a549 cells  (Millipore)


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    A549 Cells GFP EGFR
    Description:
    A549 GFP EGFR are lung carcinoma epithelial cells from a human 58 year old male caucasion having a ZFN modification creating an EGFR GFP transgene expressed from the endogenous EGFR gene locus This cell line was derived from ATCC catalog No CCL 185
    Catalog Number:
    cll1141
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    Applications:
    A549 Cells GFP-EGFR is an A549 cell line in which the genomic EGFR gene has been endogenously tagged with a Green Fluorescent Protein gene (GFP) using CompoZr(R) Zinc Finger Nuclease technology. Integration resulted in endogenous expression of the fusion protein in which GFP is attached to the C-terminus of EGFR. Fluorescence imaging shows characteristic EGFR membrane expression. Upon the addition of EGF, the cell line shows redistribution of EGFR from the cell membrane to endosomes, making it useful for high content screening of compounds. For example, the redistribution can be abolished by a selective inhibitor of EGFR, Tyrphostin AG 1478. This stable cell line was expanded from a single clone. The target's gene regulation and corresponding protein function are preserved in contrast to cell lines with overexpression via an exogenous promoter.
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    Millipore a549 cells
    A549 Cells GFP EGFR
    A549 GFP EGFR are lung carcinoma epithelial cells from a human 58 year old male caucasion having a ZFN modification creating an EGFR GFP transgene expressed from the endogenous EGFR gene locus This cell line was derived from ATCC catalog No CCL 185
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    Average 99 stars, based on 343 article reviews
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    a549 cells - by Bioz Stars, 2020-09
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    1) Product Images from "Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle"

    Article Title: Cell-to-cell and genome-to-genome variability of Adenovirus transcription tuned by the cell cycle

    Journal: bioRxiv

    doi: 10.1101/2020.02.27.967471

    Visualization of AdV-C5 E1A, E1B-55K and protein VI transcripts in infected cells by bDNA-FISH technique. A) Correlation between input virus amounts and number of virus particles bound to cells. Virus was added to A549 cells at 37°C for 60 min (moi ∼ 54400 or 13600 virus particles per cell). Cells were fixed after removal of unbound virus, stained with mouse 9C12 anti-hexon and Alexa Fluor488-conjugated anti-mouse antibodies and imaged by confocal microscopy. The scatterplot shows number of virus particles per cell, one dot representing one cell. Horizontal bars represent median values and the number of cells analyzed per sample is indicated. B) Infected cells accumulate high numbers of viral transcripts. A549 cells were incubated with virus at moi ∼ 54400 virus particles per cell as described in (A) and fixed after 4 h (left-hand panel) or 11 h (right-hand panel) post removal of unbound virus. Fixed infected and noninfected cells were stained with probes against E1A and E1B-55K mRNAs (left-hand panel) or E1A and protein VI mRNAs (right-hand panel). RNase A treatment prior to staining removes transcript signals, as shown for E1A. C) High number of E1A transcripts can be detected in infected HDF-TERT cells. AdV-C5 was added to cells at 37°C for 12 h (moi ∼ 37500 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 10 h before cells were fixed and stained for E1A mRNAs. Alexa Fluor647 NHS Ester was used for staining of cell area (pseudo-colored green in the figure). D) Timecourse analysis of E1A and E1B-55K transcript accumulation. Virus was added to A549 cells at 37°C for 60 min (moi ∼ 13600 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 0, 1, 4, 7 or 11 h. Fixed cells were stained with probes against E1A and E1B-55K. Mean fluorescence intensity per cell was used to quantify the abundance of E1A and E1B-55K transcripts in cells at the different time points. Horizontal bars represent median values and the number of cells analyzed for each time point is indicated. Representative images from the time points are shown in S1 Fig. E) Correlation of E1A and E1B-55K transcripts in individual infected cells at the indicated time points post infection. The dataset is the same as in (D). r s denotes the Spearman’s correlation rank coefficient (approximate P values
    Figure Legend Snippet: Visualization of AdV-C5 E1A, E1B-55K and protein VI transcripts in infected cells by bDNA-FISH technique. A) Correlation between input virus amounts and number of virus particles bound to cells. Virus was added to A549 cells at 37°C for 60 min (moi ∼ 54400 or 13600 virus particles per cell). Cells were fixed after removal of unbound virus, stained with mouse 9C12 anti-hexon and Alexa Fluor488-conjugated anti-mouse antibodies and imaged by confocal microscopy. The scatterplot shows number of virus particles per cell, one dot representing one cell. Horizontal bars represent median values and the number of cells analyzed per sample is indicated. B) Infected cells accumulate high numbers of viral transcripts. A549 cells were incubated with virus at moi ∼ 54400 virus particles per cell as described in (A) and fixed after 4 h (left-hand panel) or 11 h (right-hand panel) post removal of unbound virus. Fixed infected and noninfected cells were stained with probes against E1A and E1B-55K mRNAs (left-hand panel) or E1A and protein VI mRNAs (right-hand panel). RNase A treatment prior to staining removes transcript signals, as shown for E1A. C) High number of E1A transcripts can be detected in infected HDF-TERT cells. AdV-C5 was added to cells at 37°C for 12 h (moi ∼ 37500 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 10 h before cells were fixed and stained for E1A mRNAs. Alexa Fluor647 NHS Ester was used for staining of cell area (pseudo-colored green in the figure). D) Timecourse analysis of E1A and E1B-55K transcript accumulation. Virus was added to A549 cells at 37°C for 60 min (moi ∼ 13600 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 0, 1, 4, 7 or 11 h. Fixed cells were stained with probes against E1A and E1B-55K. Mean fluorescence intensity per cell was used to quantify the abundance of E1A and E1B-55K transcripts in cells at the different time points. Horizontal bars represent median values and the number of cells analyzed for each time point is indicated. Representative images from the time points are shown in S1 Fig. E) Correlation of E1A and E1B-55K transcripts in individual infected cells at the indicated time points post infection. The dataset is the same as in (D). r s denotes the Spearman’s correlation rank coefficient (approximate P values

    Techniques Used: Infection, Fluorescence In Situ Hybridization, Staining, Confocal Microscopy, Incubation, Fluorescence

    vDNAs within the same nucleus display heterogeneous transcription and replication activities. A) A549 cells were infected with EdC-labeled AdV-C5 as described in legend to Fig. 2 except that cells were fixed after 15 h post removal of unbound virus. Cells were stained with intron probes targeting the E4 transcription unit and vDNA was visualized by a click-reaction. Colocalization of nuclear vDNA with E4 probe signal indicates a transcriptionally active viral genome and the colocalization was scored from maximum projection images of confocal stacks using CellProfiler. Examples of E4 signal-positive vDNAs are indicated by red arrowheads in the representative images. Scale bar = 10 µm. In the right-hand scatterplot, nuclear vDNA numbers are correlated to the number of E4 signal-positive vDNAs within the same nucleus, one dot representing one nucleus. Number of cells analyzed was 442. Note that acetic acid was included into the fixation buffer when analyzing colocalization of E4 signals and vDNA in the nucleus, in order to improve accessibility of the E4 intron probes to nuclear targets [ 114 ]. B) Binding of E1A bDNA-FISH probes to single-stranded vDNA identifies nuclear vDNAs that have progressed to the replication stage. A549 cells were infected with EdC-labeled AdV-C5 as described in legend to Fig. 2 except that cells were fixed after 27 h post removal of unbound virus and AraC was added to the culture medium for the last 20 h. Cells were stained with E1A bDNA-FISH probes, which mark the single-stranded vDNA byproducts of viral genome replication (S6 Fig.), and the infecting vDNAs were visualized by a click-reaction. Two representative cells are shown. The majority of nuclear vDNAs at this time point post infection had progressed to the replication stage as indicated by colocalization of vDNA dots with E1A probe signals, but vDNAs devoid of E1A signal were commonly detected as well (white arrows in the overlay image). C) vDNAs within the same nucleus are associated with different stage replication centers. A549 cells were infected with EdC-labeled AdV-C5 in the absence of AraC and viral replication centers were stained with mouse anti-DBP and secondary Alexa Fluor594-conjugated anti-mouse antibodies, and incoming vDNAs were detected by a click-reaction using azide-Alexa Fluor488. Small DBP-positive puncta indicate early-phase replication centers, and both these early-phase replication centers, as well as the later-phase globular or ring-like DBP-positive structures were associated with incoming vDNAs, thus indicating that vDNAs within the same nucleus start replication asynchronously. All images shown are maximum projections of confocal stacks. Nuclei outlines were drawn from DAPI-stained nuclei. Scale bars = 10 µm.
    Figure Legend Snippet: vDNAs within the same nucleus display heterogeneous transcription and replication activities. A) A549 cells were infected with EdC-labeled AdV-C5 as described in legend to Fig. 2 except that cells were fixed after 15 h post removal of unbound virus. Cells were stained with intron probes targeting the E4 transcription unit and vDNA was visualized by a click-reaction. Colocalization of nuclear vDNA with E4 probe signal indicates a transcriptionally active viral genome and the colocalization was scored from maximum projection images of confocal stacks using CellProfiler. Examples of E4 signal-positive vDNAs are indicated by red arrowheads in the representative images. Scale bar = 10 µm. In the right-hand scatterplot, nuclear vDNA numbers are correlated to the number of E4 signal-positive vDNAs within the same nucleus, one dot representing one nucleus. Number of cells analyzed was 442. Note that acetic acid was included into the fixation buffer when analyzing colocalization of E4 signals and vDNA in the nucleus, in order to improve accessibility of the E4 intron probes to nuclear targets [ 114 ]. B) Binding of E1A bDNA-FISH probes to single-stranded vDNA identifies nuclear vDNAs that have progressed to the replication stage. A549 cells were infected with EdC-labeled AdV-C5 as described in legend to Fig. 2 except that cells were fixed after 27 h post removal of unbound virus and AraC was added to the culture medium for the last 20 h. Cells were stained with E1A bDNA-FISH probes, which mark the single-stranded vDNA byproducts of viral genome replication (S6 Fig.), and the infecting vDNAs were visualized by a click-reaction. Two representative cells are shown. The majority of nuclear vDNAs at this time point post infection had progressed to the replication stage as indicated by colocalization of vDNA dots with E1A probe signals, but vDNAs devoid of E1A signal were commonly detected as well (white arrows in the overlay image). C) vDNAs within the same nucleus are associated with different stage replication centers. A549 cells were infected with EdC-labeled AdV-C5 in the absence of AraC and viral replication centers were stained with mouse anti-DBP and secondary Alexa Fluor594-conjugated anti-mouse antibodies, and incoming vDNAs were detected by a click-reaction using azide-Alexa Fluor488. Small DBP-positive puncta indicate early-phase replication centers, and both these early-phase replication centers, as well as the later-phase globular or ring-like DBP-positive structures were associated with incoming vDNAs, thus indicating that vDNAs within the same nucleus start replication asynchronously. All images shown are maximum projections of confocal stacks. Nuclei outlines were drawn from DAPI-stained nuclei. Scale bars = 10 µm.

    Techniques Used: Infection, Labeling, Staining, Binding Assay, Fluorescence In Situ Hybridization

    G1 cell-cycle phase favors rapid accumulation of E1A transcripts. A) Total nuclear DAPI signals can be used for accurate determination of different cell-cycle stages. Fixed noninfected HeLa-FUCCI cells were stained with DAPI and imaged by automated widefield fluorescent imaging system. The histograms show integrated nuclear DAPI intensities correlated at single-cell level to bins of increasing intensities of the G1 marker Cdt1 (Kusabira Orange) and the S/G2/M marker Geminin (Azami-Green). The increasing intensity bins of Cdt1 and Geminin are equal frequency plots in which each bin has equal number of cells. The integrated nuclear DAPI intensities are normalized to values 0-45. B) E1A transcripts accumulate more rapidly in G1 cells than in S/G2/M cells. AdV-C5 was added to A549 cells at 37°C for 60 min (moi ∼ 54400 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 3 h. Fixed cells were stained with bDNA-FISH E1A probes, Alexa Fluor647 NHS Ester was used for staining of cell area and DAPI for nucleus. Images were acquired by an automated widefield fluorescent imaging system. The mean cytoplasmic E1A signal intensities were used for estimation of E1A transcript abundancies per cell. Cells were classified as G1 or S/G2/M according to total nuclear DAPI signals and 2973 cells were randomly sampled from the total population. Of these sampled cells 1659 were G1 cells and 1314 S/G2/M cells. In the left-hand panel, the mean cytoplasmic E1A signal intensities per cell in the two groups are shown as boxplots. The difference between the groups is statistically significant (permutation test, p=0.0002). In the right-hand panel, histogram of integrated nuclear DAPI intensities of cells was drawn and mean cytoplasmic intensities of E1A transcripts were mapped on the histogram. The histogram was split into five bins of increasing E1A intensities to show the correlation between E1A transcript abundancies per cell and the cell-cycle phase. Each bin contains equal number of cells. C) G1-enriched infected cell population shows increased numbers of E1A transcripts per cell in comparison to a cell population with lower number of G1 cell-cycle stage cells. AdV-C5 was added to serum-starved A375 melanoma cells at 37°C for 60 min (moi ∼ 36250 virus particles per cell), and after removal of unbound virus, cells were further incubated in serum-free (“starved + starved” sample) or serum-containing medium (“starved + serum” sample) for further 9 h. Fixed cells were stained with E1A probes, with Alexa Fluor647 NHS Ester for cell area, with DAPI for nucleus and imaged by confocal microscopy. As judged from integrated nuclear DAPI intensities, 66% of cells were in G1 phase in the “starved + starved” sample, and 42% were G1 cells in the “starved + serum” sample (S3D Fig). Cells expressing more than 50 E1A transcripts per cell were included into the data shown in the boxplot and the number of cells analyzed is indicated. Since the limit for accurate segmentation of E1A puncta was about 200 per cell, counts over this number are estimates. Permutation test indicated that the difference between the two samples is statistically significant (p=0.0002).
    Figure Legend Snippet: G1 cell-cycle phase favors rapid accumulation of E1A transcripts. A) Total nuclear DAPI signals can be used for accurate determination of different cell-cycle stages. Fixed noninfected HeLa-FUCCI cells were stained with DAPI and imaged by automated widefield fluorescent imaging system. The histograms show integrated nuclear DAPI intensities correlated at single-cell level to bins of increasing intensities of the G1 marker Cdt1 (Kusabira Orange) and the S/G2/M marker Geminin (Azami-Green). The increasing intensity bins of Cdt1 and Geminin are equal frequency plots in which each bin has equal number of cells. The integrated nuclear DAPI intensities are normalized to values 0-45. B) E1A transcripts accumulate more rapidly in G1 cells than in S/G2/M cells. AdV-C5 was added to A549 cells at 37°C for 60 min (moi ∼ 54400 virus particles per cell). After removal of unbound virus, incubation was continued at 37°C for additional 3 h. Fixed cells were stained with bDNA-FISH E1A probes, Alexa Fluor647 NHS Ester was used for staining of cell area and DAPI for nucleus. Images were acquired by an automated widefield fluorescent imaging system. The mean cytoplasmic E1A signal intensities were used for estimation of E1A transcript abundancies per cell. Cells were classified as G1 or S/G2/M according to total nuclear DAPI signals and 2973 cells were randomly sampled from the total population. Of these sampled cells 1659 were G1 cells and 1314 S/G2/M cells. In the left-hand panel, the mean cytoplasmic E1A signal intensities per cell in the two groups are shown as boxplots. The difference between the groups is statistically significant (permutation test, p=0.0002). In the right-hand panel, histogram of integrated nuclear DAPI intensities of cells was drawn and mean cytoplasmic intensities of E1A transcripts were mapped on the histogram. The histogram was split into five bins of increasing E1A intensities to show the correlation between E1A transcript abundancies per cell and the cell-cycle phase. Each bin contains equal number of cells. C) G1-enriched infected cell population shows increased numbers of E1A transcripts per cell in comparison to a cell population with lower number of G1 cell-cycle stage cells. AdV-C5 was added to serum-starved A375 melanoma cells at 37°C for 60 min (moi ∼ 36250 virus particles per cell), and after removal of unbound virus, cells were further incubated in serum-free (“starved + starved” sample) or serum-containing medium (“starved + serum” sample) for further 9 h. Fixed cells were stained with E1A probes, with Alexa Fluor647 NHS Ester for cell area, with DAPI for nucleus and imaged by confocal microscopy. As judged from integrated nuclear DAPI intensities, 66% of cells were in G1 phase in the “starved + starved” sample, and 42% were G1 cells in the “starved + serum” sample (S3D Fig). Cells expressing more than 50 E1A transcripts per cell were included into the data shown in the boxplot and the number of cells analyzed is indicated. Since the limit for accurate segmentation of E1A puncta was about 200 per cell, counts over this number are estimates. Permutation test indicated that the difference between the two samples is statistically significant (p=0.0002).

    Techniques Used: Staining, Imaging, Marker, Incubation, Fluorescence In Situ Hybridization, Infection, Confocal Microscopy, Expressing

    E1A mRNA abundancies at single-cell level early in infection only moderately correlate with vDNA counts in the total cell area or nucleus. A) Time-dependent decrease in the number of detected vDNAs in AdV-C5-EdC-infected cells. EdC-labeled AdV-C5 was added to A549 cells at 37°C for 60 min, and, after removal of unbound virus, incubation was continued at 37°C for additional 2 h or 6 h before fixation. The incoming viral vDNA was detected by a click-reaction using azide-Alexa Fluor488, Alexa Fluor647 NHS Ester was used for staining of cell area and nuclei were stained with DAPI. The graphs show number of vDNA molecules within the total cell area or within the nucleus area at the two time points. Horizontal lines represent median values. The number of cells and vDNAs analyzed is indicated. The differences in cell-associated or nuclear vDNA numbers between 3 h and 7 h were statistically significant (cell-associated vDNA P=0.0020 and nuclear vDNA P=0.0027, Kolmogorov-Smirnov test). B) qPCR quantification of vDNA copy numbers at 3 h and 7 h post virus addition. AdV-C5 infection conditions were similar to the experiment (A), except the virus used was not labeled with EdC. The two biological replicates and the technical replicates for each sample are shown separately. Horizontal bar represents mean. NI= noninfected control sample. C) Comparison of E1A mRNA and vDNA counts in infected cells. EdC-labeled AdV-C5 was added to A549 cells (moi ∼ 23440 virus particles per cell) at 37°C for 60 min, and after removal of unbound virus, incubation was continued at 37°C for additional 7 h. Fixed cells were stained with bDNA-FISH E1A probes and the incoming viral vDNA was detected by a click-reaction using azide-Alexa Fluor488. Alexa Fluor647 NHS Ester and DAPI stains were used for determination of cell area and nucleus area, respectively. Maximum projection images of confocal stacks were analysed using custom-programmed CellProfiler pipelines and E1A mRNA counts at single-cell level were correlated to vDNA counts per total cell area or to nuclear area. One dot represents one cell. Number of analyzed cells was 523. Since the limit for accurate segmentation of E1A puncta was about 200 per cell, counts over this number are estimates. Cells with no vDNA signal were excluded from the analysis. r s denotes the Spearman’s correlation rank coefficient. Images shown are maximum projections of confocal stacks with nuclear outlines indicated. Scale bar = 10 µm.
    Figure Legend Snippet: E1A mRNA abundancies at single-cell level early in infection only moderately correlate with vDNA counts in the total cell area or nucleus. A) Time-dependent decrease in the number of detected vDNAs in AdV-C5-EdC-infected cells. EdC-labeled AdV-C5 was added to A549 cells at 37°C for 60 min, and, after removal of unbound virus, incubation was continued at 37°C for additional 2 h or 6 h before fixation. The incoming viral vDNA was detected by a click-reaction using azide-Alexa Fluor488, Alexa Fluor647 NHS Ester was used for staining of cell area and nuclei were stained with DAPI. The graphs show number of vDNA molecules within the total cell area or within the nucleus area at the two time points. Horizontal lines represent median values. The number of cells and vDNAs analyzed is indicated. The differences in cell-associated or nuclear vDNA numbers between 3 h and 7 h were statistically significant (cell-associated vDNA P=0.0020 and nuclear vDNA P=0.0027, Kolmogorov-Smirnov test). B) qPCR quantification of vDNA copy numbers at 3 h and 7 h post virus addition. AdV-C5 infection conditions were similar to the experiment (A), except the virus used was not labeled with EdC. The two biological replicates and the technical replicates for each sample are shown separately. Horizontal bar represents mean. NI= noninfected control sample. C) Comparison of E1A mRNA and vDNA counts in infected cells. EdC-labeled AdV-C5 was added to A549 cells (moi ∼ 23440 virus particles per cell) at 37°C for 60 min, and after removal of unbound virus, incubation was continued at 37°C for additional 7 h. Fixed cells were stained with bDNA-FISH E1A probes and the incoming viral vDNA was detected by a click-reaction using azide-Alexa Fluor488. Alexa Fluor647 NHS Ester and DAPI stains were used for determination of cell area and nucleus area, respectively. Maximum projection images of confocal stacks were analysed using custom-programmed CellProfiler pipelines and E1A mRNA counts at single-cell level were correlated to vDNA counts per total cell area or to nuclear area. One dot represents one cell. Number of analyzed cells was 523. Since the limit for accurate segmentation of E1A puncta was about 200 per cell, counts over this number are estimates. Cells with no vDNA signal were excluded from the analysis. r s denotes the Spearman’s correlation rank coefficient. Images shown are maximum projections of confocal stacks with nuclear outlines indicated. Scale bar = 10 µm.

    Techniques Used: Infection, Labeling, Incubation, Staining, Real-time Polymerase Chain Reaction, Fluorescence In Situ Hybridization

    2) Product Images from "Autophagy-associated Production of Antimicrobial Peptides hBD1 and LL37 Exhibits Anti-Bacillus Calmette-Guérin Effects in Lung Epithelial Cells"

    Article Title: Autophagy-associated Production of Antimicrobial Peptides hBD1 and LL37 Exhibits Anti-Bacillus Calmette-Guérin Effects in Lung Epithelial Cells

    Journal: bioRxiv

    doi: 10.1101/2020.02.21.959361

    Autophagy adapter protein p62 controls the production of active AMPs by interacting with AMP precursors. (A B) Silencing P62 affected the active peptide levels of hBD1 and LL37. A549 cells were transiently transfected with si-control or si-SQSTM1 to silence P62 and then infected with BCG. The expression of hBD1 and LL37 was detected using western blotting and real-time PCR. (C) The autophagic level of A549 cells affected the co-localization level of hBD1/LL37 precursor and p62-positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 3-MA and rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1/LL37 precursor, p62, and GFP-LC3, labeled using Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. (D) Direct interaction could be observed between p62 and hCAP18. A549 cells were infected with BCG for 24 and 48 h. The interaction between p62 and hCAP18 was detected by Co-IP with anti-p62 antibody followed by western blotting with anti-p62 and anti-hCAP18 antibodies. Data are expressed as the means ± standard deviation (s.d.) * p
    Figure Legend Snippet: Autophagy adapter protein p62 controls the production of active AMPs by interacting with AMP precursors. (A B) Silencing P62 affected the active peptide levels of hBD1 and LL37. A549 cells were transiently transfected with si-control or si-SQSTM1 to silence P62 and then infected with BCG. The expression of hBD1 and LL37 was detected using western blotting and real-time PCR. (C) The autophagic level of A549 cells affected the co-localization level of hBD1/LL37 precursor and p62-positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 3-MA and rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1/LL37 precursor, p62, and GFP-LC3, labeled using Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. (D) Direct interaction could be observed between p62 and hCAP18. A549 cells were infected with BCG for 24 and 48 h. The interaction between p62 and hCAP18 was detected by Co-IP with anti-p62 antibody followed by western blotting with anti-p62 and anti-hCAP18 antibodies. Data are expressed as the means ± standard deviation (s.d.) * p

    Techniques Used: Transfection, Infection, Expressing, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection, Fluorescence, Confocal Microscopy, Labeling, Co-Immunoprecipitation Assay, Standard Deviation

    Antimicrobial peptides hBD1 and LL37 are highly expressed in MTB-infected lung epithelial cells and exhibit anti-MTB activity. (A) High level expression of hBD1 and LL37 was detected in A549 cells. The clls were infected with BCG for 48 h and the seven AMP mRNAs were evaluated by real-time PCR. (B C) hBD1 (B) and LL37 (C) mRNA expression in BCG-infected A549 cells was detected at specified time points by real-time PCR. (D) The active peptides of hBD1 and LL37 were detected at specified time points and evaluated by western blotting. (E F) High level expression of hBD1 and LL37 mRNA in PBMCs of patients with TB. (G) High level expression of LL37 was detected in human alveolar type II pneumocytes from patients with TB via immunohistochemistry. Micrograph shows strong LL37 immunostaining in human alveolar type II pneumocytes from patients with TB compared to that in healthy tissue donors and patients with pneumoconiosis (magnification x200). (H-J) Silencing of hBD1 or/and LL37 decreased intracellular BCG killing in A549 cells. The intracellular viable bacilli were determined by CFU assays at 72 h. Survival rate was calculated compared with that at 0 h. Data are expressed as the means ± standard deviation (s.d.). * p
    Figure Legend Snippet: Antimicrobial peptides hBD1 and LL37 are highly expressed in MTB-infected lung epithelial cells and exhibit anti-MTB activity. (A) High level expression of hBD1 and LL37 was detected in A549 cells. The clls were infected with BCG for 48 h and the seven AMP mRNAs were evaluated by real-time PCR. (B C) hBD1 (B) and LL37 (C) mRNA expression in BCG-infected A549 cells was detected at specified time points by real-time PCR. (D) The active peptides of hBD1 and LL37 were detected at specified time points and evaluated by western blotting. (E F) High level expression of hBD1 and LL37 mRNA in PBMCs of patients with TB. (G) High level expression of LL37 was detected in human alveolar type II pneumocytes from patients with TB via immunohistochemistry. Micrograph shows strong LL37 immunostaining in human alveolar type II pneumocytes from patients with TB compared to that in healthy tissue donors and patients with pneumoconiosis (magnification x200). (H-J) Silencing of hBD1 or/and LL37 decreased intracellular BCG killing in A549 cells. The intracellular viable bacilli were determined by CFU assays at 72 h. Survival rate was calculated compared with that at 0 h. Data are expressed as the means ± standard deviation (s.d.). * p

