubicin-resistant T47D cells (T47D-doxR) were transfected with a control siRNA (siCont) or siHSF1, western blotting or RT-PCR was performed. c After A549 and A549-taxolR were transfected with shRNA (shCont), shHSF1, CRISPR/Cas9-Control (Cont), or HSF1 CRISPR/Cas9 knockout (KO) plasmid, western blotting (top) or RT-PCR (middle) was performed. c , bottom Luciferase assays in A549 and A549-taxolR with CRISPR/Cas9-Control or HSF1 CRISPR/Cas9 KO plasmid were performed after transfection with a luciferase reporter construct with the mdr1 promoter. Values are presented as the mean ± SD of at least three independent experiments. Statistics calculated based on one-way ANOVA, * p

    Article Snippet: ChIP assay A549 and A549-taxolR cells were fixed, nuclei were isolated, and chromatin was sheared by sonication using the ChIP Kit according to the manufacturer’s instructions (ab500; Abcam).

    Techniques: Transfection, Western Blot, Reverse Transcription Polymerase Chain Reaction, shRNA, CRISPR, Knock-Out, Plasmid Preparation, Luciferase, Construct

    Decreased expression of FBXW7 in drug-resistant cells inhibited ubiquitin protein degradation of pHSF1 at Ser303/307. a A549 and A549-taxolR cells were treated with indicated concentrations of paclitaxel with or without MG132 (10 μM) for 12 h, and western blotting was performed. b Cells of A549, A549-taxolR, HSF1−/−, and HSF1+/+ mouse embryonic fibroblasts were treated with indicated concentrations of paclitaxel for 24 h, and western blotting was performed. c , left Immunoprecipitation (IP) was performed using A549 cells stably transfected of shFBXW7. c , right Western blotting was performed using A549 and A549-taxolR cells after transfection of HSF1 CRISPR/Cas9 KO or WT-FBXW7. d A549 and A549-taxolR cells were transiently transfected with point mutants of FBXW7 at Thr205 (T205A, phospho-defective), and western blotting was performed. e Western blot analysis in the phospho-mimicking mutant of HSF1 at Ser303/307 (S303/307E)-transfected A549-taxolR cells was performed after treatment with 10 μg/mL cycloheximide (CHX) for various time periods. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the control. Results are presented as the mean ± SD of at least three independent experiments. Statistics calculated based on a Student’s t -test, * p

    Journal: Cell Death & Disease

    Article Title: Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

    doi: 10.1038/s41419-020-2600-3

    Figure Lengend Snippet: Decreased expression of FBXW7 in drug-resistant cells inhibited ubiquitin protein degradation of pHSF1 at Ser303/307. a A549 and A549-taxolR cells were treated with indicated concentrations of paclitaxel with or without MG132 (10 μM) for 12 h, and western blotting was performed. b Cells of A549, A549-taxolR, HSF1−/−, and HSF1+/+ mouse embryonic fibroblasts were treated with indicated concentrations of paclitaxel for 24 h, and western blotting was performed. c , left Immunoprecipitation (IP) was performed using A549 cells stably transfected of shFBXW7. c , right Western blotting was performed using A549 and A549-taxolR cells after transfection of HSF1 CRISPR/Cas9 KO or WT-FBXW7. d A549 and A549-taxolR cells were transiently transfected with point mutants of FBXW7 at Thr205 (T205A, phospho-defective), and western blotting was performed. e Western blot analysis in the phospho-mimicking mutant of HSF1 at Ser303/307 (S303/307E)-transfected A549-taxolR cells was performed after treatment with 10 μg/mL cycloheximide (CHX) for various time periods. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the control. Results are presented as the mean ± SD of at least three independent experiments. Statistics calculated based on a Student’s t -test, * p

    Article Snippet: ChIP assay A549 and A549-taxolR cells were fixed, nuclei were isolated, and chromatin was sheared by sonication using the ChIP Kit according to the manufacturer’s instructions (ab500; Abcam).