    Techniques Used: Infection, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Western Blot, Immunohistochemistry, Immunostaining, Standard Deviation

    The autophagic level of A549 cells affected the co-localization level of hBD1 precursor and p62 positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with 3-MA and Rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1, p62 and GFP-LC3, marked with Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The autophagic level of A549 cells affected the co-localization level of hBD1 precursor and p62 positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with 3-MA and Rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1, p62 and GFP-LC3, marked with Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection, Confocal Microscopy

    Autophagy influences the active peptide levels of hBD1 and LL37. (A) The autophagic level of A549 cells affected the active peptides level of hBD1 and LL37. A549 cells were pretreated with 4 μM rapamycin for 6 h and 5 mM 3-MA for 2 h and then infected with BCG for 24 h. The active peptide levels of hBD1 and LL37 were evaluated by western blotting. (B) The autophagic level of A549 cells did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (C D) Starvation-induced autophagy promoted the production of active hBD1 and LL37. A549 cells were cultured with EBSS culture medium at various time points. The expression of hBD1 and LL37 was detected by western blotting and real-time PCR. (E F) Silencing ATG 5 and 7 disturbed the production of active hBD1 and LL37. A549 cells were transfected with 100 nM si-control or siRNA for ATG 5 and 7 for 72 h and infected with BCG. The expression of hBD1 and LL37 was detected by western blotting and real-time PCR. (G) The autophagic level of A549 cells affected the co-localization level of hBD1/LL37 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)– tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 or LL37 precursor (hBD1/LL37 precursor-mCherry) were pretreated with rapamycin and 3-MA and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1/LL37 precursor and autophagosomes was detected by confocal microscopy. Data are expressed as the means ± standard deviation (s.d.) * p
    Figure Legend Snippet: Autophagy influences the active peptide levels of hBD1 and LL37. (A) The autophagic level of A549 cells affected the active peptides level of hBD1 and LL37. A549 cells were pretreated with 4 μM rapamycin for 6 h and 5 mM 3-MA for 2 h and then infected with BCG for 24 h. The active peptide levels of hBD1 and LL37 were evaluated by western blotting. (B) The autophagic level of A549 cells did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (C D) Starvation-induced autophagy promoted the production of active hBD1 and LL37. A549 cells were cultured with EBSS culture medium at various time points. The expression of hBD1 and LL37 was detected by western blotting and real-time PCR. (E F) Silencing ATG 5 and 7 disturbed the production of active hBD1 and LL37. A549 cells were transfected with 100 nM si-control or siRNA for ATG 5 and 7 for 72 h and infected with BCG. The expression of hBD1 and LL37 was detected by western blotting and real-time PCR. (G) The autophagic level of A549 cells affected the co-localization level of hBD1/LL37 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)– tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 or LL37 precursor (hBD1/LL37 precursor-mCherry) were pretreated with rapamycin and 3-MA and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1/LL37 precursor and autophagosomes was detected by confocal microscopy. Data are expressed as the means ± standard deviation (s.d.) * p

    Techniques Used: Infection, Western Blot, Real-time Polymerase Chain Reaction, Cell Culture, Expressing, Transfection, Stable Transfection, Fluorescence, Confocal Microscopy, Standard Deviation

    The autophagic level of A549 cells affected the co-localization level of hBD1 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with rapamycin and 3-MA as described above and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1 precursor and autophagosomes was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The autophagic level of A549 cells affected the co-localization level of hBD1 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with rapamycin and 3-MA as described above and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of hBD1 precursor and autophagosomes was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection, Confocal Microscopy

    hBD1 and LL37 production was affected by the function of lysosomes. (A) The formation of autolysosomes affected the active peptide levels of hBD1 and LL37. A549 cells were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. The active peptide levels of hBD1 and LL37 were evaluated using western blotting. (B) The formation of autolysosomes did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (C) The function of lysosomes affected the active peptide levels of hBD1 and LL37. A549 cells were pretreated with 10 mg/mL NH4Cl and infected with BCG for 48 h. The active peptide levels of hBD1 and LL37 were evaluated by western blotting. (D) The function of lysosomes did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (E F) The formation of autolysosomes did not affect the co-localization rates between hBD1/LL37 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 or LL37 precursor (hBD1/LL37 precursor-mCherry) were pretreated and infected with BCG as described as (A). Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. Co-localization of hBD1/LL37 precursor and autolysosomes was detected by confocal microscopy. Data are expressed as the means ± standard deviation (s.d.) * p
    Figure Legend Snippet: hBD1 and LL37 production was affected by the function of lysosomes. (A) The formation of autolysosomes affected the active peptide levels of hBD1 and LL37. A549 cells were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. The active peptide levels of hBD1 and LL37 were evaluated using western blotting. (B) The formation of autolysosomes did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (C) The function of lysosomes affected the active peptide levels of hBD1 and LL37. A549 cells were pretreated with 10 mg/mL NH4Cl and infected with BCG for 48 h. The active peptide levels of hBD1 and LL37 were evaluated by western blotting. (D) The function of lysosomes did not affect the mRNA level of hBD1 and LL37. A549 cells were pretreated and infected with BCG as described above. The mRNA levels of hBD1 and LL37 were evaluated using real-time PCR. (E F) The formation of autolysosomes did not affect the co-localization rates between hBD1/LL37 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 or LL37 precursor (hBD1/LL37 precursor-mCherry) were pretreated and infected with BCG as described as (A). Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. Co-localization of hBD1/LL37 precursor and autolysosomes was detected by confocal microscopy. Data are expressed as the means ± standard deviation (s.d.) * p

    Techniques Used: Infection, Western Blot, Real-time Polymerase Chain Reaction, Stable Transfection, Expressing, Fluorescence, Confocal Microscopy, Standard Deviation

    The autophagic level of A549 cells affected the co-localization level of LL37 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with rapamycin and 3-MA as described above and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of LL37 precursor and autophagosomes was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The autophagic level of A549 cells affected the co-localization level of LL37 precursor and autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with rapamycin and 3-MA as described above and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of LL37 precursor and autophagosomes was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection, Confocal Microscopy

    The formation of autolysosomes could not affect the co-localization rates between LL37 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. The function of lysosomes could not affect the co-localization rates between LL37 precursor and autolysosomes. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The formation of autolysosomes could not affect the co-localization rates between LL37 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. The function of lysosomes could not affect the co-localization rates between LL37 precursor and autolysosomes. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection

    Autophagy-related production of antimicrobial peptides is a novel mechanism of autophagy-mediated BCG killing. (A-C) Silencing of hBD1 (A) or/and LL37 (B C) weakened the autophagic killing of intracellular BCG. A549 cells were transfected with si-NC, Si-DEFB1, or/and si-CAMP for 24 h then infected with BCG (MOI = 5) for 1 h. In the rapamycin (Rapa) group the cells were pretreated by 4 μM rapamycin for 6 h prior to BCG infection. The intracellular viable bacilli were determined by CFU assays at 72 h. Survival rate was calculated compared with that at 0 h. Data are expressed as the means ± standard deviation (s.d.) * p
    Figure Legend Snippet: Autophagy-related production of antimicrobial peptides is a novel mechanism of autophagy-mediated BCG killing. (A-C) Silencing of hBD1 (A) or/and LL37 (B C) weakened the autophagic killing of intracellular BCG. A549 cells were transfected with si-NC, Si-DEFB1, or/and si-CAMP for 24 h then infected with BCG (MOI = 5) for 1 h. In the rapamycin (Rapa) group the cells were pretreated by 4 μM rapamycin for 6 h prior to BCG infection. The intracellular viable bacilli were determined by CFU assays at 72 h. Survival rate was calculated compared with that at 0 h. Data are expressed as the means ± standard deviation (s.d.) * p

    Techniques Used: Transfection, Infection, Standard Deviation

    The autophagic level of A549 cells affected the co-localization level of LL37 precursor and p62 positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 3-MA and Rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of LL37, p62 and GFP-LC3, marked with Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The autophagic level of A549 cells affected the co-localization level of LL37 precursor and p62 positive autophagosomes. A549 cells stably expressing green fluorescent protein (GFP)-tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged LL37 precursor (LL37 precursor-mCherry) were pretreated with 3-MA and Rapamycin and infected with BCG as described above. GFP-LC3 puncta ( > 1 μm) were observed and counted under confocal microscopy. Co-localization of LL37, p62 and GFP-LC3, marked with Alexa Fluor 647-coupled antibody against p62, was detected by confocal microscopy. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection, Confocal Microscopy

    The formation of autolysosomes could not affect the co-localization rates between hBD1 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. The function of lysosomes could not affect the co-localization rates between hBD1 precursor and autolysosomes. These experiments were performed independently at least thrice with similar results.
    Figure Legend Snippet: The formation of autolysosomes could not affect the co-localization rates between hBD1 precursor and autolysosomes. A549 cells stably expressing GFP–tagged LC3 (GFP-LC3) and mCherry Fluorescence Protein (mCherry)-tagged hBD1 precursor (hBD1 precursor-mCherry) were pretreated with 0.1 mM Baf A1 or/and 4 μM rapamycin and infected with BCG for 48 h. Lysosome was labelled with lyso tracker, GFP-LC3 ( > 1 μm) and lyso tracker double positive puncta were determined as autolysosomes. The function of lysosomes could not affect the co-localization rates between hBD1 precursor and autolysosomes. These experiments were performed independently at least thrice with similar results.

    Techniques Used: Stable Transfection, Expressing, Fluorescence, Infection

    3) Product Images from "Low basal expression and slow induction of IFITM3 puts immune cells at risk of influenza A infection"

    Article Title: Low basal expression and slow induction of IFITM3 puts immune cells at risk of influenza A infection

    Journal: bioRxiv

    doi: 10.1101/2019.12.20.885590

    IFITM3 is preferentially induced by Type I IFN (a) Expression of IFITM3 in HEK293 cells following 24 hours IFN stimulation measured by Western blot and expressed as a percentage of GAPDH expression. (b) Expression of IFITM3 in A549 cells following 24 hours IFN stimulation measured by Western blot and expressed as a percentage of GAPDH expression. (c) Adult blood PBMC were cultured for 48 hours with IFN stimulation prior to measurement of IFITM3 expression by mass cytometry. (d) Expression of IFITM3 in myeloid and lymphoid cells following IFN stimulation. (e) IFITM3 expression is expressed as a fold change in expression compared to the basal level of expression in order to show induction. Data is expressed ±SEM and analysed by one-way ANOVA. *p
    Figure Legend Snippet: IFITM3 is preferentially induced by Type I IFN (a) Expression of IFITM3 in HEK293 cells following 24 hours IFN stimulation measured by Western blot and expressed as a percentage of GAPDH expression. (b) Expression of IFITM3 in A549 cells following 24 hours IFN stimulation measured by Western blot and expressed as a percentage of GAPDH expression. (c) Adult blood PBMC were cultured for 48 hours with IFN stimulation prior to measurement of IFITM3 expression by mass cytometry. (d) Expression of IFITM3 in myeloid and lymphoid cells following IFN stimulation. (e) IFITM3 expression is expressed as a fold change in expression compared to the basal level of expression in order to show induction. Data is expressed ±SEM and analysed by one-way ANOVA. *p

    Techniques Used: Expressing, Western Blot, Cell Culture, Mass Cytometry

    Maximal expression of IFITM3 takes at least 24 hours (a) Timecourse of IFITM3 expression in HEK293 cells following 500 U/ml IFN induction measured by western blot and expression as a relative amount of expression compared to the level recorded at 72 hours. (b) Timecourse of IFITM3 expression in A549 cells following 500 U/ml IFN induction measured by western blot and expression as a relative amount of expression compared to the level recorded at 72 hours.
    Figure Legend Snippet: Maximal expression of IFITM3 takes at least 24 hours (a) Timecourse of IFITM3 expression in HEK293 cells following 500 U/ml IFN induction measured by western blot and expression as a relative amount of expression compared to the level recorded at 72 hours. (b) Timecourse of IFITM3 expression in A549 cells following 500 U/ml IFN induction measured by western blot and expression as a relative amount of expression compared to the level recorded at 72 hours.

    Techniques Used: Expressing, Western Blot

    4) Product Images from "3′-Terminal 2′-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute 2"

    Article Title: 3′-Terminal 2′-O-methylation of lung cancer miR-21-5p enhances its stability and association with Argonaute 2

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkaa504

    HEMT1-knockout impairs 3′-terminal 2′Ome of miRNAs in A549 cells and mice. ( A ) HENMT1 expression in human tissues. ( B ) HENMT1 expression and location in A549 cell. Left: localization of HENMT1 detected by immunofluorescence analysis; right: localization of HENMT1 detected by Western blot. ( C ) HENMT1 expression detected by Western blot in WT A549 cells (WT), scramble gDNA (control cells) and HEMT1-knockout A549 cells (HEMT1 Knockout) via Crisper/cas9 technique. (D–F) Levels of miRNAs in A549 or HENMT1-knockout A549 (A549KO) cells detected by high-through sequencing after oxidation ( D ), qRT-PCR after oxidation ( E ) and Northern blot ( F ) after Oxid/β-elim procedure. ( G ) HENMT1 expression detected by western blot in lung derived from wild type mice (WT) and HEMT1-knockout mice (KO). ( H ) Ratio of piR_020485, miR-21-5p and miR-26a-5p levels with oxidation versus piR_020485, miR-21-5p and miR-26a-5p levels without oxidation in lung tissues from WT and HENMT1-KO (KO) mice by qRT-PCR. ( I ) Northern blot analysis of piR_020485 and miR-26a-5p expression in lung tissues from WT and KO mice after Oxid/β-elim procedure.
    Figure Legend Snippet: HEMT1-knockout impairs 3′-terminal 2′Ome of miRNAs in A549 cells and mice. ( A ) HENMT1 expression in human tissues. ( B ) HENMT1 expression and location in A549 cell. Left: localization of HENMT1 detected by immunofluorescence analysis; right: localization of HENMT1 detected by Western blot. ( C ) HENMT1 expression detected by Western blot in WT A549 cells (WT), scramble gDNA (control cells) and HEMT1-knockout A549 cells (HEMT1 Knockout) via Crisper/cas9 technique. (D–F) Levels of miRNAs in A549 or HENMT1-knockout A549 (A549KO) cells detected by high-through sequencing after oxidation ( D ), qRT-PCR after oxidation ( E ) and Northern blot ( F ) after Oxid/β-elim procedure. ( G ) HENMT1 expression detected by western blot in lung derived from wild type mice (WT) and HEMT1-knockout mice (KO). ( H ) Ratio of piR_020485, miR-21-5p and miR-26a-5p levels with oxidation versus piR_020485, miR-21-5p and miR-26a-5p levels without oxidation in lung tissues from WT and HENMT1-KO (KO) mice by qRT-PCR. ( I ) Northern blot analysis of piR_020485 and miR-26a-5p expression in lung tissues from WT and KO mice after Oxid/β-elim procedure.

    Techniques Used: Knock-Out, Mouse Assay, Expressing, Immunofluorescence, Western Blot, Sequencing, Quantitative RT-PCR, Northern Blot, Derivative Assay

    3′-Terminal 2′Ome of human miR-21-5p increases its association with AGO2 complex and inhibition on PDCD4 expression. ( A ) Depict of experimental strategy of assaying binding affinity of miR-21-5p or miR-21-5p CH3 with AGO2 complex using Cy3- or Cy5-labeled miRNAs. ( B ) Binding affinity of miR-21-5p and miR-21-5p CH3 with AGO2 complex detected by fluorescence intensity measurement. ( C ) Kinetics of binding of miR-21-5p and miR-21-5p CH3 with AGO2 detected by microscale thermophoresis. ( D ) Binding of miR-21-5p or miR-26-5p with AGO2 detected by the luciferase activity in A549 or A549KO cells transfected with miR-21-5p or miR-26-5p reporter plasmid. ( E ) PDCD4 and ULK1 protein level in A549 and A549KO cells at different time points after serum depletion. ( F ) Apoptosis of A549 and A549KO cells induced by serum depletion.
    Figure Legend Snippet: 3′-Terminal 2′Ome of human miR-21-5p increases its association with AGO2 complex and inhibition on PDCD4 expression. ( A ) Depict of experimental strategy of assaying binding affinity of miR-21-5p or miR-21-5p CH3 with AGO2 complex using Cy3- or Cy5-labeled miRNAs. ( B ) Binding affinity of miR-21-5p and miR-21-5p CH3 with AGO2 complex detected by fluorescence intensity measurement. ( C ) Kinetics of binding of miR-21-5p and miR-21-5p CH3 with AGO2 detected by microscale thermophoresis. ( D ) Binding of miR-21-5p or miR-26-5p with AGO2 detected by the luciferase activity in A549 or A549KO cells transfected with miR-21-5p or miR-26-5p reporter plasmid. ( E ) PDCD4 and ULK1 protein level in A549 and A549KO cells at different time points after serum depletion. ( F ) Apoptosis of A549 and A549KO cells induced by serum depletion.

    Techniques Used: Inhibition, Expressing, Binding Assay, Labeling, Fluorescence, Microscale Thermophoresis, Luciferase, Activity Assay, Transfection, Plasmid Preparation, Serum Depletion

    3′-terminal 2′Ome of human miR-21-5p increases its stability. ( A ) The half-life of miR-21-5p and miR-26-5p in A549 cells and A549KO (HENMT1-knockout) cells. (B, C) Direct degradation of synthetic miR-21-5p/miR-21-5p CH3 and miR-26-5p/miR-26-5p CH3 by PNPT1 recombination protein in a time-dependent ( B ) and concentration-dependent ( C ) manner.
    Figure Legend Snippet: 3′-terminal 2′Ome of human miR-21-5p increases its stability. ( A ) The half-life of miR-21-5p and miR-26-5p in A549 cells and A549KO (HENMT1-knockout) cells. (B, C) Direct degradation of synthetic miR-21-5p/miR-21-5p CH3 and miR-26-5p/miR-26-5p CH3 by PNPT1 recombination protein in a time-dependent ( B ) and concentration-dependent ( C ) manner.

    Techniques Used: Knock-Out, Concentration Assay

    5) 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

    6) Product Images from "The lncRNA LINC01194/miR-486-5p Axis Facilitates Malignancy in Non-Small Cell Lung Cancer via Regulating CDK4"

    Article Title: The lncRNA LINC01194/miR-486-5p Axis Facilitates Malignancy in Non-Small Cell Lung Cancer via Regulating CDK4

    Journal: OncoTargets and therapy

    doi: 10.2147/OTT.S235037

    LINC01194 exerted a biological effect on NSCLC cells via miR-486-5p. ( A ) CCK8 measured the viability of A549 cells. ( B, C ) colony formation assay cell proliferation. ( D, E ) Transwell measured the number of cell invasions. ( F, G ) Transwell measured the amount of cell migration. * P
    Figure Legend Snippet: LINC01194 exerted a biological effect on NSCLC cells via miR-486-5p. ( A ) CCK8 measured the viability of A549 cells. ( B, C ) colony formation assay cell proliferation. ( D, E ) Transwell measured the number of cell invasions. ( F, G ) Transwell measured the amount of cell migration. * P

    Techniques Used: Colony Assay, Migration

    LINC01194 regulated the expression of miR-486-5p in NSCLC cells. ( A ) Expression of miR146a-5p mRNA levels in NSCLC cell lines. ( B ) Putative target sequence of miR-486-5p on the 3ʹ-UTR of LINC01194. ( C ) miR-486-5p mRNA levels in A549 cells under different treatment conditions. ( D ) Detection of luciferase activity by luciferase reporter assay. ( E ) LINC01194 expression levels in samples by biotinylated miR-486-5p or negative control. (F) Correlation between LINC01194 and miR-486-5p levels was using detecting RNA pull down. (G) Pearson’s correlation analysis of LINC01194and miR-486-5p in NSCLC tissues (n=26) (r=-0.672, P
    Figure Legend Snippet: LINC01194 regulated the expression of miR-486-5p in NSCLC cells. ( A ) Expression of miR146a-5p mRNA levels in NSCLC cell lines. ( B ) Putative target sequence of miR-486-5p on the 3ʹ-UTR of LINC01194. ( C ) miR-486-5p mRNA levels in A549 cells under different treatment conditions. ( D ) Detection of luciferase activity by luciferase reporter assay. ( E ) LINC01194 expression levels in samples by biotinylated miR-486-5p or negative control. (F) Correlation between LINC01194 and miR-486-5p levels was using detecting RNA pull down. (G) Pearson’s correlation analysis of LINC01194and miR-486-5p in NSCLC tissues (n=26) (r=-0.672, P

    Techniques Used: Expressing, Sequencing, Luciferase, Activity Assay, Reporter Assay, Negative Control

    7) Product Images from "Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins"

    Article Title: Defective Influenza A Virus RNA Products Mediate MAVS-Dependent Upregulation of Human Leukocyte Antigen Class I Proteins

    Journal: Journal of Virology

    doi: 10.1128/JVI.00165-20

    IAV infection alters cell surface expression of ligands for NK cell receptors. A549 cells were infected with FM-MA, PR8, or CA/07 at an MOI of 1. At 17 h, cells were fixed and immunostained to determine cell surface levels of NK cell ligands; cells were subsequently permeabilized for immunostaining of intracellular IAV proteins. (A) Flow cytometry analysis of cells immunostained with a pan-HLA-A/B/C antibody, or antibodies specific for class I HLA proteins HLA-B, HLA-C, or HLA-E or isotype antibody controls. (B) Flow cytometry analysis for cells immunostained with antibodies to detect NK cell-activating ligands MICA/B and CD155/PVR, or inhibitory ligand CD113/NECTIN3, or isotype antibody controls. Representative histograms (top) show results of a single experiment; the vertical lines represent the expression level of the target in uninfected cells. (Bottom) Mean fluorescence intensity (MFI) relative to uninfected cells. Each data point represents an independent experiment. Means ± SD are shown. *, P
    Figure Legend Snippet: IAV infection alters cell surface expression of ligands for NK cell receptors. A549 cells were infected with FM-MA, PR8, or CA/07 at an MOI of 1. At 17 h, cells were fixed and immunostained to determine cell surface levels of NK cell ligands; cells were subsequently permeabilized for immunostaining of intracellular IAV proteins. (A) Flow cytometry analysis of cells immunostained with a pan-HLA-A/B/C antibody, or antibodies specific for class I HLA proteins HLA-B, HLA-C, or HLA-E or isotype antibody controls. (B) Flow cytometry analysis for cells immunostained with antibodies to detect NK cell-activating ligands MICA/B and CD155/PVR, or inhibitory ligand CD113/NECTIN3, or isotype antibody controls. Representative histograms (top) show results of a single experiment; the vertical lines represent the expression level of the target in uninfected cells. (Bottom) Mean fluorescence intensity (MFI) relative to uninfected cells. Each data point represents an independent experiment. Means ± SD are shown. *, P

    Techniques Used: Infection, Expressing, Immunostaining, Flow Cytometry, Fluorescence

    Defective viral RNAs increase ISRE-dependent luciferase activity in A549 cells. (A) FM-MA inoculum was exposed to UV light prior to infection of A549 cells at MOI of 1. At 17 hpi, cells were fixed and immunostained with a pan-anti-HLA-A/B/C antibody or an anti-HLA-B antibody and processed for flow cytometry. The vertical lines indicate the HLA expression level in uninfected cells. Representative data from one out of two independent experiments is shown. (B) (Top) Cartoon compares RNAs derived from the indicated genome segments and expressed from pPolI-based minireplicon plasmids, including full-length (FL) vRNA, defective interfering (DI) vRNA, or mini-viral RNA (mvRNA); dashed lines mark internal deletions on the DI RNAs and mvRNAs. (Bottom) A549 cells were transfected with IAV minireplicons expressing the indicated FL vRNAs, DI RNAs, or mvRNAs derived from the indicated genome segments. An ISRE-driven firefly luciferase reporter plasmid was cotransfected with minireplicon plasmids to measure IFN signaling, along with a Renilla luciferase plasmid that served as normalization control. Poly(I·C) and an empty pUC19 plasmid served as positive and negative controls, respectively. Firefly luciferase activity was normalized to Renilla luciferase control for each sample, and data were expressed as fold change compared with pUC19 plasmid transfection ( n = 6; *, P
    Figure Legend Snippet: Defective viral RNAs increase ISRE-dependent luciferase activity in A549 cells. (A) FM-MA inoculum was exposed to UV light prior to infection of A549 cells at MOI of 1. At 17 hpi, cells were fixed and immunostained with a pan-anti-HLA-A/B/C antibody or an anti-HLA-B antibody and processed for flow cytometry. The vertical lines indicate the HLA expression level in uninfected cells. Representative data from one out of two independent experiments is shown. (B) (Top) Cartoon compares RNAs derived from the indicated genome segments and expressed from pPolI-based minireplicon plasmids, including full-length (FL) vRNA, defective interfering (DI) vRNA, or mini-viral RNA (mvRNA); dashed lines mark internal deletions on the DI RNAs and mvRNAs. (Bottom) A549 cells were transfected with IAV minireplicons expressing the indicated FL vRNAs, DI RNAs, or mvRNAs derived from the indicated genome segments. An ISRE-driven firefly luciferase reporter plasmid was cotransfected with minireplicon plasmids to measure IFN signaling, along with a Renilla luciferase plasmid that served as normalization control. Poly(I·C) and an empty pUC19 plasmid served as positive and negative controls, respectively. Firefly luciferase activity was normalized to Renilla luciferase control for each sample, and data were expressed as fold change compared with pUC19 plasmid transfection ( n = 6; *, P

    Techniques Used: Luciferase, Activity Assay, Infection, Flow Cytometry, Expressing, Derivative Assay, Transfection, Plasmid Preparation