    Techniques: Expressing, Western Blot, Immunoprecipitation, Stable Transfection, Transfection, CRISPR, Mutagenesis, Software

    Phosphorylation patterns of HSF1 in drug-resistant cancer cells. a , b Protein levels in A549 lung cancer cells, paclitaxel-resistant A549 cells (A549-taxolR), T47D breast cancer cells, and doxorubicin-resistant T47D cells (T47D-doxR) after treatment for indicated times and with indicated concentrations of paclitaxel were examined by western blot analysis. c A549-taxolR cells and T47D-doxR cells were transiently transfected with FLAG-tagged point mutants of HSF1 at Ser230 (S230A, phospho-defective), Ser326 (S326E, phospho-mimicking), and Ser303/307 (S303/307A, phospho-defective), and western blotting was performed. d , left A549-taxolR cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A and S303/307E, phospho-defective and mimicking, respectively) were treated with indicated concentrations of paclitaxel for 24 h. d , right A549-taxolR cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A, phospho-defective) or Ser326 (S326E, phospho-mimicking) were treated paclitaxel for indicated times. e Western blotting was performed using cytosolic and nuclear fractions from A549 cells and A549-taxolR cells. Fraction purity and equal loading were assessed by western blots for lamin B1 and β-actin. f HEK293T cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A and S303/307E, phospho-defective and mimicking, respectively) were fractionated. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the untreated control.

    Journal: Cell Death & Disease

    Article Title: Decreased expression of FBXW7 by ERK1/2 activation in drug-resistant cancer cells confers transcriptional activation of MDR1 by suppression of ubiquitin degradation of HSF1

    doi: 10.1038/s41419-020-2600-3

    Figure Lengend Snippet: Phosphorylation patterns of HSF1 in drug-resistant cancer cells. a , b Protein levels in A549 lung cancer cells, paclitaxel-resistant A549 cells (A549-taxolR), T47D breast cancer cells, and doxorubicin-resistant T47D cells (T47D-doxR) after treatment for indicated times and with indicated concentrations of paclitaxel were examined by western blot analysis. c A549-taxolR cells and T47D-doxR cells were transiently transfected with FLAG-tagged point mutants of HSF1 at Ser230 (S230A, phospho-defective), Ser326 (S326E, phospho-mimicking), and Ser303/307 (S303/307A, phospho-defective), and western blotting was performed. d , left A549-taxolR cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A and S303/307E, phospho-defective and mimicking, respectively) were treated with indicated concentrations of paclitaxel for 24 h. d , right A549-taxolR cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A, phospho-defective) or Ser326 (S326E, phospho-mimicking) were treated paclitaxel for indicated times. e Western blotting was performed using cytosolic and nuclear fractions from A549 cells and A549-taxolR cells. Fraction purity and equal loading were assessed by western blots for lamin B1 and β-actin. f HEK293T cells after transfection with FLAG-tagged point mutants of HSF1 at Ser303/307 (S303/307A and S303/307E, phospho-defective and mimicking, respectively) were fractionated. Protein levels were quantified using Image J software, and data are expressed as the fold change relative to the untreated control.

    Article Snippet: ChIP assay A549 and A549-taxolR cells were fixed, nuclei were isolated, and chromatin was sheared by sonication using the ChIP Kit according to the manufacturer’s instructions (ab500; Abcam).

    Techniques: Western Blot, Transfection, Software

    NOLC1 knockdown significantly promoted cell apoptosis in A549/MDR cells. a Flow cytometry analysis of A549/MDR cells stained with Annexin V and PI. The effect of siRNA-mediated NOLC1 knockdown on apoptosis was analyzed. Data were expressed as means ± SD of at least three experiments. b Hoechst staining assay was used to analyze the percentage of cells with fragmented nuclei in A549/MDR cells. ** p

    Journal: Cellular & Molecular Biology Letters

    Article Title: Identification and validation of NOLC1 as a potential target for enhancing sensitivity in multidrug resistant non-small cell lung cancer cells

    doi: 10.1186/s11658-018-0119-8

    Figure Lengend Snippet: NOLC1 knockdown significantly promoted cell apoptosis in A549/MDR cells. a Flow cytometry analysis of A549/MDR cells stained with Annexin V and PI. The effect of siRNA-mediated NOLC1 knockdown on apoptosis was analyzed. Data were expressed as means ± SD of at least three experiments. b Hoechst staining assay was used to analyze the percentage of cells with fragmented nuclei in A549/MDR cells. ** p

    Article Snippet: Flow cytometric analysis of cell apoptosis Flow cytometry was performed on the A549/MDR cells using the Annexin V-FITC Kit (ab14085, Abcam, Cambridge, UK).