    NS1 protein limits cell-intrinsic and paracrine upregulation of class I HLA proteins. (A) A cartoon representing wild-type and mutant NS1 proteins used in this study. A carboxy-terminal disordered tail region present in PR8 NS1 and absent in FM-MA NS1 is shown in gray. Positions of alanine substitutions in R38A, K41A and E96A, E97A mutant proteins are indicated as “AA.” Amino-terminal double-stranded RNA (dsRNA) binding domain is in orange; effector domain is in teal. (B) A549 cells were infected with the indicated viruses at an MOI of 1 or mock infected. At 17 hpi, cell supernatants were collected prior to cell fixation and transferred to naive A549 cells for an additional 17 h of incubation prior to fixation. Donor and recipient cells were immunostained with the indicated anti-HLA antibodies to determine cell surface levels of NK cell ligands; cells were subsequently permeabilized for immunostaining of intracellular IAV proteins and analyzed by flow cytometry. (Top) Data from donor-infected or mock-infected cells. (Bottom) Data from cells exposed to conditioned media. Data are presented as MFI relative to uninfected cells or conditioned media treatment from uninfected cells. Each data point represents an independent experiment. Means ± SD are shown. *, P
    Figure Legend Snippet: NS1 protein limits cell-intrinsic and paracrine upregulation of class I HLA proteins. (A) A cartoon representing wild-type and mutant NS1 proteins used in this study. A carboxy-terminal disordered tail region present in PR8 NS1 and absent in FM-MA NS1 is shown in gray. Positions of alanine substitutions in R38A, K41A and E96A, E97A mutant proteins are indicated as “AA.” Amino-terminal double-stranded RNA (dsRNA) binding domain is in orange; effector domain is in teal. (B) A549 cells were infected with the indicated viruses at an MOI of 1 or mock infected. At 17 hpi, cell supernatants were collected prior to cell fixation and transferred to naive A549 cells for an additional 17 h of incubation prior to fixation. Donor and recipient cells were immunostained with the indicated anti-HLA antibodies to determine cell surface levels of NK cell ligands; cells were subsequently permeabilized for immunostaining of intracellular IAV proteins and analyzed by flow cytometry. (Top) Data from donor-infected or mock-infected cells. (Bottom) Data from cells exposed to conditioned media. Data are presented as MFI relative to uninfected cells or conditioned media treatment from uninfected cells. Each data point represents an independent experiment. Means ± SD are shown. *, P

    Techniques Used: Mutagenesis, Binding Assay, Infection, Incubation, Immunostaining, Flow Cytometry

    Defective IAV RNAs elicit cell-intrinsic and paracrine upregulation of class I HLA proteins. (A) A549 cells were treated with conditioned medium containing UV-inactivated culture supernatant from FM-MA-infected cells. Surface HLA levels on recipient cells (17 h posttreatment) and infected donor cells (17 hpi) were determined by flow cytometry. Histograms from a representative experiment are shown. Vertical dashed-lines indicate the expression level in uninfected cells. (B) MFI of cell surface HLA proteins on recipient cells from A relative to cells treated with conditioned media from mock-infected cells. Each data point represents an independent experiment. (C) A549 cells were treated with conditioned medium from cells transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 or from control untransfected cells or pUC19 vector-transfected cells. After 24 h, cells were fixed and immunostained with a pan-anti-HLA-A/B/C antibody ( n = 3). Histograms from a representative experiment are shown on the left; vertical lines indicate the expression level of targets in uninfected cells. On the right, relative MFI values from at least 3 independent experiments are shown (*, P
    Figure Legend Snippet: Defective IAV RNAs elicit cell-intrinsic and paracrine upregulation of class I HLA proteins. (A) A549 cells were treated with conditioned medium containing UV-inactivated culture supernatant from FM-MA-infected cells. Surface HLA levels on recipient cells (17 h posttreatment) and infected donor cells (17 hpi) were determined by flow cytometry. Histograms from a representative experiment are shown. Vertical dashed-lines indicate the expression level in uninfected cells. (B) MFI of cell surface HLA proteins on recipient cells from A relative to cells treated with conditioned media from mock-infected cells. Each data point represents an independent experiment. (C) A549 cells were treated with conditioned medium from cells transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 or from control untransfected cells or pUC19 vector-transfected cells. After 24 h, cells were fixed and immunostained with a pan-anti-HLA-A/B/C antibody ( n = 3). Histograms from a representative experiment are shown on the left; vertical lines indicate the expression level of targets in uninfected cells. On the right, relative MFI values from at least 3 independent experiments are shown (*, P

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

    Defective viral RNAs increase surface HLA presentation in a MAVS-dependent manner. (A) A549 cells or A549-MAVS-KO cells were transfected with IAV minireplicon expressing mvRNA from genome segment 5 or empty pUC19 control for 24 h prior to harvest of protein lysates and immunoblotting with antibodies for the indicated target proteins. (B) A549 cells or A549 MAVS-KO cells were transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 and analyzed by flow cytometry at 48 h posttransfection via surface immunostaining with a pan-anti-HLA-A/B/C antibody ( n = 3). Histograms from a representative experiment are shown on the left; the vertical lines indicate the expression level of the target in uninfected cells. On the right, relative MFI values from at least 3 independent experiments are shown. *, P
    Figure Legend Snippet: Defective viral RNAs increase surface HLA presentation in a MAVS-dependent manner. (A) A549 cells or A549-MAVS-KO cells were transfected with IAV minireplicon expressing mvRNA from genome segment 5 or empty pUC19 control for 24 h prior to harvest of protein lysates and immunoblotting with antibodies for the indicated target proteins. (B) A549 cells or A549 MAVS-KO cells were transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 and analyzed by flow cytometry at 48 h posttransfection via surface immunostaining with a pan-anti-HLA-A/B/C antibody ( n = 3). Histograms from a representative experiment are shown on the left; the vertical lines indicate the expression level of the target in uninfected cells. On the right, relative MFI values from at least 3 independent experiments are shown. *, P

    Techniques Used: Transfection, Expressing, Flow Cytometry, Immunostaining

    IAV infection of epithelial cells increases class I HLA gene expression. (A) Expression of NK cell ligands from 18 publicly available gene expression data sets from in vitro IAV infection of A549 cells and primary human lung cells. NK ligands are classified as activating (green), ambiguous function (orange), and inhibitory (red). Class I HLA proteins are indicated in blue. Data are presented as the log 2 fold change relative to uninfected controls for each data set; median values with interquartile range (IQR) are shown. Vertical dashed lines indicate 2-fold change thresholds. (B) A549 cells were infected with PR8 or FM-MA or mock-infected for 17 h, and RNA was harvested for RT-qPCR. The relative expression of NK cell ligands was expressed as log 2 fold change relative to mock-infected controls. Vertical dashed lines indicate 2-fold change thresholds. N = 3; *, P
    Figure Legend Snippet: IAV infection of epithelial cells increases class I HLA gene expression. (A) Expression of NK cell ligands from 18 publicly available gene expression data sets from in vitro IAV infection of A549 cells and primary human lung cells. NK ligands are classified as activating (green), ambiguous function (orange), and inhibitory (red). Class I HLA proteins are indicated in blue. Data are presented as the log 2 fold change relative to uninfected controls for each data set; median values with interquartile range (IQR) are shown. Vertical dashed lines indicate 2-fold change thresholds. (B) A549 cells were infected with PR8 or FM-MA or mock-infected for 17 h, and RNA was harvested for RT-qPCR. The relative expression of NK cell ligands was expressed as log 2 fold change relative to mock-infected controls. Vertical dashed lines indicate 2-fold change thresholds. N = 3; *, P

    Techniques Used: Infection, Expressing, In Vitro, Quantitative RT-PCR

    HLA upregulation in response to defective IAV RNAs is dependent on IFN signaling. (A) A549 cells or A549-MAVS-KO cells were infected with FM-MA for 17 h, and relative levels of IFN-β and IFN-λ1 transcripts compared with uninfected controls were analyzed by RT-qPCR ( n = 3). (B) A549 cells or A549-MAVS-KO cells were treated with recombinant IFN-β, IFN-λ1, or IFN-λ2, and RNA was harvested over a 12-h time course. The relative expression of HLA-A, -B, and –C transcripts was analyzed by RT-qPCR. (C) The surface expression of HLA-A/B/C was determined by immunostaining and flow cytometry of cells harvested over the time course of IFN treatment described in B ( n = 3). (D) Analysis of HLA surface expression on A549 cells transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 or from control pUC19 vector-transfected cells. At 6 h posttransfection, cells were treated with ruxolitinib (Rux) or mock treated. *, P
    Figure Legend Snippet: HLA upregulation in response to defective IAV RNAs is dependent on IFN signaling. (A) A549 cells or A549-MAVS-KO cells were infected with FM-MA for 17 h, and relative levels of IFN-β and IFN-λ1 transcripts compared with uninfected controls were analyzed by RT-qPCR ( n = 3). (B) A549 cells or A549-MAVS-KO cells were treated with recombinant IFN-β, IFN-λ1, or IFN-λ2, and RNA was harvested over a 12-h time course. The relative expression of HLA-A, -B, and –C transcripts was analyzed by RT-qPCR. (C) The surface expression of HLA-A/B/C was determined by immunostaining and flow cytometry of cells harvested over the time course of IFN treatment described in B ( n = 3). (D) Analysis of HLA surface expression on A549 cells transfected with IAV minireplicon expressing defective vRNAs from genome segment 5 or from control pUC19 vector-transfected cells. At 6 h posttransfection, cells were treated with ruxolitinib (Rux) or mock treated. *, P

    Techniques Used: Infection, Quantitative RT-PCR, Recombinant, Expressing, Immunostaining, Flow Cytometry, Transfection, Plasmid Preparation

    Class I HLA upregulation in IAV-infected cells is MAVS dependent. A549 cells or A549-MAVS-KO cells were infected with FM-MA at an MOI of 1. RNA was harvested for RT-qPCR at 3 hpi or 17 hpi. (A) Relative fold change in HLA-A, -B, and -C transcript levels in A549 cells or A549-MAVS-KO cells at 17 hpi ( n = 3). (B) Relative fold change in B2M, TAP, and PSMB8 transcript levels in A549 and A549 MAVS-KO cells at 3 hpi or 17 hpi ( n = 3). (C) Relative MFI of cell surface HLA proteins in FM-MA-infected A549 cells and A549-MAVS-KO cells at indicated times, relative to uninfected controls. *, P
    Figure Legend Snippet: Class I HLA upregulation in IAV-infected cells is MAVS dependent. A549 cells or A549-MAVS-KO cells were infected with FM-MA at an MOI of 1. RNA was harvested for RT-qPCR at 3 hpi or 17 hpi. (A) Relative fold change in HLA-A, -B, and -C transcript levels in A549 cells or A549-MAVS-KO cells at 17 hpi ( n = 3). (B) Relative fold change in B2M, TAP, and PSMB8 transcript levels in A549 and A549 MAVS-KO cells at 3 hpi or 17 hpi ( n = 3). (C) Relative MFI of cell surface HLA proteins in FM-MA-infected A549 cells and A549-MAVS-KO cells at indicated times, relative to uninfected controls. *, P

    Techniques Used: Infection, Quantitative RT-PCR

    8) Product Images from "Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL-6/STAT3 signaling pathway"

    Article Title: Shikonin blocks human lung adenocarcinoma cell migration and invasion in the inflammatory microenvironment via the IL-6/STAT3 signaling pathway

    Journal: Oncology Reports

    doi: 10.3892/or.2020.7683

    Shikonin suppresses THP-1-CM-induced epithelial-mesenchymal transition (EMT) in A549 and H1299 cells. (A) Immunofluorescence was used to visualize the protein expression levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. Left, representative immunofluorescence images of E-cadherin, N-cadherin and vimentin (magnification ×800). Right, quantitative image analysis of the signal intensity of E-cadherin, N-cadherin and vimentin. (B) Western blot assays were used to detect the protein expression levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. (C) Real-time PCR was used to detect the mRNA levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. β-actin was used as an internal control. Data are shown as the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: Shikonin suppresses THP-1-CM-induced epithelial-mesenchymal transition (EMT) in A549 and H1299 cells. (A) Immunofluorescence was used to visualize the protein expression levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. Left, representative immunofluorescence images of E-cadherin, N-cadherin and vimentin (magnification ×800). Right, quantitative image analysis of the signal intensity of E-cadherin, N-cadherin and vimentin. (B) Western blot assays were used to detect the protein expression levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. (C) Real-time PCR was used to detect the mRNA levels of E-cadherin, N-cadherin and vimentin after treatment with THP-1-CM and/or shikonin for 24 h. β-actin was used as an internal control. Data are shown as the mean ± SD of three independent experiments. *P

    Techniques Used: Immunofluorescence, Expressing, Western Blot, Real-time Polymerase Chain Reaction

    Inhibitory effects of shikonin on THP-1-CM-induced migration and invasion of A549 and H1299 cells. (A) Chemical structure of shikonin. (B) Effect of shikonin on the viability of lung adenocarcinoma cells. A549 and H1299 cells were treated with serial doses of shikonin in normal medium for 24 h, and the viability of cells was detected by CCK-8 assay. (C) A549 and H1299 cells were incubated with THP-1-CM and/or shikonin for 24 h, and the viability of cells was examined by CCK-8 assay. (D) Wound healing assays. A549 and H1299 cells were scratched and incubated with THP-1-CM and/or shikonin, and images were captured at 0 and 24 h after wounding. (E) Invasion assays were performed by Transwells covered with Matrigel. After treatment with THP-1-CM and/or shikonin for 24 h, the cell suspensions were subjected to Transwell assays. Images (magnification ×200) are representative of three independent experiments. Data are shown as the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: Inhibitory effects of shikonin on THP-1-CM-induced migration and invasion of A549 and H1299 cells. (A) Chemical structure of shikonin. (B) Effect of shikonin on the viability of lung adenocarcinoma cells. A549 and H1299 cells were treated with serial doses of shikonin in normal medium for 24 h, and the viability of cells was detected by CCK-8 assay. (C) A549 and H1299 cells were incubated with THP-1-CM and/or shikonin for 24 h, and the viability of cells was examined by CCK-8 assay. (D) Wound healing assays. A549 and H1299 cells were scratched and incubated with THP-1-CM and/or shikonin, and images were captured at 0 and 24 h after wounding. (E) Invasion assays were performed by Transwells covered with Matrigel. After treatment with THP-1-CM and/or shikonin for 24 h, the cell suspensions were subjected to Transwell assays. Images (magnification ×200) are representative of three independent experiments. Data are shown as the mean ± SD of three independent experiments. *P

    Techniques Used: Migration, CCK-8 Assay, Incubation

    Effects of shikonin on the migration and epithelial-mesenchymal transition (EMT) of A549 and H1299 cells induced by IL-6 and TNF-α. (A) Transwell chamber migration assay. After treatment with shikonin and IL-6 (50 ng/ml) or TNF-α (20 ng/ml) for 24 h, A549 and H1299 cell suspensions were subjected to a Transwell chamber migration assay. (B) Western blot and (C) real-time PCR assays were used to detect the protein and mRNA levels of E-cadherin, N-cadherin, vimentin and Snail in A549 and H1299 cells after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. (D) Western blot and (E) real-time PCR assays were used to detect the protein and mRNA levels of E-cadherin, N-cadherin, vimentin and Snail in A549 and H1299 cells after treatment with shikonin and TNF-α (20 ng/ml) for 24 h. β-actin was used as an internal control. Data are shown as the mean ± SD of three independent experiments. *P
    Figure Legend Snippet: Effects of shikonin on the migration and epithelial-mesenchymal transition (EMT) of A549 and H1299 cells induced by IL-6 and TNF-α. (A) Transwell chamber migration assay. After treatment with shikonin and IL-6 (50 ng/ml) or TNF-α (20 ng/ml) for 24 h, A549 and H1299 cell suspensions were subjected to a Transwell chamber migration assay. (B) Western blot and (C) real-time PCR assays were used to detect the protein and mRNA levels of E-cadherin, N-cadherin, vimentin and Snail in A549 and H1299 cells after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. (D) Western blot and (E) real-time PCR assays were used to detect the protein and mRNA levels of E-cadherin, N-cadherin, vimentin and Snail in A549 and H1299 cells after treatment with shikonin and TNF-α (20 ng/ml) for 24 h. β-actin was used as an internal control. Data are shown as the mean ± SD of three independent experiments. *P

    Techniques Used: Migration, Western Blot, Real-time Polymerase Chain Reaction

    Effects of shikonin on tumor metastasis and growth in vivo . (A) Representative images of lung tissue and hematoxylin and eosin (H E)-stained metastatic lung nodules. (B) Quantification of the number of metastatic nodules on lung surface. (C) Tumor volume and (D) weight of the control and shikonin treatment groups. (E) Representative immunohistochemistry images of p-STAT3, E-cadherin, N-cadherin and vimentin expression in the tumor tissues derived from the A549 ×enografts. Data are presented as mean ± SD, *P
    Figure Legend Snippet: Effects of shikonin on tumor metastasis and growth in vivo . (A) Representative images of lung tissue and hematoxylin and eosin (H E)-stained metastatic lung nodules. (B) Quantification of the number of metastatic nodules on lung surface. (C) Tumor volume and (D) weight of the control and shikonin treatment groups. (E) Representative immunohistochemistry images of p-STAT3, E-cadherin, N-cadherin and vimentin expression in the tumor tissues derived from the A549 ×enografts. Data are presented as mean ± SD, *P

    Techniques Used: In Vivo, Staining, Immunohistochemistry, Expressing, Derivative Assay

    Effect of IL-6 and TNF-α on the migration and epithelial-mesenchymal transition (EMT) of A549 and H1299 cells. (A) IL-6 and TNF-α secreted into the culture supernatants were measured using ELISA kits after treatment with LPS and anti-IL-6 or anti-TNF-α antibodies for 24 h in THP-1-CM. **P
    Figure Legend Snippet: Effect of IL-6 and TNF-α on the migration and epithelial-mesenchymal transition (EMT) of A549 and H1299 cells. (A) IL-6 and TNF-α secreted into the culture supernatants were measured using ELISA kits after treatment with LPS and anti-IL-6 or anti-TNF-α antibodies for 24 h in THP-1-CM. **P

    Techniques Used: Migration, Enzyme-linked Immunosorbent Assay

    Effects of shikonin on IL-6-induced STAT3 activation in A549 and H1299 cells. (A) Expression levels of phosphorylated (p)-STAT3 and STAT3 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. Protein expression levels were semi-quantified by densitometric analysis. *P
    Figure Legend Snippet: Effects of shikonin on IL-6-induced STAT3 activation in A549 and H1299 cells. (A) Expression levels of phosphorylated (p)-STAT3 and STAT3 were detected by western blotting after treatment with shikonin and IL-6 (50 ng/ml) for 24 h. Protein expression levels were semi-quantified by densitometric analysis. *P

    Techniques Used: Activation Assay, Expressing, Western Blot

    9) Product Images from "Anticancer Activity of Pyrimethamine via Ubiquitin Mediated Degradation of AIMP2-DX2"

    Article Title: Anticancer Activity of Pyrimethamine via Ubiquitin Mediated Degradation of AIMP2-DX2

    Journal: Molecules

    doi: 10.3390/molecules25122763

    Significance of the DX2 level on pyrimethamine-mediated suppression of cancer cell viability. DX2- or EV-inducible (ind) A549 cells were treated with doxycycline (Dox) and pyrimethamine (Pyr) at the indicated time to induce the expression of DX2 and to reduce cell viability, respectively. The values on the graph were calculated against cell viability of empty vector (EV)-inducible cell lines at time 0 and presented as a relative cell viability. The experiments were independently repeated three times. Student’s two-tailed t-test was performed for statistical analysis (* p
    Figure Legend Snippet: Significance of the DX2 level on pyrimethamine-mediated suppression of cancer cell viability. DX2- or EV-inducible (ind) A549 cells were treated with doxycycline (Dox) and pyrimethamine (Pyr) at the indicated time to induce the expression of DX2 and to reduce cell viability, respectively. The values on the graph were calculated against cell viability of empty vector (EV)-inducible cell lines at time 0 and presented as a relative cell viability. The experiments were independently repeated three times. Student’s two-tailed t-test was performed for statistical analysis (* p

    Techniques Used: Expressing, Plasmid Preparation, Two Tailed Test

    Screening to identify DX2 inhibitors. 763 chemicals (5 µM) from KCB at KRICT was used for first screening. Thirty chemicals showing greater than 50% inhibition were subjected to secondary screening via nanoluciferase and cell viability assays using A549 and WI-26 cells.
    Figure Legend Snippet: Screening to identify DX2 inhibitors. 763 chemicals (5 µM) from KCB at KRICT was used for first screening. Thirty chemicals showing greater than 50% inhibition were subjected to secondary screening via nanoluciferase and cell viability assays using A549 and WI-26 cells.

    Techniques Used: Inhibition

    10) Product Images from "Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin"

    Article Title: Nocodazole-Induced Expression and Phosphorylation of Anillin and Other Mitotic Proteins Are Decreased in DNA-Dependent Protein Kinase Catalytic Subunit-Deficient Cells and Rescued by Inhibition of the Anaphase-Promoting Complex/Cyclosome with proTAME but Not Apcin

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.00191-19

    Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient A549 cells. Experiments were carried out exactly as for Fig. 7 , except that A549-CRISPR-control and CRISPR–DNA-PKcs cells were used. The error bars indicate standard deviations. *, P
    Figure Legend Snippet: Inhibition of APC/C with proTAME rescues expression and phosphorylation of mitotic proteins in nocodazole-treated DNA-PKcs-deficient A549 cells. Experiments were carried out exactly as for Fig. 7 , except that A549-CRISPR-control and CRISPR–DNA-PKcs cells were used. The error bars indicate standard deviations. *, P

    Techniques Used: Inhibition, Expressing, CRISPR

    Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient A549 cells. The experiment was carried out exactly as for Fig. 5 , but A549–CRISPR–DNA-PKcs cells were used. The asterisk on the securin blot indicates a nonspecific band. The error bars indicate standard deviations.
    Figure Legend Snippet: Depletion of PP6 does not rescue nocodazole-induced downregulation of mitotic proteins in DNA-PKcs-deficient A549 cells. The experiment was carried out exactly as for Fig. 5 , but A549–CRISPR–DNA-PKcs cells were used. The asterisk on the securin blot indicates a nonspecific band. The error bars indicate standard deviations.

    Techniques Used: CRISPR

    A549 cells with CRISPR depletion of DNA-PKcs have reduced and/or delayed nocodazole-induced upregulation of anillin, cyclin B1, and securin and reduced phosphorylation of Aurora A, PLK1, and TPX2. (A) A549-CRISPR-control, A549–CRISPR–DNA-PKcs, and A549-CRISPR-ATM cells were treated with nocodazole for 6 or 16 h, and then extracts were run on SDS-PAGE and analyzed by Western blotting as for Fig. 2 . (B to M) Quantitation from 3 separate experiments. (B, D, E, G, H, J, K, L, and M) Proteins were normalized to Ku80. (C, F, and I) Phosphoproteins were normalized to their respective total proteins. Both are shown in arbitrary units ( y axis). Statistical analysis was carried out by one-way ANOVA, with P values of
    Figure Legend Snippet: A549 cells with CRISPR depletion of DNA-PKcs have reduced and/or delayed nocodazole-induced upregulation of anillin, cyclin B1, and securin and reduced phosphorylation of Aurora A, PLK1, and TPX2. (A) A549-CRISPR-control, A549–CRISPR–DNA-PKcs, and A549-CRISPR-ATM cells were treated with nocodazole for 6 or 16 h, and then extracts were run on SDS-PAGE and analyzed by Western blotting as for Fig. 2 . (B to M) Quantitation from 3 separate experiments. (B, D, E, G, H, J, K, L, and M) Proteins were normalized to Ku80. (C, F, and I) Phosphoproteins were normalized to their respective total proteins. Both are shown in arbitrary units ( y axis). Statistical analysis was carried out by one-way ANOVA, with P values of

    Techniques Used: CRISPR, SDS Page, Western Blot, Quantitation Assay

    11) Product Images from "Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus"

    Article Title: Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus

    Journal: Frontiers in Microbiology

    doi: 10.3389/fmicb.2016.00357

    Influence of recombinant NanA and NanB on expression of SA on the surface of A549 cells . Cells were treated with different neuraminidase dilutions for 48 h. The lectins MAA (A) and SNA (B) were used to detect SAα2-3Gal and SAα2-6Gal, respectively, by immunocytochemical staining. Control (Co) was stained without using lectins.
    Figure Legend Snippet: Influence of recombinant NanA and NanB on expression of SA on the surface of A549 cells . Cells were treated with different neuraminidase dilutions for 48 h. The lectins MAA (A) and SNA (B) were used to detect SAα2-3Gal and SAα2-6Gal, respectively, by immunocytochemical staining. Control (Co) was stained without using lectins.

    Techniques Used: Recombinant, Expressing, Staining

    Spread of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) in the absence and the presence of different dilutions of recombinant NanA and NanB . The effect of NanA (A) and NanB (B) on virus spread in A549 cells was analyzed by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection with Jena/8178 at MOI of 0.1 TCID 50 /cell.
    Figure Legend Snippet: Spread of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) in the absence and the presence of different dilutions of recombinant NanA and NanB . The effect of NanA (A) and NanB (B) on virus spread in A549 cells was analyzed by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection with Jena/8178 at MOI of 0.1 TCID 50 /cell.

    Techniques Used: Recombinant, Staining, Infection

    Inhibition of replication of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) by neuraminidase inhibitors (NAIs) . A549 cells infected with Jena/8178 (MOI of 0.1 TCID 50 /cells) were treated with oseltamivir, zanamivir, DANA, artocarpin, and katsumadain A in absence of pneumococcal NAs (A) , presence of rNanA (B) or presence of rNanB (C) . Virus-infected cells were detected by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection to visualize the inhibitor effect on virus spread. To analyze the effect of NAIs on virus yield (D) , virus titers in pfu/mL were determined with plaque assay 48 h after infection. Virus control titer was set to 100% and inhibition of the control titer by NAIs in % was calculated. Experiments were performed at least three times, and one representative assay is exemplarily shown. Significant values were calculated with non-parametric Wilcoxon–Mann–Whitney test ( ∗ p
    Figure Legend Snippet: Inhibition of replication of A(H1N1)pdm09 strain A/Jena/8178/09 (Jena/8178) by neuraminidase inhibitors (NAIs) . A549 cells infected with Jena/8178 (MOI of 0.1 TCID 50 /cells) were treated with oseltamivir, zanamivir, DANA, artocarpin, and katsumadain A in absence of pneumococcal NAs (A) , presence of rNanA (B) or presence of rNanB (C) . Virus-infected cells were detected by immunocytochemical staining of viral nucleoprotein (shown in red) 48 h after infection to visualize the inhibitor effect on virus spread. To analyze the effect of NAIs on virus yield (D) , virus titers in pfu/mL were determined with plaque assay 48 h after infection. Virus control titer was set to 100% and inhibition of the control titer by NAIs in % was calculated. Experiments were performed at least three times, and one representative assay is exemplarily shown. Significant values were calculated with non-parametric Wilcoxon–Mann–Whitney test ( ∗ p

    Techniques Used: Inhibition, Infection, Staining, Plaque Assay, MANN-WHITNEY

    12) Product Images from "Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells"

    Article Title: Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-018-22862-0

    Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.
    Figure Legend Snippet: Microscopic images of A549 cells stained with an anti-β-tubulin antibody (red) and counterstained with DAPI (blue). The cells were treated for 24 h with scoulerine or a solvent (0.1% DMSO) as vehicle control. Nocodazole, an antineoplastic agent that disrupts microtubule function by binding to tubulin was used as a reference compound in this assay. Scale bar: 10 µm. Experiments were performed in triplicate using epi-fluorescence microscopy. Photographs from representative chambers are shown. Compared with controls, thicker and denser microtubule bundles were evident in scoulerine-treated cells.

    Techniques Used: Staining, Binding Assay, Fluorescence, Microscopy

    Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.
    Figure Legend Snippet: Dynamic real-time monitoring of proliferation and cytotoxicity using the xCELLigence system dedicated to adherent cell lines. Growth kinetics of human A549 lung carcinoma ( A ), A2780 ovarian carcinoma ( B ), SK-BR-3 breast adenocarcinoma ( C ) and MCF-7 breast adenocarcinoma ( D ) cells treated with scoulerine. Cells treated with 0.1% DMSO were used as vehicle control and 5% DMSO treated cells were used as positive control. The normalized cell index was measured over 72 h. Plots shown are representative of at least three replicate experiments in each case.

    Techniques Used: Positive Control

    13) Product Images from "p53-independent structure-activity relationships of 3-ring mesogenic compounds’ activity as cytotoxic effects against human non-small cell lung cancer lines"

    Article Title: p53-independent structure-activity relationships of 3-ring mesogenic compounds’ activity as cytotoxic effects against human non-small cell lung cancer lines

    Journal: BMC Cancer

    doi: 10.1186/s12885-016-2585-6

    Involvement of caspase in mesogenic compound-induced cell death. a – b A549 cells were preincubated with Z-VAD-fmk for 1 h and then cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h. Cells were then harvested, and analyses of active caspase-3 expression and sub-G1 fractions were performed. a Representative histograms of three different experiments are shown, and inset numbers indicate percentages of active caspase-3-positive cells. Z-VAD indicates Z-VAD-fmk. b Sub-G1 fractions are presented as mean ± SE of 4 independent experiments. * indicates p
    Figure Legend Snippet: Involvement of caspase in mesogenic compound-induced cell death. a – b A549 cells were preincubated with Z-VAD-fmk for 1 h and then cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h. Cells were then harvested, and analyses of active caspase-3 expression and sub-G1 fractions were performed. a Representative histograms of three different experiments are shown, and inset numbers indicate percentages of active caspase-3-positive cells. Z-VAD indicates Z-VAD-fmk. b Sub-G1 fractions are presented as mean ± SE of 4 independent experiments. * indicates p

    Techniques Used: Cell Culture, Expressing

    Involvement of Fas in compound-induced cell death of A549 cells. a A549 cells were treated with C1 or C2 for 72 h and harvested for western blotting of caspase-8 and caspase-9. Representative results of two different experiments are shown. b A549 cells cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h were harvested, and cell surface Fas expression was analyzed. Representative histograms of three different experiments are shown. The dotted line histogram indicates cells stained with isotype control. The broken line and filled gray histograms indicate Fas expression in cells treated with vehicle and compound, respectively. Inset numbers indicate mean fluorescence intensities relative to the vehicle control. c A549 cells were preincubated with the Fas/Fas ligand antagonist kp7-6 and were cultured in the presence of 10 μM C1 for 48 h [ left panel ] or C2 for 72 h [ right panel ]. Cells were harvested and annexin V/propidium iodide staining was performed. Percentages of annexin V(+) cells are presented as the mean ± SE of 3 independent experiments; * indicates p
    Figure Legend Snippet: Involvement of Fas in compound-induced cell death of A549 cells. a A549 cells were treated with C1 or C2 for 72 h and harvested for western blotting of caspase-8 and caspase-9. Representative results of two different experiments are shown. b A549 cells cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h were harvested, and cell surface Fas expression was analyzed. Representative histograms of three different experiments are shown. The dotted line histogram indicates cells stained with isotype control. The broken line and filled gray histograms indicate Fas expression in cells treated with vehicle and compound, respectively. Inset numbers indicate mean fluorescence intensities relative to the vehicle control. c A549 cells were preincubated with the Fas/Fas ligand antagonist kp7-6 and were cultured in the presence of 10 μM C1 for 48 h [ left panel ] or C2 for 72 h [ right panel ]. Cells were harvested and annexin V/propidium iodide staining was performed. Percentages of annexin V(+) cells are presented as the mean ± SE of 3 independent experiments; * indicates p

    Techniques Used: Western Blot, Cell Culture, Expressing, Staining, Fluorescence

    Involvement of DNA damage-signaling pathway and p53 in compounds-induced cell death of A549 cells. a A549 cells cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h were harvested, and intracellular phospho-ATM expression was analyzed. Representative histograms of three different experiments are shown. The dotted line histogram indicates cells stained with Alexa Fluor® 488-conjugated secondary antibody alone. The broken line and filled gray histograms indicate phosphor-ATM expression in cells treated with vehicle and compound, respectively. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 30 min after irradiation. b A549 cells preincubated with the caffeine (2 mM) were cultured in the presence of 10 μM C1 or C2 for 72 h. The cells were harvested, and annexin V/propidium iodide staining was performed. Percentages of annexin V(+) cells are presented as the mean ± SE of three independent experiments; * and ** indicate p
    Figure Legend Snippet: Involvement of DNA damage-signaling pathway and p53 in compounds-induced cell death of A549 cells. a A549 cells cultured in the presence of 10 μM C1 for 48 h or C2 for 72 h were harvested, and intracellular phospho-ATM expression was analyzed. Representative histograms of three different experiments are shown. The dotted line histogram indicates cells stained with Alexa Fluor® 488-conjugated secondary antibody alone. The broken line and filled gray histograms indicate phosphor-ATM expression in cells treated with vehicle and compound, respectively. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 30 min after irradiation. b A549 cells preincubated with the caffeine (2 mM) were cultured in the presence of 10 μM C1 or C2 for 72 h. The cells were harvested, and annexin V/propidium iodide staining was performed. Percentages of annexin V(+) cells are presented as the mean ± SE of three independent experiments; * and ** indicate p

    Techniques Used: Cell Culture, Expressing, Staining, Positive Control, Irradiation

    Effects of test compounds on the growth of non-small cell lung cancer (NSCLC) cells. a The structures of the test compounds are shown. b , d – e NSCLC cells cultured in the presence of compounds C1–C7 at 10 μM for 3 days were harvested, and viable cells were counted using trypan blue exclusion assays. Dotted lines indicate the dimethyl sulfoxide control. Data are presented as the mean ± SE of 3 independent experiments. c A549 cells cultured in the presence of compounds C1–C2 at 10 μM for 1–3 days were harvested, and viable cells were counted using trypan blue exclusion assays. Results are shown as relative value against input cell number. Data are presented as the mean ± SE of 3 independent experiments. f Structural formulae of C1 and its derivatives with differing alkyl chain lengths and cytotoxic effects against A549 cells [ left panel ] and the other NSCLC cells [ light panel ]; data are presented as mean ± SE of 3 independent experiments. g The logP values of test compounds are shown
    Figure Legend Snippet: Effects of test compounds on the growth of non-small cell lung cancer (NSCLC) cells. a The structures of the test compounds are shown. b , d – e NSCLC cells cultured in the presence of compounds C1–C7 at 10 μM for 3 days were harvested, and viable cells were counted using trypan blue exclusion assays. Dotted lines indicate the dimethyl sulfoxide control. Data are presented as the mean ± SE of 3 independent experiments. c A549 cells cultured in the presence of compounds C1–C2 at 10 μM for 1–3 days were harvested, and viable cells were counted using trypan blue exclusion assays. Results are shown as relative value against input cell number. Data are presented as the mean ± SE of 3 independent experiments. f Structural formulae of C1 and its derivatives with differing alkyl chain lengths and cytotoxic effects against A549 cells [ left panel ] and the other NSCLC cells [ light panel ]; data are presented as mean ± SE of 3 independent experiments. g The logP values of test compounds are shown

    Techniques Used: Cell Culture

    Effects of test compounds on cell cycle progression. a Non-small cell lung cancer (NSCLC) cells cultured in the presence of test compounds (C1–C2) at 10 μM for 24–48 h were harvested, and then cell cycle profiles were analyzed. Representative histograms of three different experiments are shown. b NSCLC cells cultured in the presence of test compounds (C1–C5) at 10 μM for 24–48 h were harvested, and then cell cycle profiles were analyzed. Fractions of G2/M at 24 h in NSCLC cells (A549, LU99, EBC-1, and H1299 cells) [ left panel ] and G1 at 48 h in A549 and H1299 cells [ light panel ] are shown. Data are presented as mean ± SE of 3 independent experiments. * and ** indicate p
    Figure Legend Snippet: Effects of test compounds on cell cycle progression. a Non-small cell lung cancer (NSCLC) cells cultured in the presence of test compounds (C1–C2) at 10 μM for 24–48 h were harvested, and then cell cycle profiles were analyzed. Representative histograms of three different experiments are shown. b NSCLC cells cultured in the presence of test compounds (C1–C5) at 10 μM for 24–48 h were harvested, and then cell cycle profiles were analyzed. Fractions of G2/M at 24 h in NSCLC cells (A549, LU99, EBC-1, and H1299 cells) [ left panel ] and G1 at 48 h in A549 and H1299 cells [ light panel ] are shown. Data are presented as mean ± SE of 3 independent experiments. * and ** indicate p

    Techniques Used: Cell Culture

    Involvement of p53 in compound C1–induced G2/M arrest. a A549 cells were treated with C1 for 12 h and harvested for western blotting of p53 and p21 and cell cycle analyses. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 12 h after irradiation for Western blotting analyses of p53 and p21; actin was used as a loading control. Representative blots of two different experiments are shown [ upper panel ]. Representative histograms of two different experiments are shown [ lower panel ]. b A549 cells preincubated with the caffeine were cultured in the presence of 10 μM C1 for 24 h [ left panel ] or cultured for 12 h after 10 Gy-irradiation [ right panel ]. The cells were harvested, and cell cycle analysis was performed. Representative histograms of two different experiments are shown. c – d A549 cells treated with p53 siRNA were cultured in the presence of C1 at 10 μM for 12 h, and then western blotting of p53 ( c ) and cell cycle profiles ( d ) were analyzed. Representative results of two different experiments are shown. e A549 cells were treated with C1 for 12–24 h and harvested for western blotting of phospho-cdc2 (p-cdc2), cyclin B1, and phospho-histone H3 (p-H3). Representative results of two different experiments are shown
    Figure Legend Snippet: Involvement of p53 in compound C1–induced G2/M arrest. a A549 cells were treated with C1 for 12 h and harvested for western blotting of p53 and p21 and cell cycle analyses. As a positive control, A549 cells were irradiated with 10 Gy X-ray and harvested 12 h after irradiation for Western blotting analyses of p53 and p21; actin was used as a loading control. Representative blots of two different experiments are shown [ upper panel ]. Representative histograms of two different experiments are shown [ lower panel ]. b A549 cells preincubated with the caffeine were cultured in the presence of 10 μM C1 for 24 h [ left panel ] or cultured for 12 h after 10 Gy-irradiation [ right panel ]. The cells were harvested, and cell cycle analysis was performed. Representative histograms of two different experiments are shown. c – d A549 cells treated with p53 siRNA were cultured in the presence of C1 at 10 μM for 12 h, and then western blotting of p53 ( c ) and cell cycle profiles ( d ) were analyzed. Representative results of two different experiments are shown. e A549 cells were treated with C1 for 12–24 h and harvested for western blotting of phospho-cdc2 (p-cdc2), cyclin B1, and phospho-histone H3 (p-H3). Representative results of two different experiments are shown

    Techniques Used: Western Blot, Positive Control, Irradiation, Cell Culture, Cell Cycle Assay

    14) Product Images from "Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis"

    Article Title: Lassa Virus Cell Entry via Dystroglycan Involves an Unusual Pathway of Macropinocytosis

    Journal: Journal of Virology

    doi: 10.1128/JVI.00257-16

    Entry of rLCMV-LASVGP into A549 epithelial cells shows hallmarks of macropinocytosis. (A) Schema of the inhibitor experiment. For details, see the text. LE, late endosome. (B, C) Entry of rLCMV-LASVGP is independent of dynamin and clathrin. A549 cells
    Figure Legend Snippet: Entry of rLCMV-LASVGP into A549 epithelial cells shows hallmarks of macropinocytosis. (A) Schema of the inhibitor experiment. For details, see the text. LE, late endosome. (B, C) Entry of rLCMV-LASVGP is independent of dynamin and clathrin. A549 cells

    Techniques Used:

    Entry of rLCMV-LASVGP does not affect either overall cellular actin dynamics or bulk fluid uptake. (A) Changes in cell morphology during virus entry. Subconfluent A549 cells were mock treated or exposed to rLCMV-LASVGP (100 PFU/cell) or VACV (3 PFU/cell)
    Figure Legend Snippet: Entry of rLCMV-LASVGP does not affect either overall cellular actin dynamics or bulk fluid uptake. (A) Changes in cell morphology during virus entry. Subconfluent A549 cells were mock treated or exposed to rLCMV-LASVGP (100 PFU/cell) or VACV (3 PFU/cell)

    Techniques Used:

    Infection of rLCMV -LASVGP in A549 cells depleted of HGFR. (A) Depletion of HGFR by RNAi. A549 cells were transfected with siRNAs specific for HGFR and the corresponding control siRNA (C911). An initial transfection (First) was performed 16 h after plating.
    Figure Legend Snippet: Infection of rLCMV -LASVGP in A549 cells depleted of HGFR. (A) Depletion of HGFR by RNAi. A549 cells were transfected with siRNAs specific for HGFR and the corresponding control siRNA (C911). An initial transfection (First) was performed 16 h after plating.

    Techniques Used: Infection, Transfection

    Conserved profile of cellular factors involved in rLCMV-LASVGP entry. (A) Inhibition of rLCMV-LASVGP infection with selected inhibitors in different epithelial cell lines. Monolayers of A549, WI-26VA4, and Caco-2 cells were treated with the indicated
    Figure Legend Snippet: Conserved profile of cellular factors involved in rLCMV-LASVGP entry. (A) Inhibition of rLCMV-LASVGP infection with selected inhibitors in different epithelial cell lines. Monolayers of A549, WI-26VA4, and Caco-2 cells were treated with the indicated

    Techniques Used: Inhibition, Infection

    Identification of cellular kinases involved in rLCMV-LASVGP entry. (A) Screening of a library of kinase inhibitors against rLCMV-LASVGP entry. Monolayers of A549 cells were pretreated with inhibitors for 30 min. Infection with rLCMV-LASVGP (MOI = 0.5)
    Figure Legend Snippet: Identification of cellular kinases involved in rLCMV-LASVGP entry. (A) Screening of a library of kinase inhibitors against rLCMV-LASVGP entry. Monolayers of A549 cells were pretreated with inhibitors for 30 min. Infection with rLCMV-LASVGP (MOI = 0.5)

    Techniques Used: Infection

    Antiviral activity of the kinase inhibitor EMD 1214063 (EMD). (A) Inhibition of different viruses with EMD 1214063. Monolayers of A549 cells were pretreated with the indicated concentration of EMD 1214063 for 30 min, followed by infection with the different
    Figure Legend Snippet: Antiviral activity of the kinase inhibitor EMD 1214063 (EMD). (A) Inhibition of different viruses with EMD 1214063. Monolayers of A549 cells were pretreated with the indicated concentration of EMD 1214063 for 30 min, followed by infection with the different

    Techniques Used: Activity Assay, Inhibition, Concentration Assay, Infection

    Conserved profile of cellular factors involved in rLCMV-LASVGP entry into polarized cells. (A) Detection of functional DG at the apical and basolateral surfaces of polarized A549 and Caco-2 cells. Cells were cultured on Transwell filters to obtain polarized
    Figure Legend Snippet: Conserved profile of cellular factors involved in rLCMV-LASVGP entry into polarized cells. (A) Detection of functional DG at the apical and basolateral surfaces of polarized A549 and Caco-2 cells. Cells were cultured on Transwell filters to obtain polarized

    Techniques Used: Functional Assay, Cell Culture

    Additive antiviral activity of EMD 1214063 and ribavirin. (A) Inhibition of rLCMV-LASVGP infection with Rib. A549 cells were infected with rLCMV-LASVGP at an MOI of 0.01. After removal of unbound virus, fresh medium containing the indicated concentrations
    Figure Legend Snippet: Additive antiviral activity of EMD 1214063 and ribavirin. (A) Inhibition of rLCMV-LASVGP infection with Rib. A549 cells were infected with rLCMV-LASVGP at an MOI of 0.01. After removal of unbound virus, fresh medium containing the indicated concentrations

    Techniques Used: Activity Assay, Inhibition, Infection

    15) Product Images from "Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection"

    Article Title: Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection

    Journal: PLoS Pathogens

    doi: 10.1371/journal.ppat.1001109

    Protein phosphorylation and transcriptomic response to r-ceramide or LPS+sCD14. Panel A shows a phosphoproteomic heat map of A549 cells stimulated for 60 min with r-ceramide (SMase, 1 U/ml) or LPS+sCD14 (10+1 µg/ml) compared to untreated cells. Fold changes in protein phosphorylation levels were determined, using the Human Phospho-Kinase Array Kit. Panels B and C show gene expression heat maps of human epithelial cell RNAs. A549 or A498 cells were stimulated for 1 h or 3 h with r-ceramide or LPS+sCD14. Panels D and E show CREB phosphorylation (CREB-P) activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human CREB-P primary antibodies). Fos-P activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human Fos-P primary antibodies). JNK phosphorylation (JNK-P) by r-ceramide and LPS+sCD14 (rabbit anti-human JNK-P primary antibodies). Fluorescence intensities were quantified with LSM 510 software. Mean ± SEM, n = 60 cells/treatment, * p
    Figure Legend Snippet: Protein phosphorylation and transcriptomic response to r-ceramide or LPS+sCD14. Panel A shows a phosphoproteomic heat map of A549 cells stimulated for 60 min with r-ceramide (SMase, 1 U/ml) or LPS+sCD14 (10+1 µg/ml) compared to untreated cells. Fold changes in protein phosphorylation levels were determined, using the Human Phospho-Kinase Array Kit. Panels B and C show gene expression heat maps of human epithelial cell RNAs. A549 or A498 cells were stimulated for 1 h or 3 h with r-ceramide or LPS+sCD14. Panels D and E show CREB phosphorylation (CREB-P) activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human CREB-P primary antibodies). Fos-P activated by r-ceramide but not by LPS+sCD14 (rabbit anti-human Fos-P primary antibodies). JNK phosphorylation (JNK-P) by r-ceramide and LPS+sCD14 (rabbit anti-human JNK-P primary antibodies). Fluorescence intensities were quantified with LSM 510 software. Mean ± SEM, n = 60 cells/treatment, * p

    Techniques Used: Expressing, Fluorescence, Software

    Ceramide-TLR4 interactions, adaptors and TRAM phosphorylation. TLR4 and native ceramide were labeled with specific primary antibodies, followed by Alexa fluor-488 (donor) and Alexa fluor-568 (acceptor)-labeled secondary antibodies, respectively. Acceptor bleaching was quantified by confocal microscopy, comparing target membrane regions (inset) and cytoplasmic control areas. Panel A shows membrane and cytoplasmic staining for TLR4 (green) and ceramide (red). Panel B shows recording of donor and acceptor channels, ten times before and after acceptor bleaching (arrow). The intensity at time 0 was set to 100%. Panel C compared cells exposed to LPS+sCD14 (10+1µg/ml) or r-ceramide (SMase, 1 U/ml, 1 h). Panel D shows an increase in membrane FRET signals after ceramide release, but not after LPS+sCD14 treatment. Panel E shows siRNA silencing ( black , 72 h transfection) of TLR4, TRAM or MyD88 and resulting inhibition of IL-8 responses in A549 cells, stimulated with SMase (1 U/ml) or LPS+sCD14 (10+1µg/ml). Cytokine responses (Medians±SEMs ≥3 experiments) were compared to irrelevant siRNA-transfected cells ( grey , * p
    Figure Legend Snippet: Ceramide-TLR4 interactions, adaptors and TRAM phosphorylation. TLR4 and native ceramide were labeled with specific primary antibodies, followed by Alexa fluor-488 (donor) and Alexa fluor-568 (acceptor)-labeled secondary antibodies, respectively. Acceptor bleaching was quantified by confocal microscopy, comparing target membrane regions (inset) and cytoplasmic control areas. Panel A shows membrane and cytoplasmic staining for TLR4 (green) and ceramide (red). Panel B shows recording of donor and acceptor channels, ten times before and after acceptor bleaching (arrow). The intensity at time 0 was set to 100%. Panel C compared cells exposed to LPS+sCD14 (10+1µg/ml) or r-ceramide (SMase, 1 U/ml, 1 h). Panel D shows an increase in membrane FRET signals after ceramide release, but not after LPS+sCD14 treatment. Panel E shows siRNA silencing ( black , 72 h transfection) of TLR4, TRAM or MyD88 and resulting inhibition of IL-8 responses in A549 cells, stimulated with SMase (1 U/ml) or LPS+sCD14 (10+1µg/ml). Cytokine responses (Medians±SEMs ≥3 experiments) were compared to irrelevant siRNA-transfected cells ( grey , * p

    Techniques Used: Labeling, Confocal Microscopy, Staining, Transfection, Inhibition

    Nuclear IRF3 translocation in response to ceramide/TLR4. Panels A and C show IRF3 and panels B and D show NF-κB p65 translocation in 70% confluent A549 cells exposed to r-ceramide (SMase (1U/ml), C6 ceramide (30 µg/ml) or LPS+sCD14 (10+1 µg/ml) for 90 min (mean ± SEM of 50 cells/sample, *** p
    Figure Legend Snippet: Nuclear IRF3 translocation in response to ceramide/TLR4. Panels A and C show IRF3 and panels B and D show NF-κB p65 translocation in 70% confluent A549 cells exposed to r-ceramide (SMase (1U/ml), C6 ceramide (30 µg/ml) or LPS+sCD14 (10+1 µg/ml) for 90 min (mean ± SEM of 50 cells/sample, *** p

    Techniques Used: Translocation Assay

    16) Product Images from "Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F"

    Article Title: Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F

    Journal: Oncogene

    doi: 10.1038/s41388-019-1009-x

    EIF3F overexpression alters lung tumor growth in vivo but promotes hepatic metastasis. a Protocol used for mice orthotopic human lung tumors generation and tumor imaging and sampling. b Immunostaining of A549 human cells in the lung parenchyma using anti-HLA antibodies allowed verifying the presence of human orthotopic tumor in NSG-mice lung (4 weeks time point). Observation was performed on control mice that received no cell injection and on mice injected with EIF3F-A549 human cancer cells. The lung tissue was stained with HES staining. c in vivo determination of the volume of luciferase-expressing mice orthotopic human lung tumors in NSG mice (4 weeks time point). Results are expressed as number of photons per second per steradian. d CTL and EIF3F lung tumors volume follow-up during the course of the experiment (20 mice per group). e Lung tumors volume after 28 days of growth determined in vivo using bioluminescence imaging ( N = 20). f Identification and quantification of hepatic metastasis in the two groups of mice. g Quantification of the volume of hepatic metastasis was performed ex vivo ( N = 20). h EIF3F gene expression was analyzed in silico on a cohort of 584 lung adenocarcinoma tumors (RNAseqV2 data from the TCGA LUAD cohort). EIF3F high and EIF3F low populations were determined by setting the cut-off for EIF3F expression at 11.8, based on calculations using the Cbioportal bioinformatic platform. This cut-off value of 11.8 segregates two groups of patients with a significant difference in survival ( p = 0.018). The group of patients with EIF3F expression > 11.8 accounted for 10% of the total population. i Patients survival curves were obtained for the two subgroups stratified by EIF3F expression (high or low; as obtained from h ). j The presence of metastasis (M1 population) was compared in EIF3F high and EIF3F low LUAD patients (TCGA data). k Genetic alterations of the EIF3F gene in the TCGA LUAD cohort of human lung tumors (1144 samples; obtained from Cbioportal). * P
    Figure Legend Snippet: EIF3F overexpression alters lung tumor growth in vivo but promotes hepatic metastasis. a Protocol used for mice orthotopic human lung tumors generation and tumor imaging and sampling. b Immunostaining of A549 human cells in the lung parenchyma using anti-HLA antibodies allowed verifying the presence of human orthotopic tumor in NSG-mice lung (4 weeks time point). Observation was performed on control mice that received no cell injection and on mice injected with EIF3F-A549 human cancer cells. The lung tissue was stained with HES staining. c in vivo determination of the volume of luciferase-expressing mice orthotopic human lung tumors in NSG mice (4 weeks time point). Results are expressed as number of photons per second per steradian. d CTL and EIF3F lung tumors volume follow-up during the course of the experiment (20 mice per group). e Lung tumors volume after 28 days of growth determined in vivo using bioluminescence imaging ( N = 20). f Identification and quantification of hepatic metastasis in the two groups of mice. g Quantification of the volume of hepatic metastasis was performed ex vivo ( N = 20). h EIF3F gene expression was analyzed in silico on a cohort of 584 lung adenocarcinoma tumors (RNAseqV2 data from the TCGA LUAD cohort). EIF3F high and EIF3F low populations were determined by setting the cut-off for EIF3F expression at 11.8, based on calculations using the Cbioportal bioinformatic platform. This cut-off value of 11.8 segregates two groups of patients with a significant difference in survival ( p = 0.018). The group of patients with EIF3F expression > 11.8 accounted for 10% of the total population. i Patients survival curves were obtained for the two subgroups stratified by EIF3F expression (high or low; as obtained from h ). j The presence of metastasis (M1 population) was compared in EIF3F high and EIF3F low LUAD patients (TCGA data). k Genetic alterations of the EIF3F gene in the TCGA LUAD cohort of human lung tumors (1144 samples; obtained from Cbioportal). * P