    Techniques: Flow Cytometry, Cytometry, Staining

    Effects of NOLC1 knockdown on drug resistance-associated expression in A549/MDR cells. The mRNA ( a ) and protein ( b ) expression of NOLC1, LRP, MRP and Beclin was determined by qRT-PCR and western blot analysis, respectively. GAPDH was used as internal controls. Data were expressed as means ± SD of at least three experiments. * p

    Journal: Cellular & Molecular Biology Letters

    Article Title: Identification and validation of NOLC1 as a potential target for enhancing sensitivity in multidrug resistant non-small cell lung cancer cells

    doi: 10.1186/s11658-018-0119-8

    Figure Lengend Snippet: Effects of NOLC1 knockdown on drug resistance-associated expression in A549/MDR cells. The mRNA ( a ) and protein ( b ) expression of NOLC1, LRP, MRP and Beclin was determined by qRT-PCR and western blot analysis, respectively. GAPDH was used as internal controls. Data were expressed as means ± SD of at least three experiments. * p

    Article Snippet: Flow cytometric analysis of cell apoptosis Flow cytometry was performed on the A549/MDR cells using the Annexin V-FITC Kit (ab14085, Abcam, Cambridge, UK).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Heat map generated by hierarchical clustering of differentially expressed genes between A549/DDP and A549/MDR cell lines. Rows: genes; Columns: cell lines

    Journal: Cellular & Molecular Biology Letters

    Article Title: Identification and validation of NOLC1 as a potential target for enhancing sensitivity in multidrug resistant non-small cell lung cancer cells

    doi: 10.1186/s11658-018-0119-8

    Figure Lengend Snippet: Heat map generated by hierarchical clustering of differentially expressed genes between A549/DDP and A549/MDR cell lines. Rows: genes; Columns: cell lines

    Article Snippet: Flow cytometric analysis of cell apoptosis Flow cytometry was performed on the A549/MDR cells using the Annexin V-FITC Kit (ab14085, Abcam, Cambridge, UK).

    Techniques: Generated

    NOLC1 knockdown significantly decreased cell viability of A549/MDR cells after multidrug treatments. A549/MDR cells were transfected with siNOLC1 or NC for 48 h after treatment with 5-FU, paclitaxel, mitomycin, vinorelbine tartrate, DDP and gemcitabine hydrochloride. CCK-8 assay was used to assess the drug cytotoxicities in A549/MDR cells. Data were expressed as means ± SD of at least three experiments. * p

    Journal: Cellular & Molecular Biology Letters

    Article Title: Identification and validation of NOLC1 as a potential target for enhancing sensitivity in multidrug resistant non-small cell lung cancer cells

    doi: 10.1186/s11658-018-0119-8

    Figure Lengend Snippet: NOLC1 knockdown significantly decreased cell viability of A549/MDR cells after multidrug treatments. A549/MDR cells were transfected with siNOLC1 or NC for 48 h after treatment with 5-FU, paclitaxel, mitomycin, vinorelbine tartrate, DDP and gemcitabine hydrochloride. CCK-8 assay was used to assess the drug cytotoxicities in A549/MDR cells. Data were expressed as means ± SD of at least three experiments. * p

    Article Snippet: Flow cytometric analysis of cell apoptosis Flow cytometry was performed on the A549/MDR cells using the Annexin V-FITC Kit (ab14085, Abcam, Cambridge, UK).

    Techniques: Transfection, CCK-8 Assay

    Quantitative real-time PCR was used to confirm the microarray results in A549/DDP and A549/MDR lines. The expression levels of upregulated DEGs ( a ) and downregulated DEGs ( b ) were determined by qRT-PCR in A549/DDP and A549/MDR cell lines. GAPDH was used as an internal control. * p

    Journal: Cellular & Molecular Biology Letters

    Article Title: Identification and validation of NOLC1 as a potential target for enhancing sensitivity in multidrug resistant non-small cell lung cancer cells

    doi: 10.1186/s11658-018-0119-8

    Figure Lengend Snippet: Quantitative real-time PCR was used to confirm the microarray results in A549/DDP and A549/MDR lines. The expression levels of upregulated DEGs ( a ) and downregulated DEGs ( b ) were determined by qRT-PCR in A549/DDP and A549/MDR cell lines. GAPDH was used as an internal control. * p

    Article Snippet: Flow cytometric analysis of cell apoptosis Flow cytometry was performed on the A549/MDR cells using the Annexin V-FITC Kit (ab14085, Abcam, Cambridge, UK).