    Techniques Used: Over Expression, In Vivo, Mouse Assay, Imaging, Sampling, Immunostaining, Injection, Staining, Luciferase, Expressing, CTL Assay, Ex Vivo, In Silico

    Identification of the ‘EIF3F gene cluster’. a Representative images of nuclear immuno-staining of EIF3F in CTL-A549 cells and EIF3F-A549 cells. b EIF3F gene cluster identification methods using ChIP-seq and proteomics. Following chromatin immuno-precipitation using two different antibodies, the DNA fragments associated to EIF3F were sequenced and analyzed for peak annotation. The peaks common to the two sets of experiments (134 reads) were compared to the group of proteins altered at the proteomic level (see Fig. 3 ) to identify the ‘EIF3F gene cluster’ composed of 34 genes. c Components of the EIF3F gene cluster were organized according to the Hallmarks of cancer based on their cellular function. d The expression of the EIF3F gene cluster components was analyzed by qPCR in A549 cells, EIF3F-A549 cells, and in A549-EIF3F cells treated with FZM1 (Frizzled-4 dependent β-catenin pathway inhibitor) and Nifuroxazide (STAT3 inhibitor) ( N = 3). e Representative images, and f quantification of cell migration (transwell) assay performed in CTL-A549 cells and EIF3F-A549 cells using FZD4, SNAI2, NDP, and STAT3 siRNAs ( N = 3). g Summary of the findings showing the implication of the EIF3F-FZD4-STAT3 signaling pathway in Snai2 expression and metastasis gene regulation. * P
    Figure Legend Snippet: Identification of the ‘EIF3F gene cluster’. a Representative images of nuclear immuno-staining of EIF3F in CTL-A549 cells and EIF3F-A549 cells. b EIF3F gene cluster identification methods using ChIP-seq and proteomics. Following chromatin immuno-precipitation using two different antibodies, the DNA fragments associated to EIF3F were sequenced and analyzed for peak annotation. The peaks common to the two sets of experiments (134 reads) were compared to the group of proteins altered at the proteomic level (see Fig. 3 ) to identify the ‘EIF3F gene cluster’ composed of 34 genes. c Components of the EIF3F gene cluster were organized according to the Hallmarks of cancer based on their cellular function. d The expression of the EIF3F gene cluster components was analyzed by qPCR in A549 cells, EIF3F-A549 cells, and in A549-EIF3F cells treated with FZM1 (Frizzled-4 dependent β-catenin pathway inhibitor) and Nifuroxazide (STAT3 inhibitor) ( N = 3). e Representative images, and f quantification of cell migration (transwell) assay performed in CTL-A549 cells and EIF3F-A549 cells using FZD4, SNAI2, NDP, and STAT3 siRNAs ( N = 3). g Summary of the findings showing the implication of the EIF3F-FZD4-STAT3 signaling pathway in Snai2 expression and metastasis gene regulation. * P

    Techniques Used: Immunostaining, CTL Assay, Chromatin Immunoprecipitation, Cell Function Assay, Expressing, Real-time Polymerase Chain Reaction, Migration, Transwell Assay

    EIF3F reprograms the proteome of mice orthotopic human lung tumors and promotes cell migration. a Proteome analysis of EIF3F lung tumors compared to CTL lung tumors ( N = 4). Ingenuity pathway core analysis (IPA; Qiagen). The bar graph of a was generated using Ingenuity Pathway analysis (IPA; Qiagen) software from the analysis of the raw data of the differential proteomics study, obtained between EIF3F-A549 and A549 orthtotopic lung tumors. The proteins with a different expression between the two types of tumors were organized based on pre-defined categories suggested by IPA (as for example ‘EIF2 signaling’). Then, proteins with different expression were assigned to these IPA-categories based on the IPA-experts curated database, and the categories were ranked according to their frequency of identification in the proteome (the –log( p value); top axis value). Then, the directionality of the change per category was given by a color code. Orange means that the pathway was increased, blue that it was inhibited, and gray that no directionality could be calculated (some proteins were increased but other were decreased). The ‘ratio’ given in the bottom axis indicates the % of proteins from the predetermined IPA-category that were identified in the differential proteome. For instance 62% of the proteins composing the ‘EIF2 signaling’ group were found to be differentially expressed between EIF3F-A549 and A549 cells. b KEGG pathways analysis of the differential proteome analysis data showing the changes in metabolic pathways induced by EIF3F overexpression in orthotopic mice tumors. These data were obtained using String ( https://string-db.org/ ). The pie chart was obtained by plotting the number of genes in each category, expressed as percentage of the total. c Cellular functions impacted by EIF3F expression in the mice orthotopic human lung tumors. d Representative images and e quantification of transwell migration experiments performed in vitro on CTL-A549 cells, EIF3F-A549 cells and EIF3F-A549 cells treated with a EIF3F-siRNA ( n = 3). f Representative images of CTL-A549 at t = 0 h plus CTL-A549 and EIF3F-A549 at t = 24 h. g Quantification of wound healing assay performed in vitro on CTL-A549 cells and EIF3F-A549 cells ( N = 3). The area wound was measured after 24 h of proliferation in vitro. h and i Transwell migration experiments were performed in vitro on A549 cells and EIF3F-A549 cells ( N = 6) in the presence or absence of treatment with the STAT3 inhibitor Nifuroxazide 10 µM and siRNA against STAT3. * P
    Figure Legend Snippet: EIF3F reprograms the proteome of mice orthotopic human lung tumors and promotes cell migration. a Proteome analysis of EIF3F lung tumors compared to CTL lung tumors ( N = 4). Ingenuity pathway core analysis (IPA; Qiagen). The bar graph of a was generated using Ingenuity Pathway analysis (IPA; Qiagen) software from the analysis of the raw data of the differential proteomics study, obtained between EIF3F-A549 and A549 orthtotopic lung tumors. The proteins with a different expression between the two types of tumors were organized based on pre-defined categories suggested by IPA (as for example ‘EIF2 signaling’). Then, proteins with different expression were assigned to these IPA-categories based on the IPA-experts curated database, and the categories were ranked according to their frequency of identification in the proteome (the –log( p value); top axis value). Then, the directionality of the change per category was given by a color code. Orange means that the pathway was increased, blue that it was inhibited, and gray that no directionality could be calculated (some proteins were increased but other were decreased). The ‘ratio’ given in the bottom axis indicates the % of proteins from the predetermined IPA-category that were identified in the differential proteome. For instance 62% of the proteins composing the ‘EIF2 signaling’ group were found to be differentially expressed between EIF3F-A549 and A549 cells. b KEGG pathways analysis of the differential proteome analysis data showing the changes in metabolic pathways induced by EIF3F overexpression in orthotopic mice tumors. These data were obtained using String ( https://string-db.org/ ). The pie chart was obtained by plotting the number of genes in each category, expressed as percentage of the total. c Cellular functions impacted by EIF3F expression in the mice orthotopic human lung tumors. d Representative images and e quantification of transwell migration experiments performed in vitro on CTL-A549 cells, EIF3F-A549 cells and EIF3F-A549 cells treated with a EIF3F-siRNA ( n = 3). f Representative images of CTL-A549 at t = 0 h plus CTL-A549 and EIF3F-A549 at t = 24 h. g Quantification of wound healing assay performed in vitro on CTL-A549 cells and EIF3F-A549 cells ( N = 3). The area wound was measured after 24 h of proliferation in vitro. h and i Transwell migration experiments were performed in vitro on A549 cells and EIF3F-A549 cells ( N = 6) in the presence or absence of treatment with the STAT3 inhibitor Nifuroxazide 10 µM and siRNA against STAT3. * P

    Techniques Used: Mouse Assay, Migration, CTL Assay, Indirect Immunoperoxidase Assay, Generated, Software, Expressing, Over Expression, In Vitro, Wound Healing Assay

    EIF3F overexpression alters A549 human lung cancer cells proliferation. a Study workflow including in vitro and in vivo analyses of A549 cells overexpressing EIF3F. b Verification of the ectopic expression of EIF3F in A549 human lung adenocarcinoma cells using quantitative proteomics ( N = 3) and c – e immunoblot analysis of the M2-FLAG and EIF3F protein expression level in control and EIF3F cells. β-actin was used as loading control for EIF3F expression quantification ( N = 3 and N = 4, respectively). f EIF3 complex subunits expression was determined by qPCR ( N = 3). g Expression of PIC (pre initiation complex) components was analyzed using qPCR ( N = 3). h The global rate of protein synthesis was assessed by immunostaining of HPG Alexa Fluor® 488 ( N = 10) and i corresponding quantification. L-homopropargylglycine (HPG) is an amino acid analog of methionine containing an alkyne moiety and Alexa Fluor® 488. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. j Cell proliferation rate was determined using the BrdU incorporation assay ( N = 12). k Growth curves of A549, CTL-A549, and EIF3F-A549 cells were obtained by performing cell enumeration studies ( N = 6). l Apoptosis was investigated by measuring caspases 3 and 7 activities using a fluorescent substrate ( N = 4). All data were expressed as mean ± SEM from ‘n’ independent cell cultures. * P
    Figure Legend Snippet: EIF3F overexpression alters A549 human lung cancer cells proliferation. a Study workflow including in vitro and in vivo analyses of A549 cells overexpressing EIF3F. b Verification of the ectopic expression of EIF3F in A549 human lung adenocarcinoma cells using quantitative proteomics ( N = 3) and c – e immunoblot analysis of the M2-FLAG and EIF3F protein expression level in control and EIF3F cells. β-actin was used as loading control for EIF3F expression quantification ( N = 3 and N = 4, respectively). f EIF3 complex subunits expression was determined by qPCR ( N = 3). g Expression of PIC (pre initiation complex) components was analyzed using qPCR ( N = 3). h The global rate of protein synthesis was assessed by immunostaining of HPG Alexa Fluor® 488 ( N = 10) and i corresponding quantification. L-homopropargylglycine (HPG) is an amino acid analog of methionine containing an alkyne moiety and Alexa Fluor® 488. The HPG is fed to cultured cells and incorporated into proteins during active protein synthesis. j Cell proliferation rate was determined using the BrdU incorporation assay ( N = 12). k Growth curves of A549, CTL-A549, and EIF3F-A549 cells were obtained by performing cell enumeration studies ( N = 6). l Apoptosis was investigated by measuring caspases 3 and 7 activities using a fluorescent substrate ( N = 4). All data were expressed as mean ± SEM from ‘n’ independent cell cultures. * P

    Techniques Used: Over Expression, In Vitro, In Vivo, Expressing, Real-time Polymerase Chain Reaction, Immunostaining, Cell Culture, BrdU Incorporation Assay, CTL Assay

    EIF3F overexpression promotes an oxidative shift in human lung adenocarcinoma A549 cells. a The mitochondrial proteins involved in energy metabolism and upregulated in the proteome of EIF3F-A549 mice orthotopic lung tumors are shown in red in the respiratory chain schematic representation. b Representative and c quantitative expression level of p-AMPk and total AMPK ( N = 8). d Citrate synthase enzymatic activity measurement ( N = 3) indicative of the cellular mitochondrial content in A549 and EIF3F-A549 cells. e Mitochondrial respiration measured in different respiratory states: basal (DMEM 1 g/L), leak (oligomycin), maximal (CCCP); see ‘Methods’. The reserve capacity was calculated as the difference between the maximal and the basal respiratory rates. The different rates were corrected for nonmitochondrial respiration determined after potassium cyanide addition ( N = 7). f Mitochondrial respiration measurement in permeabilized cells (A549 and EIF3F-A549) in presence of pyruvate-malate as energy substrate. g Steady-state ATP content in A549 and A549-EIF3F cells. The part of ATP produced by mitochondria was inhibited using a cocktail of OXPHOS inhibitors (see ‘Methods’) ( N = 8). h Extracellular acidification rate of A549 and A549-EIF3F cells ( N = 5). i Quantification, and j representative images of cell migration (transwell) assay performed in CTL-A549 and EIF3F-A549 cells using compound C (5 μM), an AMPK inhibitor and metformin (5 mM), a complex I inhibitor ( N = 3). For each condition the respective treated (or untreated) control was used. * P
    Figure Legend Snippet: EIF3F overexpression promotes an oxidative shift in human lung adenocarcinoma A549 cells. a The mitochondrial proteins involved in energy metabolism and upregulated in the proteome of EIF3F-A549 mice orthotopic lung tumors are shown in red in the respiratory chain schematic representation. b Representative and c quantitative expression level of p-AMPk and total AMPK ( N = 8). d Citrate synthase enzymatic activity measurement ( N = 3) indicative of the cellular mitochondrial content in A549 and EIF3F-A549 cells. e Mitochondrial respiration measured in different respiratory states: basal (DMEM 1 g/L), leak (oligomycin), maximal (CCCP); see ‘Methods’. The reserve capacity was calculated as the difference between the maximal and the basal respiratory rates. The different rates were corrected for nonmitochondrial respiration determined after potassium cyanide addition ( N = 7). f Mitochondrial respiration measurement in permeabilized cells (A549 and EIF3F-A549) in presence of pyruvate-malate as energy substrate. g Steady-state ATP content in A549 and A549-EIF3F cells. The part of ATP produced by mitochondria was inhibited using a cocktail of OXPHOS inhibitors (see ‘Methods’) ( N = 8). h Extracellular acidification rate of A549 and A549-EIF3F cells ( N = 5). i Quantification, and j representative images of cell migration (transwell) assay performed in CTL-A549 and EIF3F-A549 cells using compound C (5 μM), an AMPK inhibitor and metformin (5 mM), a complex I inhibitor ( N = 3). For each condition the respective treated (or untreated) control was used. * P

    Techniques Used: Over Expression, Mouse Assay, Expressing, Activity Assay, Produced, Migration, Transwell Assay, CTL Assay

    17) Product Images from "Cardiac mitochondrial function depends on BUD23 mediated ribosome programming"

    Article Title: Cardiac mitochondrial function depends on BUD23 mediated ribosome programming

    Journal: eLife

    doi: 10.7554/eLife.50705

    Further analysis of ribosomal deficiency in A549 cells. ( A and B ) Further analysis of the data in Figure 2C : Basal respiration was determined from average OCR measurements of A549 cells in an unstimulated state. LEAK respiration was determined by subtracting the average OCR after addition of OA from the average OCR after addition of AA+R. ( C ) qPCR analysis of mitochondrial gene expression (MT-ATP6 and MT-ATP8) in A549 cells treated with control siRNA (neg1) or siRNA specifically targeting BUD23, RPS27A, LTV1 or RIOK2. ( D ) Example RNA screentape gel showing 28S and 18S bands for A549 cells treated with either control siRNA or BUD23 specific siRNA.
    Figure Legend Snippet: Further analysis of ribosomal deficiency in A549 cells. ( A and B ) Further analysis of the data in Figure 2C : Basal respiration was determined from average OCR measurements of A549 cells in an unstimulated state. LEAK respiration was determined by subtracting the average OCR after addition of OA from the average OCR after addition of AA+R. ( C ) qPCR analysis of mitochondrial gene expression (MT-ATP6 and MT-ATP8) in A549 cells treated with control siRNA (neg1) or siRNA specifically targeting BUD23, RPS27A, LTV1 or RIOK2. ( D ) Example RNA screentape gel showing 28S and 18S bands for A549 cells treated with either control siRNA or BUD23 specific siRNA.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing

    Network analysis of up-regulated proteins. ( A ) The up-regulated proteins from proteomic analysis of BUD23-deficient A549 cells are visualised as a STRING10 network.
    Figure Legend Snippet: Network analysis of up-regulated proteins. ( A ) The up-regulated proteins from proteomic analysis of BUD23-deficient A549 cells are visualised as a STRING10 network.

    Techniques Used:

    Further analysis of BUD23-dependent ribosome function in A549 cells. ( A ) Global translation rates in A549 cells after siRNA treatment were measured by 35-S-methionine incorporation assay. Equal protein loading was confirmed by Coomassie. ( B ) Data-set enrichment analysis using the PANTHER DB GO-Slim Biological processes database, of transcripts with > 2 fold decrease in TE in the BUD23 -depleted condition relative to control. These transcripts form a highly inter-connected network with a PPI enrichment value
    Figure Legend Snippet: Further analysis of BUD23-dependent ribosome function in A549 cells. ( A ) Global translation rates in A549 cells after siRNA treatment were measured by 35-S-methionine incorporation assay. Equal protein loading was confirmed by Coomassie. ( B ) Data-set enrichment analysis using the PANTHER DB GO-Slim Biological processes database, of transcripts with > 2 fold decrease in TE in the BUD23 -depleted condition relative to control. These transcripts form a highly inter-connected network with a PPI enrichment value

    Techniques Used:

    5'UTR motif analysis. ( A and B ) De-novo motif analysis of 5’UTRs from the highly down-regulated ( A ) and up-regulated ( B ) transcripts identified by comparing TE from control and BUD23 knockdown A549 cells.
    Figure Legend Snippet: 5'UTR motif analysis. ( A and B ) De-novo motif analysis of 5’UTRs from the highly down-regulated ( A ) and up-regulated ( B ) transcripts identified by comparing TE from control and BUD23 knockdown A549 cells.

    Techniques Used:

    18) Product Images from "CDK16 Phosphorylates and Degrades p53 to Promote Radioresistance and Predicts Prognosis in Lung Cancer"

    Article Title: CDK16 Phosphorylates and Degrades p53 to Promote Radioresistance and Predicts Prognosis in Lung Cancer

    Journal: Theranostics

    doi: 10.7150/thno.21963

    CDK16 regulates p53 stability via the ubiquitin/proteasome pathway. A. A549 cells were transfected with the indicated siRNA for 48 h, then incubated with 10 μM MG132 for 4 h prior to harvesting. Cell lysates were analyzed by Western blotting using indicated antibodies. The ratio shows relative p53 protein expression normalized for GAPDH (scramble, set at 1). B. Left panel: A549 cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting. Right panel: quantification of p53 band intensities is shown. C. A549 cells were transfected with indicated constructs after cells were transfected with CDK16 siRNA for 24 h. Cells were harvested after treatment with MG132 (10 μM) for 4 h. The samples were subjected to immunoprecipitation using S-protein agarose beads followed by Western blotting analysis as indicated. D and E. A549 cells were transfected with indicated constructs for 24 h and collected after treatment with MG132 (10 μM) for 4 h. The samples were subjected to immunoprecipitation using S-protein agarose beads and analyzed by Western blotting using indicated antibodies.
    Figure Legend Snippet: CDK16 regulates p53 stability via the ubiquitin/proteasome pathway. A. A549 cells were transfected with the indicated siRNA for 48 h, then incubated with 10 μM MG132 for 4 h prior to harvesting. Cell lysates were analyzed by Western blotting using indicated antibodies. The ratio shows relative p53 protein expression normalized for GAPDH (scramble, set at 1). B. Left panel: A549 cells transfected with indicated siRNA for 48 h were treated with CHX (20 mg/mL) for the indicated times and then analyzed by Western blotting. Right panel: quantification of p53 band intensities is shown. C. A549 cells were transfected with indicated constructs after cells were transfected with CDK16 siRNA for 24 h. Cells were harvested after treatment with MG132 (10 μM) for 4 h. The samples were subjected to immunoprecipitation using S-protein agarose beads followed by Western blotting analysis as indicated. D and E. A549 cells were transfected with indicated constructs for 24 h and collected after treatment with MG132 (10 μM) for 4 h. The samples were subjected to immunoprecipitation using S-protein agarose beads and analyzed by Western blotting using indicated antibodies.

    Techniques Used: Transfection, Incubation, Western Blot, Expressing, Construct, Immunoprecipitation

    p53 mediates the biological effects of CDK16 depletion in lung cancer cells. A. A549 cells were transfected with indicated siRNAs for 48 h. Cell lysates were analyzed by immunoblotting as indicated. B. A549 cells transfected with indicated siRNAs were seeded and cultured for two weeks. The colonies were stained and then counted. Representative pictures are shown. *** P
    Figure Legend Snippet: p53 mediates the biological effects of CDK16 depletion in lung cancer cells. A. A549 cells were transfected with indicated siRNAs for 48 h. Cell lysates were analyzed by immunoblotting as indicated. B. A549 cells transfected with indicated siRNAs were seeded and cultured for two weeks. The colonies were stained and then counted. Representative pictures are shown. *** P

    Techniques Used: Transfection, Cell Culture, Staining

    CDK16 knockdown leads to p53 accumulation. A. A549 cells were transfected with indicated siRNAs for 48 h, and samples were collected and analyzed by Western blotting with the indicated antibodies. The ratio shows relative p53 protein expression normalized for GAPDH (scramble, set at 1). B . A549 cells infected with lenti-CRISPR sgRNAs targeting CDK16 or control were incubated with puromycin for one week, and endogenous CDK16 and p53 were analyzed by western blotting. C. A549 and H292 cells were transfected with constructs encoding SFB-CDK16. Cells were collected 24 h later and cell lysates were analyzed by Western blotting using indicated antibodies. D. H292, U2OS, and MCF-7 cells were transfected with indicated siRNAs for 48 h, and analyzed as described in (A). E. A549 and H292 cells were transfected with scrambled or CDK16 siRNAs for 48 h. The mRNA levels for the indicated genes were determined by Real-time quantitative PCR (n=3). F. HCC827 and H1975 cells were transfected with scrambled or CDK16 siRNAs as indicated for 48 h and analyzed as described in (A).
    Figure Legend Snippet: CDK16 knockdown leads to p53 accumulation. A. A549 cells were transfected with indicated siRNAs for 48 h, and samples were collected and analyzed by Western blotting with the indicated antibodies. The ratio shows relative p53 protein expression normalized for GAPDH (scramble, set at 1). B . A549 cells infected with lenti-CRISPR sgRNAs targeting CDK16 or control were incubated with puromycin for one week, and endogenous CDK16 and p53 were analyzed by western blotting. C. A549 and H292 cells were transfected with constructs encoding SFB-CDK16. Cells were collected 24 h later and cell lysates were analyzed by Western blotting using indicated antibodies. D. H292, U2OS, and MCF-7 cells were transfected with indicated siRNAs for 48 h, and analyzed as described in (A). E. A549 and H292 cells were transfected with scrambled or CDK16 siRNAs for 48 h. The mRNA levels for the indicated genes were determined by Real-time quantitative PCR (n=3). F. HCC827 and H1975 cells were transfected with scrambled or CDK16 siRNAs as indicated for 48 h and analyzed as described in (A).

    Techniques Used: Transfection, Western Blot, Expressing, Infection, CRISPR, Incubation, Construct, Real-time Polymerase Chain Reaction

    CDK16 directly interacts with p53 in vivo and in vitro . A. HEK293T cells transfected with the indicated constructs were collected 24 h later. Cells were lysed with NETN buffer. Immunoprecipitation (IP) using S-protein agarose were performed and Western blotted with the indicated antibodies. WCL: whole cell lysate. B. Endogenous CDK16 associated with p53 in A549 cells. A549 cells were lysed and subjected to immunoprecipitation using anti-IgG or anti-p53 as indicated, and were analyzed by Western blotting using indicated antibodies. C. Beads coated with GST or GST-p53 fusion proteins were incubated with SFB-CDK16 protein overnight. GST pulldown was immunoblotted with indicated antibodies. D. Schematic description of p53 domains and deletion mutants used in this study. E. HEK293T cells co-transfected with constructs encoding SFB-CDK16 and indicated p53 mutants were collected 24 h later. Cells were lysed, the supernatants were incubated with S-protein agarose beads, and then analyzed by Western blotting using antibodies as indicated.
    Figure Legend Snippet: CDK16 directly interacts with p53 in vivo and in vitro . A. HEK293T cells transfected with the indicated constructs were collected 24 h later. Cells were lysed with NETN buffer. Immunoprecipitation (IP) using S-protein agarose were performed and Western blotted with the indicated antibodies. WCL: whole cell lysate. B. Endogenous CDK16 associated with p53 in A549 cells. A549 cells were lysed and subjected to immunoprecipitation using anti-IgG or anti-p53 as indicated, and were analyzed by Western blotting using indicated antibodies. C. Beads coated with GST or GST-p53 fusion proteins were incubated with SFB-CDK16 protein overnight. GST pulldown was immunoblotted with indicated antibodies. D. Schematic description of p53 domains and deletion mutants used in this study. E. HEK293T cells co-transfected with constructs encoding SFB-CDK16 and indicated p53 mutants were collected 24 h later. Cells were lysed, the supernatants were incubated with S-protein agarose beads, and then analyzed by Western blotting using antibodies as indicated.