    Techniques: Real-time Polymerase Chain Reaction, Microarray, Expressing, Quantitative RT-PCR

    Increased expression of TNC following IAV infection of A549 cells. ( a ) The abundance of tnc transcripts was measured with qRT-PCR (open bars) 24 hours after IAV infection (IAV+) of A549 cells and compared to uninfected A549 cells (IAV−). The closed bar shows the results obtained with DNA microarrays. The y-axis shows the fold-increase in transcripts associated with IAV-infected cells compared to uninfected controls. ( b ) The abundance of TNC was measured in A549 cells 24 hours after IAV infection (IAV+) and compared to uninfected A549 cells (IAV−). Additionally, TNC was similarly measured in BALF and plasma collected from mice 8 days after being inoculated with IAV (IAV+; n = 4 mice) or allantoic fluid (IAV−; n = 4 mice). The mean and sem are indicated. A student’s t-test was used to measure statistical significance.

    Journal: Scientific Reports

    Article Title: The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection

    doi: 10.1038/s41598-018-29714-x

    Figure Lengend Snippet: Increased expression of TNC following IAV infection of A549 cells. ( a ) The abundance of tnc transcripts was measured with qRT-PCR (open bars) 24 hours after IAV infection (IAV+) of A549 cells and compared to uninfected A549 cells (IAV−). The closed bar shows the results obtained with DNA microarrays. The y-axis shows the fold-increase in transcripts associated with IAV-infected cells compared to uninfected controls. ( b ) The abundance of TNC was measured in A549 cells 24 hours after IAV infection (IAV+) and compared to uninfected A549 cells (IAV−). Additionally, TNC was similarly measured in BALF and plasma collected from mice 8 days after being inoculated with IAV (IAV+; n = 4 mice) or allantoic fluid (IAV−; n = 4 mice). The mean and sem are indicated. A student’s t-test was used to measure statistical significance.

    Article Snippet: To quantify TNC expression in A549 cell-culture supernatants, mouse BALF and plasma, an ELISA was used according to the manufactures instructions (Abcam; Cambridge, United Kingdom).

    Techniques: Expressing, Infection, Quantitative RT-PCR, Mouse Assay

    Inactivation of prtF.2 decreased the adherence of GAS to IAV-infected A549 cells. The number of wild-type (wt), prtF.2 mutant ( prtF.2 − ), or the complemented prtF.2 mutant ( prtF.2 −/+ ) bacteria adhered to A549 cells following 24 hours of IAV infection (IAV+) or not (IAV−) was determined by dilution plating. The means and sem are reported. Two-way ANOVA with Tukey’s post-test was used to measure the significance of the differences among the strains.

    Journal: Scientific Reports

    Article Title: The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection

    doi: 10.1038/s41598-018-29714-x

    Figure Lengend Snippet: Inactivation of prtF.2 decreased the adherence of GAS to IAV-infected A549 cells. The number of wild-type (wt), prtF.2 mutant ( prtF.2 − ), or the complemented prtF.2 mutant ( prtF.2 −/+ ) bacteria adhered to A549 cells following 24 hours of IAV infection (IAV+) or not (IAV−) was determined by dilution plating. The means and sem are reported. Two-way ANOVA with Tukey’s post-test was used to measure the significance of the differences among the strains.

    Article Snippet: To quantify TNC expression in A549 cell-culture supernatants, mouse BALF and plasma, an ELISA was used according to the manufactures instructions (Abcam; Cambridge, United Kingdom).

    Techniques: Infection, Mutagenesis

    Inactivation of prtF.2 decreased co-localization of GAS to TNC expressed by A549 cells. The co-localization of adhered MGAS315 wild-type (wt), prtF.2 mutant ( prtF.2 − ), or the complemented prtF.2 mutant strain ( prtF.2 −/+ ) to TNC expressed by A549 cells infected with IAV (IAV+) or not (IAV−) was determined. Cells were fixed and indirect immunofluorescence was used to detect GAS (green) or TNC (red), with antibodies specific to each protein. Coverslips were imaged with an Olympus BX 60 fluorescent scope (60X magnification) with a Nikon DS camera. Co-localization of GAS and TNC was detected in the merged images.