    Techniques Used: In Vivo, In Vitro, Transfection, Construct, Immunoprecipitation, Western Blot, Incubation

    CDK16 phosphorylates p53 at Ser315 site and inhibits p53 transcriptional activity. A. A549 cells were transfected with the indicated siRNA for 48 h, then incubated with 10 μM MG132 for 4 h prior to collection. Cell lysates were analyzed by Western blotting using indicated antibodies. The ratio shows relative phosphorylated p53 protein expression normalized for GAPDH (scramble, set at 1). B. Alignment of CDK16 candidate phosphorylation site in p53 from different species. C. CDK16 phosphorylates p53 at Ser315 site in vitro . HA-p53-WT or S315A proteins were incubated in vitro with immunoprecipitates isolated from HEK293T cells transfected with constructs encoding SFB-CDK16 and then analyzed by Western blotting using indicated antibodies. D. CDK16 depletion increases p53 abundance in the nucleus. A549 cells were fractionated into cytoplasmic and nuclear fractions, and the indicated proteins in each compartment were analyzed by Western blotting. α-tubulin and LaminB1 were used respectively as cytoplasmic and nuclear loading controls. E. The mRNA levels of the indicated genes were analyzed by Real time quantitative PCR in A549 cells transfected with scramble or CDK16 siRNAs. *** P
    Figure Legend Snippet: CDK16 phosphorylates p53 at Ser315 site and inhibits p53 transcriptional activity. A. A549 cells were transfected with the indicated siRNA for 48 h, then incubated with 10 μM MG132 for 4 h prior to collection. Cell lysates were analyzed by Western blotting using indicated antibodies. The ratio shows relative phosphorylated p53 protein expression normalized for GAPDH (scramble, set at 1). B. Alignment of CDK16 candidate phosphorylation site in p53 from different species. C. CDK16 phosphorylates p53 at Ser315 site in vitro . HA-p53-WT or S315A proteins were incubated in vitro with immunoprecipitates isolated from HEK293T cells transfected with constructs encoding SFB-CDK16 and then analyzed by Western blotting using indicated antibodies. D. CDK16 depletion increases p53 abundance in the nucleus. A549 cells were fractionated into cytoplasmic and nuclear fractions, and the indicated proteins in each compartment were analyzed by Western blotting. α-tubulin and LaminB1 were used respectively as cytoplasmic and nuclear loading controls. E. The mRNA levels of the indicated genes were analyzed by Real time quantitative PCR in A549 cells transfected with scramble or CDK16 siRNAs. *** P

    Techniques Used: Activity Assay, Transfection, Incubation, Western Blot, Expressing, In Vitro, Isolation, Construct, Real-time Polymerase Chain Reaction

    CDK16 silencing results in enhanced radiosensitivity of lung cancer cells. A. Colony formation ability was significantly reduced in CDK16-depleted A549 cells. *** P
    Figure Legend Snippet: CDK16 silencing results in enhanced radiosensitivity of lung cancer cells. A. Colony formation ability was significantly reduced in CDK16-depleted A549 cells. *** P

    Techniques Used:

    19) Product Images from "Dioscorea japonica extract down-regulates prostaglandin E2 synthetic pathway and induces apoptosis in lung cancer cells"

    Article Title: Dioscorea japonica extract down-regulates prostaglandin E2 synthetic pathway and induces apoptosis in lung cancer cells

    Journal: Journal of Clinical Biochemistry and Nutrition

    doi: 10.3164/jcbn.14-25

    Dose-dependent effect of DJE on the expression of COX-2 and mPGES-1 in A549 cells. DJE by 50% ethanol (0–100 µg/ml) was added to A549 cells. mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured by real-time PCR, as described in the Materials and Methods section. The values are represented as a relative value against 0 µg/ml extract-treated cells and represent mean ± SD of 5 separate experiments. * p
    Figure Legend Snippet: Dose-dependent effect of DJE on the expression of COX-2 and mPGES-1 in A549 cells. DJE by 50% ethanol (0–100 µg/ml) was added to A549 cells. mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured by real-time PCR, as described in the Materials and Methods section. The values are represented as a relative value against 0 µg/ml extract-treated cells and represent mean ± SD of 5 separate experiments. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Effect of water-soluble and Ethanol extract of D. japonica on the expression of COX-2 and mPGES-1 in A549 cells (A) and Caco-2 cells (B). Water-soluble or Ethanol (20, 50, and 80%) extract of D. japonica (100 µg/ml) was added to A549 cells (A), and the DJE by 50% ethanol (100 µg/ml) was added to Caco-2 (B). mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured using real-time PCR. The values are represented as a relative value control cells and represent mean ± SD of 5 separate experiments. ** p
    Figure Legend Snippet: Effect of water-soluble and Ethanol extract of D. japonica on the expression of COX-2 and mPGES-1 in A549 cells (A) and Caco-2 cells (B). Water-soluble or Ethanol (20, 50, and 80%) extract of D. japonica (100 µg/ml) was added to A549 cells (A), and the DJE by 50% ethanol (100 µg/ml) was added to Caco-2 (B). mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured using real-time PCR. The values are represented as a relative value control cells and represent mean ± SD of 5 separate experiments. ** p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Decrease in COX activity (A) and PGE 2 production (B) after treatment with DJE. COX activity after the treatment without (open column) or with (closed column) DJE (100 µg/ml) was measured. Supernatants obtained from the centrifugation (10,000 × g ) of lysates from A549 and Caco-2 cells were used as enzyme sources for measurement of COX activity. The supernatants were incubated with 25 mM linoleic acid at 24°C for 5 min in the standard COX reaction mixture and the products were quantified using reversed-phase HPLC, as described in the Materials and Methods section. PGE 2 released in the culture medium of A549 cells was quantified by enzyme immunoassay. The values represent mean ± SD of 5 separate experiments. * p
    Figure Legend Snippet: Decrease in COX activity (A) and PGE 2 production (B) after treatment with DJE. COX activity after the treatment without (open column) or with (closed column) DJE (100 µg/ml) was measured. Supernatants obtained from the centrifugation (10,000 × g ) of lysates from A549 and Caco-2 cells were used as enzyme sources for measurement of COX activity. The supernatants were incubated with 25 mM linoleic acid at 24°C for 5 min in the standard COX reaction mixture and the products were quantified using reversed-phase HPLC, as described in the Materials and Methods section. PGE 2 released in the culture medium of A549 cells was quantified by enzyme immunoassay. The values represent mean ± SD of 5 separate experiments. * p

    Techniques Used: Activity Assay, Centrifugation, Incubation, High Performance Liquid Chromatography, Enzyme-linked Immunosorbent Assay

    Effect of diosgenin on the expression of COX-2 and mPGES-1 in A549 cells. Diosgenin (0.1–10 µM) was added to A549 cells. mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured by real-time PCR, as described in the Materials and Methods section. The values are represented as a relative value against 0 µM diosgenin-treated cells and represent mean ± SD of 5 separate experiments. * p
    Figure Legend Snippet: Effect of diosgenin on the expression of COX-2 and mPGES-1 in A549 cells. Diosgenin (0.1–10 µM) was added to A549 cells. mRNA expression of COX-2 (open column) and mPGES-1 (closed column) was measured by real-time PCR, as described in the Materials and Methods section. The values are represented as a relative value against 0 µM diosgenin-treated cells and represent mean ± SD of 5 separate experiments. * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Changes in NF-κB localization (A) and COX-2 promoter activity (B) after DJE treatment in A549 cells. Localization of the transcription factor NF-κB (red) and DAPI (blue, nuclear counterstain) was visualized by confocal laser scanning microscopy (A). Scale bar indicates 50 µm. Cluc reporter plasmid with COX-2 promoter was used for the promoter activity assay, as described in the Materials and Methods section (B). TPA (0.1 M) was used as an inducer of COX-2 expression. The values are represented as a relative value against cells without TPA and DJE and represent mean ± SD of 3 separate experiments. a p
    Figure Legend Snippet: Changes in NF-κB localization (A) and COX-2 promoter activity (B) after DJE treatment in A549 cells. Localization of the transcription factor NF-κB (red) and DAPI (blue, nuclear counterstain) was visualized by confocal laser scanning microscopy (A). Scale bar indicates 50 µm. Cluc reporter plasmid with COX-2 promoter was used for the promoter activity assay, as described in the Materials and Methods section (B). TPA (0.1 M) was used as an inducer of COX-2 expression. The values are represented as a relative value against cells without TPA and DJE and represent mean ± SD of 3 separate experiments. a p

    Techniques Used: Activity Assay, Confocal Laser Scanning Microscopy, Plasmid Preparation, Expressing

    20) Product Images from "Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis"

    Article Title: Disruption of STAT3 signalling promotes KRAS-induced lung tumorigenesis

    Journal: Nature Communications

    doi: 10.1038/ncomms7285

    STAT3 alters tumour microenvironment and angiogenesis. ( a ) IHC analysis of von Willebrand Factor (vWF) and CD31 is shown in consecutive sections of murine lung tumours of the indicated genotype. On right, CD31 + counts per tumour area (mm 2 ) quantification at indicated time points. Data were analysed by Student’s t -test and displayed as mean±s.e.m. n ≥5 tumours per mouse with n ≥4 mice per genotype and time point. Scale bar, 50 μm. ( b ) ELISA of VEGFA levels in lung tumour lysates at indicated time points. Data were analysed by Student’s t -test and displayed as mean±s.d., n ≥6 mice per genotype and time point. ( c ) Expression levels of Pdgfa in total lungs were measured by quantitative real-time PCR at indicated time points. Values are presented as fold change of relative mRNA expression compared with Stat3 ΔLep/ΔLep :Kras +/+ mice. Data were analysed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test and are shown as mean± s.d., n ≥5 animals/genotype. ( d ) Flow cytometric analysis of Cd11b + Gr1 + granulocytes and Cd11b + F4/80 + macrophages in bronchoalveolar lavage (BAL) at 6 and 13 weeks post AdCre. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are shown as mean±s.e.m. ( e ) Flow cytometric analysis of myeloid-derived suppressor cell (MDSC) subsets at 6 and 13 weeks post AdCre. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are shown as mean± s.e.m. ( f ) Ratio of CD4 + /CD8 + T-cell counts in BAL are shown. Data were analysed by Kruskal–Wallis test with Dunn’s multiple comparison testing and are shown as mean±s.e.m. Data displayed in d – f are n ≥6 mice per genotype and time point, 13-week group represents two independent experiments. ( g ) Flow cytometric analysis of Cd11b + Gr1 + and Cd11b + F4/80 + cells of A549 sh Control versus A549-sh STAT3 xenograft tumours. Data were analysed by Student’s t -test and are displayed as mean±s.e.m. ( n ≥8 tumours; ≥4 mice per group). ( h ) IHC analysis and representative pictures of CD31 + counts per xenograft tumour area (mm 2 ). Scale bar, 100 μm ( n =8 tumours/≥4 mice). Data were analysed by Student’s t -test and are shown as mean±s.e.m. For all graphs: * P
    Figure Legend Snippet: STAT3 alters tumour microenvironment and angiogenesis. ( a ) IHC analysis of von Willebrand Factor (vWF) and CD31 is shown in consecutive sections of murine lung tumours of the indicated genotype. On right, CD31 + counts per tumour area (mm 2 ) quantification at indicated time points. Data were analysed by Student’s t -test and displayed as mean±s.e.m. n ≥5 tumours per mouse with n ≥4 mice per genotype and time point. Scale bar, 50 μm. ( b ) ELISA of VEGFA levels in lung tumour lysates at indicated time points. Data were analysed by Student’s t -test and displayed as mean±s.d., n ≥6 mice per genotype and time point. ( c ) Expression levels of Pdgfa in total lungs were measured by quantitative real-time PCR at indicated time points. Values are presented as fold change of relative mRNA expression compared with Stat3 ΔLep/ΔLep :Kras +/+ mice. Data were analysed by one-way analysis of variance (ANOVA) with Tukey’s multiple comparison test and are shown as mean± s.d., n ≥5 animals/genotype. ( d ) Flow cytometric analysis of Cd11b + Gr1 + granulocytes and Cd11b + F4/80 + macrophages in bronchoalveolar lavage (BAL) at 6 and 13 weeks post AdCre. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are shown as mean±s.e.m. ( e ) Flow cytometric analysis of myeloid-derived suppressor cell (MDSC) subsets at 6 and 13 weeks post AdCre. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are shown as mean± s.e.m. ( f ) Ratio of CD4 + /CD8 + T-cell counts in BAL are shown. Data were analysed by Kruskal–Wallis test with Dunn’s multiple comparison testing and are shown as mean±s.e.m. Data displayed in d – f are n ≥6 mice per genotype and time point, 13-week group represents two independent experiments. ( g ) Flow cytometric analysis of Cd11b + Gr1 + and Cd11b + F4/80 + cells of A549 sh Control versus A549-sh STAT3 xenograft tumours. Data were analysed by Student’s t -test and are displayed as mean±s.e.m. ( n ≥8 tumours; ≥4 mice per group). ( h ) IHC analysis and representative pictures of CD31 + counts per xenograft tumour area (mm 2 ). Scale bar, 100 μm ( n =8 tumours/≥4 mice). Data were analysed by Student’s t -test and are shown as mean±s.e.m. For all graphs: * P

    Techniques Used: Immunohistochemistry, Mouse Assay, Enzyme-linked Immunosorbent Assay, Expressing, Real-time Polymerase Chain Reaction, Flow Cytometry, Derivative Assay

    STAT3 suppresses Kras -induced lung tumorigenesis. ( a ) Kaplan–Meier plot showing overall survival of male mice with the indicated genotypes infected with AdCre ( n ≥10 male mice per genotype; log-rank test). ( b ) Representative haematoxylin and eosin stainings and quantification of tumour area/lung area in mice at indicated time points. Data were analysed by Student’s t -test and are shown as mean±s.d., n =6–10. Scale bar, 800 μm. ( c ) Tumour grade quantification at 6 weeks post AdCre infection in Stat3 ΔLep/ΔLep :Kras G12D/+ compared with Stat3 +/+ :Kras G12D/+ mice. Data were analysed by Student’s t -test and are shown as mean±s.e.m., n ≥7 mice per genotype. ( d ) Human A549 lung AC cells were transducted with scrambled shRNA (sh Control ) or shRNA against STAT3 (sh STAT3 ) and 2 × 10 6 cells were injected into male nude mice ( n =5 per group). Data were analysed by two-way analysis of variance with Bonferroni correction and are shown as mean±s.e.m. ( e ) IHC evaluation of total STAT3 in human mucinous adenocarcinomas with or without KRAS mutation. Intensity of staining (0/+1/+2/+3) was multiplied by percentage of stained tumour cells. Data are shown as mean±s.d. n ≥28 per group. (Mann–Whitney U -test, P =0.0138) ( f ) STAT3 mRNA expression of lung adenocarcinoma patients harbouring smoking history at different tumour grades is shown ( n =139; grade I, low metastatic potential; grade II, intermediate metastatic potential; grade III, high metastatic potential 25 ). (Kruskal–Wallis test with Dunn’s multiple comparison testing P
    Figure Legend Snippet: STAT3 suppresses Kras -induced lung tumorigenesis. ( a ) Kaplan–Meier plot showing overall survival of male mice with the indicated genotypes infected with AdCre ( n ≥10 male mice per genotype; log-rank test). ( b ) Representative haematoxylin and eosin stainings and quantification of tumour area/lung area in mice at indicated time points. Data were analysed by Student’s t -test and are shown as mean±s.d., n =6–10. Scale bar, 800 μm. ( c ) Tumour grade quantification at 6 weeks post AdCre infection in Stat3 ΔLep/ΔLep :Kras G12D/+ compared with Stat3 +/+ :Kras G12D/+ mice. Data were analysed by Student’s t -test and are shown as mean±s.e.m., n ≥7 mice per genotype. ( d ) Human A549 lung AC cells were transducted with scrambled shRNA (sh Control ) or shRNA against STAT3 (sh STAT3 ) and 2 × 10 6 cells were injected into male nude mice ( n =5 per group). Data were analysed by two-way analysis of variance with Bonferroni correction and are shown as mean±s.e.m. ( e ) IHC evaluation of total STAT3 in human mucinous adenocarcinomas with or without KRAS mutation. Intensity of staining (0/+1/+2/+3) was multiplied by percentage of stained tumour cells. Data are shown as mean±s.d. n ≥28 per group. (Mann–Whitney U -test, P =0.0138) ( f ) STAT3 mRNA expression of lung adenocarcinoma patients harbouring smoking history at different tumour grades is shown ( n =139; grade I, low metastatic potential; grade II, intermediate metastatic potential; grade III, high metastatic potential 25 ). (Kruskal–Wallis test with Dunn’s multiple comparison testing P

    Techniques Used: Mouse Assay, Infection, shRNA, Injection, Immunohistochemistry, Mutagenesis, Staining, MANN-WHITNEY, Expressing

    STAT3 regulates chemoattractive CXCL1 expression. ( a ) ELISA of CXCL1 levels in lung lysates and BAL at 6 and 13 weeks post AdCre. Data were analysed by Student’s t -test and shown as mean±s.d., n ≥7 mice per genotype and time point. ( b ) Representative in situ hybridization with probes specific for murine Cxcl1 (AS) in lung tumours at 13 weeks post AdCre. Sense probes (S) were used as negative control and surfactant protein C (SP-C) as lung epithelial maker. Scale bar, 100 μm. ( c ) Kaplan–Meier plot showing overall survival of lung adenocarcinoma patient samples harbouring smoking history with high or low IL-8 mRNA expression (expression values of upper quartile, log-rank test) n ≥35–104 per group. ( d ) Human A549 lung cancer cell line ( KRAS G12S mutated) was stimulated with designated cytokines for indicated time points. IL-8 or CXCL1 expression was analysed via qRT–PCR. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.d. ( e ) A549 cells transducted with lentiviral vectors expressing nonspecific scrambled small hairpin (sh)RNA or shRNA against STAT3 were stimulated with indicated cytokines for 60 min. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.d. ( f ) Primary pneumocytes were isolated and infected with AdCre. 120 h post infection cells were stimulated with indicated cytokines for 60 min. Cxcl1 expression was analysed by qRT–PCR. Data are shown as mean±s.e.m. ( g ) Murine 3T3 fibroblasts and STAT3 overexpressing 3T3 fibroblasts were stimulated as in f . Cxcl1 expression was analysed by qRT–PCR. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean± s.d. Values in d – g are presented as fold change of relative mRNA expression compared with each unstimulated individual cell line. At least two independent experiments with three individual plates per stimulation were performed. For all graphs: * P
    Figure Legend Snippet: STAT3 regulates chemoattractive CXCL1 expression. ( a ) ELISA of CXCL1 levels in lung lysates and BAL at 6 and 13 weeks post AdCre. Data were analysed by Student’s t -test and shown as mean±s.d., n ≥7 mice per genotype and time point. ( b ) Representative in situ hybridization with probes specific for murine Cxcl1 (AS) in lung tumours at 13 weeks post AdCre. Sense probes (S) were used as negative control and surfactant protein C (SP-C) as lung epithelial maker. Scale bar, 100 μm. ( c ) Kaplan–Meier plot showing overall survival of lung adenocarcinoma patient samples harbouring smoking history with high or low IL-8 mRNA expression (expression values of upper quartile, log-rank test) n ≥35–104 per group. ( d ) Human A549 lung cancer cell line ( KRAS G12S mutated) was stimulated with designated cytokines for indicated time points. IL-8 or CXCL1 expression was analysed via qRT–PCR. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.d. ( e ) A549 cells transducted with lentiviral vectors expressing nonspecific scrambled small hairpin (sh)RNA or shRNA against STAT3 were stimulated with indicated cytokines for 60 min. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.d. ( f ) Primary pneumocytes were isolated and infected with AdCre. 120 h post infection cells were stimulated with indicated cytokines for 60 min. Cxcl1 expression was analysed by qRT–PCR. Data are shown as mean±s.e.m. ( g ) Murine 3T3 fibroblasts and STAT3 overexpressing 3T3 fibroblasts were stimulated as in f . Cxcl1 expression was analysed by qRT–PCR. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean± s.d. Values in d – g are presented as fold change of relative mRNA expression compared with each unstimulated individual cell line. At least two independent experiments with three individual plates per stimulation were performed. For all graphs: * P

    Techniques Used: Expressing, Enzyme-linked Immunosorbent Assay, Mouse Assay, In Situ Hybridization, Negative Control, Quantitative RT-PCR, shRNA, Isolation, Infection

    CXCL1 inhibiton reverts oncogenic effects of STAT3 ablation. ( a ) Mice were treated with the CXCR2 antagonist SB225002 or vehicle control starting 1 week after tumour induction and euthanized after 5 weeks (treatment 1 shown in Supplementary Fig. 5b ). Tumour area/lung area was quantified within each group and at least two sections of each lung were stained with haematoxylin and eosin and analysed in a blinded manner. Tumour grading is shown in the right panel ( n =4–7 mice per genotype). Data in both panels were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 2 mm. ( b ) Tumour vascularization was quantified by CD31 + counts per tumour area (mm 2 ). At least 3 tumours per mouse were analysed with n =4–7 mice per genotype and treatment. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 100 μm. ( c ) Short hairpin-mediated knockdown of IL-8 (sh IL-8 ) in either sh Control or sh STAT3 A549 NSCLC cells were performed and 2 × 10 6 cells were injected in both flanks of male nude mice ( n =5 per group). Xenograft tumour growth was determined at indicated time points. Data were analysed by Two-way ANOVA with Bonferroni multiple comparison test and shown as mean±s.e.m. ( d ) IHC analysis of CD31 + counts per tumour area (mm 2 ) showed reduced vascularization of A549-sh STAT3 ;sh IL-8 xenograft tumours compared with controls. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 100 μm. ( e ) Flow cytometric analysis of Cd11b + Gr1 + granulocytes and Cd11b + F4/80 + macrophages displayed reduced myeloid infiltration in A549-sh STAT3 ;sh IL-8 xenograft tumours compared with controls ( n ≥8 tumours; 5=mice per group). At least 6 tumours per mouse were analysed with n ≥7 mice per genotype and treatment. Data were analysed by Kruskal–Wallis test with Dunn’s multiple comparison testing and shown as mean±s.e.m. For all graphs: * P
    Figure Legend Snippet: CXCL1 inhibiton reverts oncogenic effects of STAT3 ablation. ( a ) Mice were treated with the CXCR2 antagonist SB225002 or vehicle control starting 1 week after tumour induction and euthanized after 5 weeks (treatment 1 shown in Supplementary Fig. 5b ). Tumour area/lung area was quantified within each group and at least two sections of each lung were stained with haematoxylin and eosin and analysed in a blinded manner. Tumour grading is shown in the right panel ( n =4–7 mice per genotype). Data in both panels were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 2 mm. ( b ) Tumour vascularization was quantified by CD31 + counts per tumour area (mm 2 ). At least 3 tumours per mouse were analysed with n =4–7 mice per genotype and treatment. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 100 μm. ( c ) Short hairpin-mediated knockdown of IL-8 (sh IL-8 ) in either sh Control or sh STAT3 A549 NSCLC cells were performed and 2 × 10 6 cells were injected in both flanks of male nude mice ( n =5 per group). Xenograft tumour growth was determined at indicated time points. Data were analysed by Two-way ANOVA with Bonferroni multiple comparison test and shown as mean±s.e.m. ( d ) IHC analysis of CD31 + counts per tumour area (mm 2 ) showed reduced vascularization of A549-sh STAT3 ;sh IL-8 xenograft tumours compared with controls. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. Scale bar, 100 μm. ( e ) Flow cytometric analysis of Cd11b + Gr1 + granulocytes and Cd11b + F4/80 + macrophages displayed reduced myeloid infiltration in A549-sh STAT3 ;sh IL-8 xenograft tumours compared with controls ( n ≥8 tumours; 5=mice per group). At least 6 tumours per mouse were analysed with n ≥7 mice per genotype and treatment. Data were analysed by Kruskal–Wallis test with Dunn’s multiple comparison testing and shown as mean±s.e.m. For all graphs: * P

    Techniques Used: Mouse Assay, Staining, Injection, Immunohistochemistry, Flow Cytometry

    STAT3 retains p65 in the cytoplasm to reduce NF-κB activity. ( a ) NF-κB subunit p65 activation status (p-p65) was analysed by IHC. n ≥5 tumour sections per mouse per genotype and time point. Data were analysed by Student’s t -test and are shown as mean±s.d. Scale bar, 100 μm. ( b ) qRT–PCR of NF-κB subunit p65 target genes ( Tnfα , c-Myc ) from total lungs at 13 weeks post AdCre. n ≥4 mice per genotype. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are displayed as mean±s.e.m. ( c ) Chromatin immunoprecipitation (ChIP) of NF-κB subunit p65 or STAT3 binding on human CXCL1 (responsive element 2, RE2). A549 cells were stimulated with indicated cytokines for 10 min. Binding of STAT3 and p65 on ICAM1 promoter served as positive control. ACTB element was chosen as negative binding region, indicated by the horizontal line. Values are presented as fold enrichment relative to chromatin input. Data of two independent experiments are shown. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. ( d ) A549 cells were stimulated as in c . Immunoprecipitation (IP) of nuclear and cytoplasmic fractions was performed with antibodies against p65 or STAT3 and subjected to STAT3 and p65 immunoblot analysis, respectively. IP inputs were subjected to STAT3 and p65 (arrow indicates unspecific binding). PARP (nuclear) and TUBA1A (cytoplasmic) were used to determine purity of input. Densitometric quantification of bands was performed (AU, arbitrary units). ( e ) A549 cells transducted with scrambled shRNA or shRNA against STAT3 were stimulated as in c and stained with antibodies for NF-κB subunit p65 (FITC). n ≥6 regions of interest were quantified. White scale bar, 20 μm. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean± s.e.m. For all graphs: * P
    Figure Legend Snippet: STAT3 retains p65 in the cytoplasm to reduce NF-κB activity. ( a ) NF-κB subunit p65 activation status (p-p65) was analysed by IHC. n ≥5 tumour sections per mouse per genotype and time point. Data were analysed by Student’s t -test and are shown as mean±s.d. Scale bar, 100 μm. ( b ) qRT–PCR of NF-κB subunit p65 target genes ( Tnfα , c-Myc ) from total lungs at 13 weeks post AdCre. n ≥4 mice per genotype. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and are displayed as mean±s.e.m. ( c ) Chromatin immunoprecipitation (ChIP) of NF-κB subunit p65 or STAT3 binding on human CXCL1 (responsive element 2, RE2). A549 cells were stimulated with indicated cytokines for 10 min. Binding of STAT3 and p65 on ICAM1 promoter served as positive control. ACTB element was chosen as negative binding region, indicated by the horizontal line. Values are presented as fold enrichment relative to chromatin input. Data of two independent experiments are shown. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean±s.e.m. ( d ) A549 cells were stimulated as in c . Immunoprecipitation (IP) of nuclear and cytoplasmic fractions was performed with antibodies against p65 or STAT3 and subjected to STAT3 and p65 immunoblot analysis, respectively. IP inputs were subjected to STAT3 and p65 (arrow indicates unspecific binding). PARP (nuclear) and TUBA1A (cytoplasmic) were used to determine purity of input. Densitometric quantification of bands was performed (AU, arbitrary units). ( e ) A549 cells transducted with scrambled shRNA or shRNA against STAT3 were stimulated as in c and stained with antibodies for NF-κB subunit p65 (FITC). n ≥6 regions of interest were quantified. White scale bar, 20 μm. Data were analysed by one-way ANOVA with Tukey’s multiple comparison test and shown as mean± s.e.m. For all graphs: * P