    Journal: Scientific Reports

    Article Title: The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection

    doi: 10.1038/s41598-018-29714-x

    Figure Lengend Snippet: Inactivation of prtF.2 decreased co-localization of GAS to TNC expressed by A549 cells. The co-localization of adhered MGAS315 wild-type (wt), prtF.2 mutant ( prtF.2 − ), or the complemented prtF.2 mutant strain ( prtF.2 −/+ ) to TNC expressed by A549 cells infected with IAV (IAV+) or not (IAV−) was determined. Cells were fixed and indirect immunofluorescence was used to detect GAS (green) or TNC (red), with antibodies specific to each protein. Coverslips were imaged with an Olympus BX 60 fluorescent scope (60X magnification) with a Nikon DS camera. Co-localization of GAS and TNC was detected in the merged images.

    Article Snippet: To quantify TNC expression in A549 cell-culture supernatants, mouse BALF and plasma, an ELISA was used according to the manufactures instructions (Abcam; Cambridge, United Kingdom).

    Techniques: Mutagenesis, Infection, Immunofluorescence

    IAV infection of A549 cells increased expression of genes encoding FnIII domains. qRT-PCR (open bars) was used to measure transcripts of select genes that were differentially expressed in response to IAV infection (X-axis), as determined by using DNA microarrays (closed bars). The y-axis shows the fold-increase in transcripts associated with IAV-infected cells compared to uninfected controls. Transcripts encoding tenascin N ( tnn ), fibronectin type III and ankyrin repeat domains 1 ( fank1) , and fibronectin type III domain containing 6 ( fndc6) were measured. The means and sem are indicated.

    Journal: Scientific Reports

    Article Title: The Streptococcus pyogenes fibronectin/tenascin-binding protein PrtF.2 contributes to virulence in an influenza superinfection

    doi: 10.1038/s41598-018-29714-x

    Figure Lengend Snippet: IAV infection of A549 cells increased expression of genes encoding FnIII domains. qRT-PCR (open bars) was used to measure transcripts of select genes that were differentially expressed in response to IAV infection (X-axis), as determined by using DNA microarrays (closed bars). The y-axis shows the fold-increase in transcripts associated with IAV-infected cells compared to uninfected controls. Transcripts encoding tenascin N ( tnn ), fibronectin type III and ankyrin repeat domains 1 ( fank1) , and fibronectin type III domain containing 6 ( fndc6) were measured. The means and sem are indicated.

    Article Snippet: To quantify TNC expression in A549 cell-culture supernatants, mouse BALF and plasma, an ELISA was used according to the manufactures instructions (Abcam; Cambridge, United Kingdom).

    Techniques: Infection, Expressing, Quantitative RT-PCR

    Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in A549 cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P

    Journal: Molecular Medicine Reports

    Article Title: MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer

    doi: 10.3892/mmr.2017.7517

    Figure Lengend Snippet: Chanti-MACC-1 induces downregulation of EMT and HGF/MET. (A) Representative image and quantification of HGF and MET protein expression levels in A549 cells, following Chanti-MACC-1 treatment. (B) Representative image and quantification of EMT markers Vimentin, E-cadherin and Slug protein expression analyzed in A549 cells, following Chanti-MACC-1 treatment. (C) Proteins that promoted tumor migration alterations were analyzed in A549 cells following Chanti-MACC-1 treatment. Student's t-test revealed a significant difference. **P

    Article Snippet: The sections were dewaxed by conventional methods and underwent microwave antigen retrieval at 95°C for 10 min. After cooling, they were washed with distilled water and blocked in normal 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. A549 cells and tumor sections were incubated for 1 h at room temperature with MACC-1 antibodies (1:2,000; cat. no. ab106579; Abcam).

    Techniques: Expressing, Migration

    Expression of MACC-1 in non-small-cell lung cancer cells and tissues. (A) RT-qPCR and (B) western blot analysis of MACC-1 mRNA and protein expression levels in NCI-H520, A549 and H358 lung cancer cells, compared with MRC-5 normal lung cells. (C) RT-qPCR and (D) western blot analysis of the mRNA and protein expression level of MACC-1 in large cell carcinoma, squamous cell carcinoma, adenocarcinoma and normal lung tissues. Data are presented as the mean ± standard error of mean, from triplicate samples. **P

    Journal: Molecular Medicine Reports

    Article Title: MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer

    doi: 10.3892/mmr.2017.7517

    Figure Lengend Snippet: Expression of MACC-1 in non-small-cell lung cancer cells and tissues. (A) RT-qPCR and (B) western blot analysis of MACC-1 mRNA and protein expression levels in NCI-H520, A549 and H358 lung cancer cells, compared with MRC-5 normal lung cells. (C) RT-qPCR and (D) western blot analysis of the mRNA and protein expression level of MACC-1 in large cell carcinoma, squamous cell carcinoma, adenocarcinoma and normal lung tissues. Data are presented as the mean ± standard error of mean, from triplicate samples. **P

    Article Snippet: The sections were dewaxed by conventional methods and underwent microwave antigen retrieval at 95°C for 10 min. After cooling, they were washed with distilled water and blocked in normal 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. A549 cells and tumor sections were incubated for 1 h at room temperature with MACC-1 antibodies (1:2,000; cat. no. ab106579; Abcam).

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    Therapeutic and metastasis-inhibitory effects of Chanti-MACC-1 in A549-bearing mice. (A) Tumor volume was analyzed following Chanti-MACC-1 treatment in a 24-day short term observation. (B) Long-term survival probability was performed in a 150-day observation between Chanti-MACC-1 and PBS treatment groups. (C) NSCLC metastasis was analyzed between Chanti-MACC-1 and PBS treatment animals. (D) MACC-1expression was analyzed via histological staining in tumors following treatment with Chanti-MACC-1. Student's t-test revealed a significant effect. *P

    Journal: Molecular Medicine Reports

    Article Title: MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer

    doi: 10.3892/mmr.2017.7517

    Figure Lengend Snippet: Therapeutic and metastasis-inhibitory effects of Chanti-MACC-1 in A549-bearing mice. (A) Tumor volume was analyzed following Chanti-MACC-1 treatment in a 24-day short term observation. (B) Long-term survival probability was performed in a 150-day observation between Chanti-MACC-1 and PBS treatment groups. (C) NSCLC metastasis was analyzed between Chanti-MACC-1 and PBS treatment animals. (D) MACC-1expression was analyzed via histological staining in tumors following treatment with Chanti-MACC-1. Student's t-test revealed a significant effect. *P

    Article Snippet: The sections were dewaxed by conventional methods and underwent microwave antigen retrieval at 95°C for 10 min. After cooling, they were washed with distilled water and blocked in normal 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. A549 cells and tumor sections were incubated for 1 h at room temperature with MACC-1 antibodies (1:2,000; cat. no. ab106579; Abcam).

    Techniques: Mouse Assay, Staining

    Inhibitory effects of Chanti-MACC-1 on MACC-1 expression and non-small-cell lung cancer cell growth in vitro . (A) MACC-1 mRNA expression levels were decreased in A549 cells following treatment with Chanti-MACC-1. (B) MACC-1 expression alterations in A549 cells transfected with Chanti-MACC-1, determined via immunofluorescence. (C) MTT assays analyzed the inhibitory effects of Chanti-MACC-1 on A549 cells. (D) Migration analysis was performed to detect the efficacy of Chanti-MACC-1 on A549 cells. Student t-tests revealed a significant difference. **P

    Journal: Molecular Medicine Reports

    Article Title: MACC-1 antibody target therapy suppresses growth and migration of non-small cell lung cancer

    doi: 10.3892/mmr.2017.7517

    Figure Lengend Snippet: Inhibitory effects of Chanti-MACC-1 on MACC-1 expression and non-small-cell lung cancer cell growth in vitro . (A) MACC-1 mRNA expression levels were decreased in A549 cells following treatment with Chanti-MACC-1. (B) MACC-1 expression alterations in A549 cells transfected with Chanti-MACC-1, determined via immunofluorescence. (C) MTT assays analyzed the inhibitory effects of Chanti-MACC-1 on A549 cells. (D) Migration analysis was performed to detect the efficacy of Chanti-MACC-1 on A549 cells. Student t-tests revealed a significant difference. **P

    Article Snippet: The sections were dewaxed by conventional methods and underwent microwave antigen retrieval at 95°C for 10 min. After cooling, they were washed with distilled water and blocked in normal 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) at room temperature for 30 min. A549 cells and tumor sections were incubated for 1 h at room temperature with MACC-1 antibodies (1:2,000; cat. no. ab106579; Abcam).

    Techniques: Expressing, In Vitro, Transfection, Immunofluorescence, MTT Assay, Migration