    Techniques Used: Activity Assay, Activation Assay, Immunohistochemistry, Quantitative RT-PCR, Mouse Assay, Chromatin Immunoprecipitation, Binding Assay, Positive Control, Immunoprecipitation, shRNA, Staining

    21) Product Images from "The PIDDosome activates p53 in response to supernumerary centrosomes"

    Article Title: The PIDDosome activates p53 in response to supernumerary centrosomes

    Journal: Genes & Development

    doi: 10.1101/gad.289728.116

    The PIDDosome is required to activate p53 after cytokinesis failure. A549 CASP2, PIDD1, or RAIDD knockout cells obtained using two different small guide RNAs (sgRNAs) were treated with ZM447439 for either 24 h and processed for immunoblotting ( A ) or up
    Figure Legend Snippet: The PIDDosome is required to activate p53 after cytokinesis failure. A549 CASP2, PIDD1, or RAIDD knockout cells obtained using two different small guide RNAs (sgRNAs) were treated with ZM447439 for either 24 h and processed for immunoblotting ( A ) or up

    Techniques Used: Knock-Out

    PIDDosome-activated p53 shares features with nongenotoxic activation via Nutlin-3 and functions via p21. ( A ) A549 knockout cells obtained using the indicated sgRNAs were treated with doxorubicin (DXR), nocodazole, or ZM447439 for 24 h and processed for
    Figure Legend Snippet: PIDDosome-activated p53 shares features with nongenotoxic activation via Nutlin-3 and functions via p21. ( A ) A549 knockout cells obtained using the indicated sgRNAs were treated with doxorubicin (DXR), nocodazole, or ZM447439 for 24 h and processed for

    Techniques Used: Activation Assay, Knock-Out

    Extra centrosomes are necessary and sufficient for PIDDosome activation. ( A ) Scheme of the protocol used to generate polyploid cells with or without extra centrosomes. A549 cells were treated with either solvent control (DMSO), ZM447439 (ZM), or nocodazole
    Figure Legend Snippet: Extra centrosomes are necessary and sufficient for PIDDosome activation. ( A ) Scheme of the protocol used to generate polyploid cells with or without extra centrosomes. A549 cells were treated with either solvent control (DMSO), ZM447439 (ZM), or nocodazole

    Techniques Used: Activation Assay

    Caspase-2 constrains polyploidization after cytokinesis failure by MDM2 processing, leading to p53 stabilization . ( A ) A549 cells transfected with the indicated siRNAs targeting either luciferase (Gl2) or Caspase-2 (C2) were treated for 24 h with reversine
    Figure Legend Snippet: Caspase-2 constrains polyploidization after cytokinesis failure by MDM2 processing, leading to p53 stabilization . ( A ) A549 cells transfected with the indicated siRNAs targeting either luciferase (Gl2) or Caspase-2 (C2) were treated for 24 h with reversine

    Techniques Used: Transfection, Luciferase

    22) Product Images from "DAPK2 is a novel modulator of TRAIL-induced apoptosis"

    Article Title: DAPK2 is a novel modulator of TRAIL-induced apoptosis

    Journal: Cell Death and Differentiation

    doi: 10.1038/cdd.2014.93

    DR5 increased expression following the knockdown of DAPK2 is transcriptionally regulated and not due to alterations on mRNA or protein stability. U2OS ( a , d and g ) and A549 ( b , e and h ) cells were transfected with either siNS or siDAPK2. Forty-eight hours
    Figure Legend Snippet: DR5 increased expression following the knockdown of DAPK2 is transcriptionally regulated and not due to alterations on mRNA or protein stability. U2OS ( a , d and g ) and A549 ( b , e and h ) cells were transfected with either siNS or siDAPK2. Forty-eight hours

    Techniques Used: Expressing, Transfection

    NF- κ B is transcriptionally active upon knockdown of DAPK2. U2OS ( a , c , e , g and i ) and A549 ( b , d , f , h and j ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection, the expression levels of NF- κ
    Figure Legend Snippet: NF- κ B is transcriptionally active upon knockdown of DAPK2. U2OS ( a , c , e , g and i ) and A549 ( b , d , f , h and j ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection, the expression levels of NF- κ

    Techniques Used: Transfection, Expressing

    DAPK2 silencing leads to the upregulation of DR5 and DR4, key receptors for TRAIL. U2OS ( a and b ) and A549 ( c – e ) cells were transfected with either siNS or siDAPK2 for 48 h. Proteins were isolated and the level of DR expression was assessed
    Figure Legend Snippet: DAPK2 silencing leads to the upregulation of DR5 and DR4, key receptors for TRAIL. U2OS ( a and b ) and A549 ( c – e ) cells were transfected with either siNS or siDAPK2 for 48 h. Proteins were isolated and the level of DR expression was assessed

    Techniques Used: Transfection, Isolation, Expressing

    In the absence of DR5, siDAPK2 can neither sensitise U2OS nor A549 to TRAIL-induced apoptosis. Double knockdowns were carried out in U2OS ( a and b ) and A549 ( c – e ) cells. For this purpose, cells were transfected with 40 nM of the following
    Figure Legend Snippet: In the absence of DR5, siDAPK2 can neither sensitise U2OS nor A549 to TRAIL-induced apoptosis. Double knockdowns were carried out in U2OS ( a and b ) and A549 ( c – e ) cells. For this purpose, cells were transfected with 40 nM of the following

    Techniques Used: Transfection

    Knockdown of DAPK2 increases apoptotic signalling and sensitises resistant cancer cell lines to TRAIL-induced cell death. U2OS ( a – e ) and A549 ( g – k ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection
    Figure Legend Snippet: Knockdown of DAPK2 increases apoptotic signalling and sensitises resistant cancer cell lines to TRAIL-induced cell death. U2OS ( a – e ) and A549 ( g – k ) cells were transfected with either siNS or DAPK2 siRNA. Forty-eight hours after transfection

    Techniques Used: Transfection

    The transcription factor NF- κ B is a critical component of DR5 expression and is necessary for the sensitisation to TRAIL-induced cell death seen after DAPK2 silencing. U2OS ( a – e , l and m ) and A549 ( f – j , n and o ) cells were transfected
    Figure Legend Snippet: The transcription factor NF- κ B is a critical component of DR5 expression and is necessary for the sensitisation to TRAIL-induced cell death seen after DAPK2 silencing. U2OS ( a – e , l and m ) and A549 ( f – j , n and o ) cells were transfected

    Techniques Used: Expressing, Transfection

    23) Product Images from "Does the Number of Irradiated Cells Influence the Spatial Distribution of Bystander Effects?"

    Article Title: Does the Number of Irradiated Cells Influence the Spatial Distribution of Bystander Effects?

    Journal: Dose-Response

    doi: 10.2203/dose-response.14-001.Belchior

    Quantification of nuclear γ-H 2 AX foci. A549 cells were exposed to 5 to 100 mGy of α-radiation. (a) Induction of DSBs in irradiated cells, by means of foci number, after 5, 10, 50 and 100 mGy of α-radiation. Data were collected
    Figure Legend Snippet: Quantification of nuclear γ-H 2 AX foci. A549 cells were exposed to 5 to 100 mGy of α-radiation. (a) Induction of DSBs in irradiated cells, by means of foci number, after 5, 10, 50 and 100 mGy of α-radiation. Data were collected

    Techniques Used: Irradiation

    24) Product Images from "Polyubiquitination of Apurinic/Apyrimidinic Endonuclease 1 by Parkin"

    Article Title: Polyubiquitination of Apurinic/Apyrimidinic Endonuclease 1 by Parkin

    Journal: Molecular carcinogenesis

    doi: 10.1002/mc.22495

    Interaction of APE1 with Parkin in the cytoplasm ( A ) Accumulation of APE1 in the presence of MG132. MEF la cells expressing the wild-type APE1 were incubated with MG132 for indicated time periods. MG132 concentrations were 40 μM (0 – 3 h) and 3 μM (overnight). APE1 was labeled with anti-APE1 antibody and AlexaFluor 488 and analyzed in fluorescence microscopy. ( B ) MEF la ). ( C ) Proximity ligation assay (PLA) between APE1 and Parkin in A549. A549 expressing APE1, Parkin, PINK1 as indicated. APE1 and Parkin specific antibodies were used to probe the interactions of the two proteins as described in Materials and Methods. Positive signals were seen only in the cells co-transfected with APE1, Parkin, PINK1 (bottom panel shown with arrows). MT: fluorescent signals by MitoTracker Red (Invitrogen). ( D ) Co-immunoprecipitation of APE1 and Parkin. Parkin and APE1 (lane 1) or Parkin and APE1-FLAG (lane 2) co-expressed in E. coli BLR were subjected to immunoprecipitation using FLAG antibody, and the total and eluted fractions were analyzed in immunoblot using APE1 and Parkin antibodies.
    Figure Legend Snippet: Interaction of APE1 with Parkin in the cytoplasm ( A ) Accumulation of APE1 in the presence of MG132. MEF la cells expressing the wild-type APE1 were incubated with MG132 for indicated time periods. MG132 concentrations were 40 μM (0 – 3 h) and 3 μM (overnight). APE1 was labeled with anti-APE1 antibody and AlexaFluor 488 and analyzed in fluorescence microscopy. ( B ) MEF la ). ( C ) Proximity ligation assay (PLA) between APE1 and Parkin in A549. A549 expressing APE1, Parkin, PINK1 as indicated. APE1 and Parkin specific antibodies were used to probe the interactions of the two proteins as described in Materials and Methods. Positive signals were seen only in the cells co-transfected with APE1, Parkin, PINK1 (bottom panel shown with arrows). MT: fluorescent signals by MitoTracker Red (Invitrogen). ( D ) Co-immunoprecipitation of APE1 and Parkin. Parkin and APE1 (lane 1) or Parkin and APE1-FLAG (lane 2) co-expressed in E. coli BLR were subjected to immunoprecipitation using FLAG antibody, and the total and eluted fractions were analyzed in immunoblot using APE1 and Parkin antibodies.

    Techniques Used: Expressing, Incubation, Labeling, Fluorescence, Microscopy, Proximity Ligation Assay, Transfection, Immunoprecipitation

    Parkin targets endogenous APE1 for degradation ( A ) Expression of Parkin and its cofactors, Uba1, UbcH7, Parkin and PINK1 in A549. A549 was transfected with (−) empty vector alone or (+) the vectors carrying Parkin, PINK1, Uba1, and UbcH7 cDNAs. β-tub: β-tubulin. Note that there was no change in the Uba1 probably due to its abundance. ( B ) A549 cells were transfected with cDNA expression vectors for ubiquitin, UBA1, UBCH7, Parkin, PINK1. Amount of each DNA: 1: 0 μg, 2: 0.17 μg, 3: 0.35 μg, 4: 0.7 μg. Empty vector DNA (vec) was added to each to make the total amount of DNA equal among samples. Cells were then incubated for 16 h, and harvested for immunoblot examination. ( C ) A549 transfected with vector alone (1) or ubiquitin, Uba1, UbcH7, Parkin and PINK1 as in (B) and incubated with MG132 (3 μM for 16 h), and APE1 (middle) and its ubiquitinated forms (top), as well as β-tubulin (bottom) were analyzed by immunoblot separately.
    Figure Legend Snippet: Parkin targets endogenous APE1 for degradation ( A ) Expression of Parkin and its cofactors, Uba1, UbcH7, Parkin and PINK1 in A549. A549 was transfected with (−) empty vector alone or (+) the vectors carrying Parkin, PINK1, Uba1, and UbcH7 cDNAs. β-tub: β-tubulin. Note that there was no change in the Uba1 probably due to its abundance. ( B ) A549 cells were transfected with cDNA expression vectors for ubiquitin, UBA1, UBCH7, Parkin, PINK1. Amount of each DNA: 1: 0 μg, 2: 0.17 μg, 3: 0.35 μg, 4: 0.7 μg. Empty vector DNA (vec) was added to each to make the total amount of DNA equal among samples. Cells were then incubated for 16 h, and harvested for immunoblot examination. ( C ) A549 transfected with vector alone (1) or ubiquitin, Uba1, UbcH7, Parkin and PINK1 as in (B) and incubated with MG132 (3 μM for 16 h), and APE1 (middle) and its ubiquitinated forms (top), as well as β-tubulin (bottom) were analyzed by immunoblot separately.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Incubation

    25) Product Images from "Tetrandrine suppresses lung cancer growth and induces apoptosis, potentially via the VEGF/HIF-1α/ICAM-1 signaling pathway"

    Article Title: Tetrandrine suppresses lung cancer growth and induces apoptosis, potentially via the VEGF/HIF-1α/ICAM-1 signaling pathway

    Journal: Oncology Letters

    doi: 10.3892/ol.2018.8190

    Tetrandrine suppresses A549 lung cancer cell viability. *P
    Figure Legend Snippet: Tetrandrine suppresses A549 lung cancer cell viability. *P

    Techniques Used:

    26) Product Images from "AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models"

    Article Title: AGI-134: a fully synthetic α-Gal glycolipid that converts tumors into in situ autologous vaccines, induces anti-tumor immunity and is synergistic with an anti-PD-1 antibody in mouse melanoma models

    Journal: Cancer Cell International

    doi: 10.1186/s12935-019-1059-8

    Anti-Gal binds to AGI-134-treated human cancer cells and activates CDC and ADCC. a Human SW480 and A549 cancer cells were treated with PBS (open histograms) or the indicated concentrations of AGI-134 (grey and black histograms). The cells were then incubated with affinity purified human anti-Gal IgG or 25% heat-inactivated human serum. Anti-Gal antibody binding was detected with fluorescently-labeled secondary antibodies and samples analyzed by flow cytometry. Representative histogram overlays from two to three independently conducted experiments for each data set are shown. b SW480 and A549 cells were treated with half-log dilutions of AGI-134 and incubated with 50% normal (NHS) or heat-inactivated (iNHS) human serum. In some experiments, SW480 cells were exposed to C7 depleted serum ± 70 µg/mL C7. Cell viability was determined using a luminescence-based cell viability assay and data normalized and expressed as percentage viability. Representative data from 3 independent experiments are shown, with mean values ± SD. c A549 cells were treated with PBS or 0.5 mg/mL AGI-134 and then co-cultured with Promega’s ADCC reporter bioassay effector cells in a 25:1 effector:target cell ratio, in the presence or absence of 30 µg/mL affinity purified human anti-Gal IgG for 6 h. Induction of ADCC over no anti-Gal antibody controls was determined by addition of Bio-Glo Luciferase reagent to quantify reporter gene expression downstream of FcγRIIIa. For assessment of target cell killing by NK cells, CHO-K1 cells were treated with PBS or 1 mg/mL AGI-134 and pre-incubated with 30 µg/mL affinity purified human anti-Gal IgG, before co-culture with IL-2-activated human NK cells. After 4–6 h of co-culture the percentage of dead CHO-K1 cells was determined by incorporation of the viability dye 7-AAD into the target cells. Data shown is the mean + SEM for three (reporter bioassay) or six (cell killing assay) independent experiments
    Figure Legend Snippet: Anti-Gal binds to AGI-134-treated human cancer cells and activates CDC and ADCC. a Human SW480 and A549 cancer cells were treated with PBS (open histograms) or the indicated concentrations of AGI-134 (grey and black histograms). The cells were then incubated with affinity purified human anti-Gal IgG or 25% heat-inactivated human serum. Anti-Gal antibody binding was detected with fluorescently-labeled secondary antibodies and samples analyzed by flow cytometry. Representative histogram overlays from two to three independently conducted experiments for each data set are shown. b SW480 and A549 cells were treated with half-log dilutions of AGI-134 and incubated with 50% normal (NHS) or heat-inactivated (iNHS) human serum. In some experiments, SW480 cells were exposed to C7 depleted serum ± 70 µg/mL C7. Cell viability was determined using a luminescence-based cell viability assay and data normalized and expressed as percentage viability. Representative data from 3 independent experiments are shown, with mean values ± SD. c A549 cells were treated with PBS or 0.5 mg/mL AGI-134 and then co-cultured with Promega’s ADCC reporter bioassay effector cells in a 25:1 effector:target cell ratio, in the presence or absence of 30 µg/mL affinity purified human anti-Gal IgG for 6 h. Induction of ADCC over no anti-Gal antibody controls was determined by addition of Bio-Glo Luciferase reagent to quantify reporter gene expression downstream of FcγRIIIa. For assessment of target cell killing by NK cells, CHO-K1 cells were treated with PBS or 1 mg/mL AGI-134 and pre-incubated with 30 µg/mL affinity purified human anti-Gal IgG, before co-culture with IL-2-activated human NK cells. After 4–6 h of co-culture the percentage of dead CHO-K1 cells was determined by incorporation of the viability dye 7-AAD into the target cells. Data shown is the mean + SEM for three (reporter bioassay) or six (cell killing assay) independent experiments

    Techniques Used: Incubation, Affinity Purification, Binding Assay, Labeling, Flow Cytometry, Cytometry, Viability Assay, Cell Culture, Luciferase, Expressing, Co-Culture Assay

    AGI-134-treated cells are phagocytosed by antigen-presenting cells and antigen cross-presented. a CFSE-labeled A549 cells were treated with PBS or 500 μg/mL AGI-134 and then incubated with or without normal human serum (NHS) to opsonize them with anti-Gal and complement. Subsequently, human macrophages were added at a A549 to macrophage ratio of 3:1. Subsequently, the co-cultures were stained with an anti-CD11 antibody and analyzed by flow cytometry. CFSE (for A549 cells) vs. CD11b (for macrophages) dot plots are shown for the various conditions. Double-positive events were assumed to be macrophages with associated (adherent or phagocytosed) A549 cells. In the bar graphs, the results of three independent experiments, specifically the average percentages of double positive events + SD are shown (* p
    Figure Legend Snippet: AGI-134-treated cells are phagocytosed by antigen-presenting cells and antigen cross-presented. a CFSE-labeled A549 cells were treated with PBS or 500 μg/mL AGI-134 and then incubated with or without normal human serum (NHS) to opsonize them with anti-Gal and complement. Subsequently, human macrophages were added at a A549 to macrophage ratio of 3:1. Subsequently, the co-cultures were stained with an anti-CD11 antibody and analyzed by flow cytometry. CFSE (for A549 cells) vs. CD11b (for macrophages) dot plots are shown for the various conditions. Double-positive events were assumed to be macrophages with associated (adherent or phagocytosed) A549 cells. In the bar graphs, the results of three independent experiments, specifically the average percentages of double positive events + SD are shown (* p

    Techniques Used: Labeling, Incubation, Staining, Flow Cytometry, Cytometry

    27) Product Images from "Transforming Growth Factor ?1 Receptor II Is Downregulated by E1A in Adenovirus-Infected Cells"

    Article Title: Transforming Growth Factor ?1 Receptor II Is Downregulated by E1A in Adenovirus-Infected Cells

    Journal: Journal of Virology

    doi: 10.1128/JVI.77.17.9324-9336.2003

    TGF-β1 suppresses adenovirus late protein synthesis and virus yields in infected cells. A549 cells were maintained in 10 or 0.2% FCS or 0.2% FCS plus 5 ng of TGF-β1/ml as indicated throughout the experiment. Following 3 days of pretreatment, cells were infected with adenovirus mutants at 10 PFU/cell. At 24 h p.i., cell lysates were collected and analyzed by Western blotting using anti-Ad5 (A) and anti-E1A (B) antibodies. Equal protein concentrations were loaded in all lanes. The signal from every lane was measured and quantified with FluorChem software (Alpha Innotech Corporation). (C) Ratio of the signal in serum-starved cells to that in cytokine-treated cells for each adenovirus mutant. (D) A549 cells were treated and infected as for panels A and B. Cell lysates and supernatants were collected, and total virus yields at the indicated times postinfection were determined.
    Figure Legend Snippet: TGF-β1 suppresses adenovirus late protein synthesis and virus yields in infected cells. A549 cells were maintained in 10 or 0.2% FCS or 0.2% FCS plus 5 ng of TGF-β1/ml as indicated throughout the experiment. Following 3 days of pretreatment, cells were infected with adenovirus mutants at 10 PFU/cell. At 24 h p.i., cell lysates were collected and analyzed by Western blotting using anti-Ad5 (A) and anti-E1A (B) antibodies. Equal protein concentrations were loaded in all lanes. The signal from every lane was measured and quantified with FluorChem software (Alpha Innotech Corporation). (C) Ratio of the signal in serum-starved cells to that in cytokine-treated cells for each adenovirus mutant. (D) A549 cells were treated and infected as for panels A and B. Cell lysates and supernatants were collected, and total virus yields at the indicated times postinfection were determined.

    Techniques Used: Infection, Western Blot, Software, Mutagenesis

    E1A inhibits TGF-β-induced signal transduction in adenovirus-infected cells as determined by using the 3TP-lux reporter plasmid. (A) HepG2 cells were transfected with 3TP-lux and pCMV-βGal plasmids. After 6 h of transfection, cells were infected with rec 700 at 50 PFU/cell or mock infected. Infections were maintained in the presence of AraC. Cells were treated with TGF-β1 from 12 to 26 h p.i.; subsequently, cells were lysed and luciferase and β-Gal activities were measured. Luciferase values were normalized against β-Gal activity for each sample. Each experimental condition was done in triplicate, and the average values are shown. (B) HepG2 cells were transfected with 3TP-lux and CMV β-Gal plasmids and an empty vector or a plasmid expressing either the 13S or 12S isoform of E1A. At 18 h posttransfection, cells were treated with recombinant TGF-β1 (3 ng/ml) for 8 h. (C) HepG2 cells were treated as described for panel A. Cells were maintained from 12 to 48 h p.i. in the presence of 5 ng of TGF-β1/ml. (D) A549 cells were infected with 50 PFU/cell of rec 700 or mock infected and maintained in the presence of AraC. At 20 (lane d) or 24 (lane e) h p.i. cells were mock treated or treated with 5 ng of TGF-β1/ml for 20 min. Levels of phospho-Smad 2 (arrow) were determined by Western analysis. Molecular weight marker positions are shown.
    Figure Legend Snippet: E1A inhibits TGF-β-induced signal transduction in adenovirus-infected cells as determined by using the 3TP-lux reporter plasmid. (A) HepG2 cells were transfected with 3TP-lux and pCMV-βGal plasmids. After 6 h of transfection, cells were infected with rec 700 at 50 PFU/cell or mock infected. Infections were maintained in the presence of AraC. Cells were treated with TGF-β1 from 12 to 26 h p.i.; subsequently, cells were lysed and luciferase and β-Gal activities were measured. Luciferase values were normalized against β-Gal activity for each sample. Each experimental condition was done in triplicate, and the average values are shown. (B) HepG2 cells were transfected with 3TP-lux and CMV β-Gal plasmids and an empty vector or a plasmid expressing either the 13S or 12S isoform of E1A. At 18 h posttransfection, cells were treated with recombinant TGF-β1 (3 ng/ml) for 8 h. (C) HepG2 cells were treated as described for panel A. Cells were maintained from 12 to 48 h p.i. in the presence of 5 ng of TGF-β1/ml. (D) A549 cells were infected with 50 PFU/cell of rec 700 or mock infected and maintained in the presence of AraC. At 20 (lane d) or 24 (lane e) h p.i. cells were mock treated or treated with 5 ng of TGF-β1/ml for 20 min. Levels of phospho-Smad 2 (arrow) were determined by Western analysis. Molecular weight marker positions are shown.

    Techniques Used: Transduction, Infection, Plasmid Preparation, Transfection, Luciferase, Activity Assay, Expressing, Recombinant, Western Blot, Molecular Weight, Marker

    The E1A 13S or 12S protein, including amino acids 2 to 36 and the CtBP binding site, is required to downregulate the TβRII protein in adenovirus-infected cells. A549 (A and B) or HepG2 (C and D) cells were infected with wild-type (wt) or mutant adenoviruses at 50 PFU/cell. Cell lysates were harvested at 24 h p.i. for the wild type and most mutants and at 48 h p.i. for dl 313, 12Swt, dC-term, Ad/E3, 12S.2-36, 12S.928, and dl 312. Cell lysates were subjected to Western blotting with anti-TβRII antibodies (Santa Cruz Biotechnology). (E) Schematic of the adenovirus (Ad) genome. The E1A proteins activate the transcription of adenovirus genes and deregulate the cell cycle by suppressing or activating cellular proteins and genes. E1B proteins suppress cellular apoptosis. E3 proteins confer a stealth function to the virus by inhibiting immune cell-mediated apoptosis. E4 proteins function in gene regulation, in part by facilitating degradation of p53; they are also required for viral mRNA transport from the nucleus. Virus DNA replication is necessary for late protein synthesis derived from the major late transcription unit. At about 24 h p.i., virions begin to assemble in the cell nucleus, and after 2 to 3 days cell lysis begins to occur, with the release of virions.
    Figure Legend Snippet: The E1A 13S or 12S protein, including amino acids 2 to 36 and the CtBP binding site, is required to downregulate the TβRII protein in adenovirus-infected cells. A549 (A and B) or HepG2 (C and D) cells were infected with wild-type (wt) or mutant adenoviruses at 50 PFU/cell. Cell lysates were harvested at 24 h p.i. for the wild type and most mutants and at 48 h p.i. for dl 313, 12Swt, dC-term, Ad/E3, 12S.2-36, 12S.928, and dl 312. Cell lysates were subjected to Western blotting with anti-TβRII antibodies (Santa Cruz Biotechnology). (E) Schematic of the adenovirus (Ad) genome. The E1A proteins activate the transcription of adenovirus genes and deregulate the cell cycle by suppressing or activating cellular proteins and genes. E1B proteins suppress cellular apoptosis. E3 proteins confer a stealth function to the virus by inhibiting immune cell-mediated apoptosis. E4 proteins function in gene regulation, in part by facilitating degradation of p53; they are also required for viral mRNA transport from the nucleus. Virus DNA replication is necessary for late protein synthesis derived from the major late transcription unit. At about 24 h p.i., virions begin to assemble in the cell nucleus, and after 2 to 3 days cell lysis begins to occur, with the release of virions.

    Techniques Used: Binding Assay, Infection, Mutagenesis, Western Blot, Derivative Assay, Lysis

    Adenovirus infection downregulates the TβRII protein. (A) A549 human lung adenocarcinoma cells were infected with rec 700 at 50 PFU/cell. Some of the infected cells were maintained in 20 μg of AraC/ml. Cell lysates collected at 19 h p.i. were subjected to Western blotting using anti-TβRII antibodies (Santa Cruz Biotechnology). Upper and lower arrows, mature and immature forms of TβRII, respectively. (B) A549 cells were infected with rec 700 at 10 PFU/cell. Cell lysates were collected at the indicated hours p.i. and analyzed by Western blotting. Equal protein amounts were loaded per lane.
    Figure Legend Snippet: Adenovirus infection downregulates the TβRII protein. (A) A549 human lung adenocarcinoma cells were infected with rec 700 at 50 PFU/cell. Some of the infected cells were maintained in 20 μg of AraC/ml. Cell lysates collected at 19 h p.i. were subjected to Western blotting using anti-TβRII antibodies (Santa Cruz Biotechnology). Upper and lower arrows, mature and immature forms of TβRII, respectively. (B) A549 cells were infected with rec 700 at 10 PFU/cell. Cell lysates were collected at the indicated hours p.i. and analyzed by Western blotting. Equal protein amounts were loaded per lane.

    Techniques Used: Infection, Western Blot

    Stability of the TβRII protein as determined by pulse-chase analysis in adenovirus-infected cells. (A and D) In two independent experiments, A549 cells were mock infected or infected with rec 700 at 50 PFU/cell and maintained in the presence of AraC throughout the infection. At 19 h p.i., cells were pulsed with [ 35 S]methionine-cysteine for 15 min and chased in 10% FCS-supplemented nonradioactive medium for the indicated time periods. Immunoprecipitated, radioactively labeled TβRII was quantified by phosphorimager at each time point of chase (B and E, respectively). (C) Calculations of the TβRII half-life ( t 1/2 ).
    Figure Legend Snippet: Stability of the TβRII protein as determined by pulse-chase analysis in adenovirus-infected cells. (A and D) In two independent experiments, A549 cells were mock infected or infected with rec 700 at 50 PFU/cell and maintained in the presence of AraC throughout the infection. At 19 h p.i., cells were pulsed with [ 35 S]methionine-cysteine for 15 min and chased in 10% FCS-supplemented nonradioactive medium for the indicated time periods. Immunoprecipitated, radioactively labeled TβRII was quantified by phosphorimager at each time point of chase (B and E, respectively). (C) Calculations of the TβRII half-life ( t 1/2 ).

    Techniques Used: Pulse Chase, Infection, Immunoprecipitation, Labeling

    28) Product Images from "miRNA-200c-3p is crucial in acute respiratory distress syndrome"

    Article Title: miRNA-200c-3p is crucial in acute respiratory distress syndrome

    Journal: Cell Discovery

    doi: 10.1038/celldisc.2017.21

    NF-κB signaling pathway mediates the upregulation of miR-200c-3p. ( a ) A549 cells were transfected with the corresponding siRNAs and then challenged with AF or H5N1 (MOI=4) for 48 h. The expression of miR-200c-3p was detected by qRT-PCR. ( b ) A549 cells were treated with CAPE or JSH-23 and then challenged with AF or H5N1 (MOI=4) for 48 h. The expression of miR-200c-3p was detected by qRT-PCR. ( c ) A549 cells were transfected with the corresponding siRNAs and then transfected with 1 μg ml −1 RNA extracted from AF- or H5N1-infected cells for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. ( d ) A549 cells were treated with CAPE or JSH-23 and then transfected with 1 μg ml −1 RNA extracted from AF- or H5N1-infected cells for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. ( e ) A549 cells were transfected with the corresponding siRNAs and then transfected with 1 μg ml −1 poly (I:C) for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, A549 cells were treated with the solvent used. ( f ) A549 cells were treated with CAPE or JSH-23 and then transfected with 1 μg ml −1 poly (I:C) for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, A549 cells were treated with the solvent used. ( g ) THP1 cells were transfected with the corresponding siRNAs and then treated with 1 μg ml −1 LPS for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( h ) THP1 cells were treated with CAPE or JSH-23 and then treated with 1 μg ml −1 LPS for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( i ) THP1 cells were transfected with the corresponding siRNAs and then treated with 10 μg ml −1 LTA for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( j ) THP1 cells were treated with CAPE or JSH-23 and then treated with 10 μg ml −1 LTA for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. The data are shown as the mean±s.e.m. * P
    Figure Legend Snippet: NF-κB signaling pathway mediates the upregulation of miR-200c-3p. ( a ) A549 cells were transfected with the corresponding siRNAs and then challenged with AF or H5N1 (MOI=4) for 48 h. The expression of miR-200c-3p was detected by qRT-PCR. ( b ) A549 cells were treated with CAPE or JSH-23 and then challenged with AF or H5N1 (MOI=4) for 48 h. The expression of miR-200c-3p was detected by qRT-PCR. ( c ) A549 cells were transfected with the corresponding siRNAs and then transfected with 1 μg ml −1 RNA extracted from AF- or H5N1-infected cells for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. ( d ) A549 cells were treated with CAPE or JSH-23 and then transfected with 1 μg ml −1 RNA extracted from AF- or H5N1-infected cells for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. ( e ) A549 cells were transfected with the corresponding siRNAs and then transfected with 1 μg ml −1 poly (I:C) for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, A549 cells were treated with the solvent used. ( f ) A549 cells were treated with CAPE or JSH-23 and then transfected with 1 μg ml −1 poly (I:C) for 12 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, A549 cells were treated with the solvent used. ( g ) THP1 cells were transfected with the corresponding siRNAs and then treated with 1 μg ml −1 LPS for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( h ) THP1 cells were treated with CAPE or JSH-23 and then treated with 1 μg ml −1 LPS for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( i ) THP1 cells were transfected with the corresponding siRNAs and then treated with 10 μg ml −1 LTA for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. ( j ) THP1 cells were treated with CAPE or JSH-23 and then treated with 10 μg ml −1 LTA for 24 h. The expression of miR-200c-3p was detected by qRT-PCR. Native, THP1 cells were treated with the solvent used. The data are shown as the mean±s.e.m. * P

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

    ACE2 is a target of miR-200c-3p that can be induced by NS1. ( a ) A549 cells were transfected with 50 nM mimics or inhibitors of miR-200c-3p or miR-141-3p for 36 h. ACE2 protein expression levels were analyzed by western blotting and were normalized to β-actin. Native, untreated A549 cells. ( b ) The wild-type 3′-UTR of the ACE2 transcript was cloned into the psiCHECK-2 vector (ACE2 3′-UTR-WT reporter vector). Luciferase activity in HEK293T cells transfected with mimics of miRNAs or the vector was detected using the Dual Luciferase Reporter Assay System. ( c ) An ACE2 3′-UTR-MUT reporter vector was constructed in which the miR-200c-3p binding site on the ACE2 3′-UTR was deleted. Luciferase activity in HEK293T cells transfected with miR-200c-3p mimics and the ACE2 3′-UTR-WT or -MUT reporter vector was detected using the Dual Luciferase Reporter Assay System. ( d ) Vectors containing H5N1 influenza viral protein coding genes were individually transfected into HEK293T cells for 48 h. The expression of miR-200c-3p was quantified by qRT-PCR. Native, untreated HEK293T cells. ( e and f ) Vectors coding for NS1 of H1N1, H5N1 and H7N9 viruses were transfected into HEK293T cells for 72 h. The expression of miR-200c-3p was detected by qRT-PCR. The expression of ACE2 and NS1 was detected by western blotting. In each graph, the data are presented as the mean±s.e.m. NS, not significant, ** P
    Figure Legend Snippet: ACE2 is a target of miR-200c-3p that can be induced by NS1. ( a ) A549 cells were transfected with 50 nM mimics or inhibitors of miR-200c-3p or miR-141-3p for 36 h. ACE2 protein expression levels were analyzed by western blotting and were normalized to β-actin. Native, untreated A549 cells. ( b ) The wild-type 3′-UTR of the ACE2 transcript was cloned into the psiCHECK-2 vector (ACE2 3′-UTR-WT reporter vector). Luciferase activity in HEK293T cells transfected with mimics of miRNAs or the vector was detected using the Dual Luciferase Reporter Assay System. ( c ) An ACE2 3′-UTR-MUT reporter vector was constructed in which the miR-200c-3p binding site on the ACE2 3′-UTR was deleted. Luciferase activity in HEK293T cells transfected with miR-200c-3p mimics and the ACE2 3′-UTR-WT or -MUT reporter vector was detected using the Dual Luciferase Reporter Assay System. ( d ) Vectors containing H5N1 influenza viral protein coding genes were individually transfected into HEK293T cells for 48 h. The expression of miR-200c-3p was quantified by qRT-PCR. Native, untreated HEK293T cells. ( e and f ) Vectors coding for NS1 of H1N1, H5N1 and H7N9 viruses were transfected into HEK293T cells for 72 h. The expression of miR-200c-3p was detected by qRT-PCR. The expression of ACE2 and NS1 was detected by western blotting. In each graph, the data are presented as the mean±s.e.m. NS, not significant, ** P

    Techniques Used: Transfection, Expressing, Western Blot, Clone Assay, Plasmid Preparation, Luciferase, Activity Assay, Reporter Assay, Construct, Binding Assay, Quantitative RT-PCR

    Elevated levels of miR-200c-3p in cells treated with poly (I:C), LPS and LTA and in the plasma of severe pneumonia patients. ( a ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells transfected with poly (I:C) for 6 h at the indicated concentration. ( b ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells treated with LPS for 24 h at the indicated concentration. ( c ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells treated with LTA for 24 h at the indicated concentration. ( d ) After transfection with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were transfected with poly (I:C) (10 μg ml −1 ). The expression of ACE2 protein in the cells was detected. Native, A549 cells were treated with the solvent used. ( e ) After transfection with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were challenged with LPS (10 μg ml −1 ). The expression of ACE2 protein in the cells was detected. Native, A549 cells were treated with the solvent used. ( f ) After transfected with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were challenged with LTA at (500 μg ml −1 ). The expression of ACE2 protein in the cells was detected. β-Actin served as an internal control. Native, A549 cells were treated with the solvent used. ( g and h ) qRT-PCR analysis of miR-200c-3p ( g ) and enzyme-linked immunosorbent assay analysis of Ang II ( h ) in the plasma of healthy controls and severe pneumonia patients. The number of the study participants for each group was as follows: healthy control group ( n =21), severe pneumonia patients ( n =56). ( i ) Kinetics of miR-200c-3p (ratio to cel-miR-39 which was used as a spike-in control) and Ang II plasma levels of one severe pneumonia patient (patient 48th). The data are shown as the mean±s.e.m. ** P
    Figure Legend Snippet: Elevated levels of miR-200c-3p in cells treated with poly (I:C), LPS and LTA and in the plasma of severe pneumonia patients. ( a ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells transfected with poly (I:C) for 6 h at the indicated concentration. ( b ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells treated with LPS for 24 h at the indicated concentration. ( c ) qRT-PCR analysis of the expression of miR-200c-3p and miR-421 in A549 cells treated with LTA for 24 h at the indicated concentration. ( d ) After transfection with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were transfected with poly (I:C) (10 μg ml −1 ). The expression of ACE2 protein in the cells was detected. Native, A549 cells were treated with the solvent used. ( e ) After transfection with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were challenged with LPS (10 μg ml −1 ). The expression of ACE2 protein in the cells was detected. Native, A549 cells were treated with the solvent used. ( f ) After transfected with inhibitors of NC or miR-200c-3p for 6 h, A549 cells were challenged with LTA at (500 μg ml −1 ). The expression of ACE2 protein in the cells was detected. β-Actin served as an internal control. Native, A549 cells were treated with the solvent used. ( g and h ) qRT-PCR analysis of miR-200c-3p ( g ) and enzyme-linked immunosorbent assay analysis of Ang II ( h ) in the plasma of healthy controls and severe pneumonia patients. The number of the study participants for each group was as follows: healthy control group ( n =21), severe pneumonia patients ( n =56). ( i ) Kinetics of miR-200c-3p (ratio to cel-miR-39 which was used as a spike-in control) and Ang II plasma levels of one severe pneumonia patient (patient 48th). The data are shown as the mean±s.e.m. ** P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection, Concentration Assay, Enzyme-linked Immunosorbent Assay

    Aberrant expression of miR-200c-3p and miR-141-3p in H5N1-infected A549 cells. ( a ) Heat map of fold changes (log 2) of miRNAs’ expression in A549 cells after infection with either H1N1 or H5N1 (MOI=4) influenza virus for 18 h. The fold changes were compared with miRNAs’ expression in cells mock infected with AF for 18 h. The upregulated miRNAs in H5N1-infected A549 cells are listed. ( b and c ) qRT-PCR analysis of the expression of miR-200c-3p and miR-141-3p in A549 cells at the indicated hours after challenge with H1N1 virus, H5N1 virus (MOI=4) or AF control. Graph shows the mean±s.e.m. ** P
    Figure Legend Snippet: Aberrant expression of miR-200c-3p and miR-141-3p in H5N1-infected A549 cells. ( a ) Heat map of fold changes (log 2) of miRNAs’ expression in A549 cells after infection with either H1N1 or H5N1 (MOI=4) influenza virus for 18 h. The fold changes were compared with miRNAs’ expression in cells mock infected with AF for 18 h. The upregulated miRNAs in H5N1-infected A549 cells are listed. ( b and c ) qRT-PCR analysis of the expression of miR-200c-3p and miR-141-3p in A549 cells at the indicated hours after challenge with H1N1 virus, H5N1 virus (MOI=4) or AF control. Graph shows the mean±s.e.m. ** P

    Techniques Used: Expressing, Infection, Quantitative RT-PCR

    29) Product Images from "Glutathione S-transferase A1 mediates nicotine-induced lung cancer cell metastasis by promoting epithelial-mesenchymal transition"

    Article Title: Glutathione S-transferase A1 mediates nicotine-induced lung cancer cell metastasis by promoting epithelial-mesenchymal transition

    Journal: Experimental and Therapeutic Medicine

    doi: 10.3892/etm.2017.4663

    Expression of GSTA1 in A549 cells following treatment with nicotine and transfection with siRNA. *P
    Figure Legend Snippet: Expression of GSTA1 in A549 cells following treatment with nicotine and transfection with siRNA. *P

    Techniques Used: Expressing, Transfection

    Adhesion of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P
    Figure Legend Snippet: Adhesion of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P

    Techniques Used: Transfection

    Invasion of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P
    Figure Legend Snippet: Invasion of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P

    Techniques Used: Transfection

    Levels of GSTA1 mRNA and protein in A549 cells following treatment with 0, 0.01, 0.1, 1 or 10 µM nicotine. (A) Relative mRNA expression of GSTA1. (B) Relative protein expression of GSTA1. *P
    Figure Legend Snippet: Levels of GSTA1 mRNA and protein in A549 cells following treatment with 0, 0.01, 0.1, 1 or 10 µM nicotine. (A) Relative mRNA expression of GSTA1. (B) Relative protein expression of GSTA1. *P

    Techniques Used: Expressing

    Levels of epithelial-mesenchymal transition markers in A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P
    Figure Legend Snippet: Levels of epithelial-mesenchymal transition markers in A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P

    Techniques Used: Transfection

    Viability of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P
    Figure Legend Snippet: Viability of A549 cells following treatment with 10 µM nicotine and transfection with siRNA. *P

    Techniques Used: Transfection

    30) Product Images from "Enhanced Mitochondrial DNA Repair of the Common Disease- Associated Variant, Ser326Cys, of hOGG1 through Small Molecule Intervention"

    Article Title: Enhanced Mitochondrial DNA Repair of the Common Disease- Associated Variant, Ser326Cys, of hOGG1 through Small Molecule Intervention

    Journal: Free radical biology & medicine

    doi: 10.1016/j.freeradbiomed.2018.05.094

    OGG1 activators attenuate paraquat-induced oxidative damage in A549 cells A549 cells were pre-treated for 4 hr with small molecule OGG1 activators or 0.1% DMSO prior to addition of 0.6 mM paraquat for 48 hr. The cells were stained and imaged for 8-oxoG content and the average fluorescence intensity within the cell was normalized based on the number of cells imaged. (A) Graphical representations of the change in 8-oxoG intensity within the cytoplasm of cells compared to 0.6 mM paraquat treated cells (dashed line). (B) Representative images of cells from panel A depict changes in mitochondrial 8-oxoG staining (Hoechst 33342, overlaid blue; MitoTracker® orange, overlaid orange; 8-oxoG-AleaFluor®-647, overlaid red; line = 40 μm). (C) The percent of the cell populations labeled as high responders is shown for A549 cells pre-incubated with OGG1 activators (4 hr) prior to exposure to a single concentration of paraquat (0.6 mM, dashed line) for 48 hr. (D) The images from panel A were analyzed further by examining morphology changes. The mean nuclear area (dashed line indicates no treatment, 0.6 mM PQ) for A549 cells was graphed depicting a change in overall cell health. Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing paraquat alone treated groups to untreated control (0 mM) and OGG1 activator treated groups to 0.6 mM paraquat control (** p
    Figure Legend Snippet: OGG1 activators attenuate paraquat-induced oxidative damage in A549 cells A549 cells were pre-treated for 4 hr with small molecule OGG1 activators or 0.1% DMSO prior to addition of 0.6 mM paraquat for 48 hr. The cells were stained and imaged for 8-oxoG content and the average fluorescence intensity within the cell was normalized based on the number of cells imaged. (A) Graphical representations of the change in 8-oxoG intensity within the cytoplasm of cells compared to 0.6 mM paraquat treated cells (dashed line). (B) Representative images of cells from panel A depict changes in mitochondrial 8-oxoG staining (Hoechst 33342, overlaid blue; MitoTracker® orange, overlaid orange; 8-oxoG-AleaFluor®-647, overlaid red; line = 40 μm). (C) The percent of the cell populations labeled as high responders is shown for A549 cells pre-incubated with OGG1 activators (4 hr) prior to exposure to a single concentration of paraquat (0.6 mM, dashed line) for 48 hr. (D) The images from panel A were analyzed further by examining morphology changes. The mean nuclear area (dashed line indicates no treatment, 0.6 mM PQ) for A549 cells was graphed depicting a change in overall cell health. Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing paraquat alone treated groups to untreated control (0 mM) and OGG1 activator treated groups to 0.6 mM paraquat control (** p

    Techniques Used: Staining, Fluorescence, Labeling, Incubation, Concentration Assay

    OGG1 activators protect against paraquat-induced loss of mitochondrial membrane potential in A549 cells A549 cells were pre-treated for 4 hours with the OGG1 small molecule activators or 0.1% DMSO prior to exposure to 0.3 mM paraquat for 24 hr. Mitochondrial membrane potential was measured from images captured as described in the methods. (A) Graphical representation of the change in the JC-1 ratio with increasing concentration of paraquat and cells pre-treated with the OGG1 activators prior to exposure to a single concentration of paraquat (0.3 mM) for 24 hr. (B) Representative images from untreated, 3 mM paraquat, 0.3 mM paraquat, and Compound D (30 μM) with 0.3 mM paraquat exposed cells (Hoechst 33342, overlaid blue; JC-1 monomer, overlaid green; JC-1 aggregate, overlaid orange; CellMask™ Deep Red, overlaid red; line = 40 μm). Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing paraquat alone treated groups to untreated control (0 mM) and OGG1 activator treated groups to 0.3 mM paraquat control (* p
    Figure Legend Snippet: OGG1 activators protect against paraquat-induced loss of mitochondrial membrane potential in A549 cells A549 cells were pre-treated for 4 hours with the OGG1 small molecule activators or 0.1% DMSO prior to exposure to 0.3 mM paraquat for 24 hr. Mitochondrial membrane potential was measured from images captured as described in the methods. (A) Graphical representation of the change in the JC-1 ratio with increasing concentration of paraquat and cells pre-treated with the OGG1 activators prior to exposure to a single concentration of paraquat (0.3 mM) for 24 hr. (B) Representative images from untreated, 3 mM paraquat, 0.3 mM paraquat, and Compound D (30 μM) with 0.3 mM paraquat exposed cells (Hoechst 33342, overlaid blue; JC-1 monomer, overlaid green; JC-1 aggregate, overlaid orange; CellMask™ Deep Red, overlaid red; line = 40 μm). Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing paraquat alone treated groups to untreated control (0 mM) and OGG1 activator treated groups to 0.3 mM paraquat control (* p

    Techniques Used: Concentration Assay

    Paraquat-induced mitochondrial DNA damage is inversely related to OGG1 expression A549 cells were treated with paraquat for 24 or 48 hr and subjected to formaldehyde fixation prior to immunofluorescence detection of 8-oxoguanine. (A) Graphical representation of the percent change in 8-oxoguanine intensity within the cytoplasm region following 24- and 48-hr treated cells with increasing concentrations of paraquat. Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing treatment groups to an untreated control (* p
    Figure Legend Snippet: Paraquat-induced mitochondrial DNA damage is inversely related to OGG1 expression A549 cells were treated with paraquat for 24 or 48 hr and subjected to formaldehyde fixation prior to immunofluorescence detection of 8-oxoguanine. (A) Graphical representation of the percent change in 8-oxoguanine intensity within the cytoplasm region following 24- and 48-hr treated cells with increasing concentrations of paraquat. Significance was determined using a 1-way ANOVA with a Dunnett’s posttest comparing treatment groups to an untreated control (* p

    Techniques Used: Expressing, Immunofluorescence

    31) Product Images from "Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice"

    Article Title: Effect of Hyperoxia on Retinoid Metabolism and Retinoid Receptor Expression in the Lungs of Newborn Mice

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0140343

    The effect of Vitamin A on the hyperoxia-induced decline of thymidine incorporation in A549 cells. Proliferation of A549 cells was assayed by thymidine [ 3 H] incorporation, which measures DNA synthesis. The cells were plated at a final concentration of 1x10 4 cells per well in a 96-well plate with or without vitamin A. Triplicates were tested for each sample. Cells were then placed in a humidified chamber with either 95% air-5% CO 2 or 95% oxygen- 5% CO 2 and cultured for 2 (A) or 3 d (B). Thymidine [ 3 H] was added for another 18 h and then the cells were harvested and measured by Matrix 96 Direct beta Counter. *P
    Figure Legend Snippet: The effect of Vitamin A on the hyperoxia-induced decline of thymidine incorporation in A549 cells. Proliferation of A549 cells was assayed by thymidine [ 3 H] incorporation, which measures DNA synthesis. The cells were plated at a final concentration of 1x10 4 cells per well in a 96-well plate with or without vitamin A. Triplicates were tested for each sample. Cells were then placed in a humidified chamber with either 95% air-5% CO 2 or 95% oxygen- 5% CO 2 and cultured for 2 (A) or 3 d (B). Thymidine [ 3 H] was added for another 18 h and then the cells were harvested and measured by Matrix 96 Direct beta Counter. *P

    Techniques Used: DNA Synthesis, Concentration Assay, Cell Culture

    Hyperoxia-induced inhibition of RAR expression in vitro . A549 cells were plated in 100-mm dishes at a density of 5x10 5 cells/mL. The cells were then cultured in a humidified chamber under normoxic (95% air-5% CO 2 ) or hyperoxic (95% oxygen- 5% CO 2 ) conditions for 2 or 3 d. The cells were then harvested and assayed for RARα and RARγ expression. The expression levels were normalized to a GAPDH internal control. *P
    Figure Legend Snippet: Hyperoxia-induced inhibition of RAR expression in vitro . A549 cells were plated in 100-mm dishes at a density of 5x10 5 cells/mL. The cells were then cultured in a humidified chamber under normoxic (95% air-5% CO 2 ) or hyperoxic (95% oxygen- 5% CO 2 ) conditions for 2 or 3 d. The cells were then harvested and assayed for RARα and RARγ expression. The expression levels were normalized to a GAPDH internal control. *P

    Techniques Used: Inhibition, Expressing, In Vitro, Cell Culture

    32) Product Images from "SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro"

    Article Title: SOCS3 inhibiting migration of A549 cells correlates with PYK2 signaling in vitro

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-8-150

    Effects of SOCS3-KIR mutant on PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells . (a) Down-regulations of PYK2 expression, Tyr402 and ERK1/2 phosphorylations by SOCS3-KIR mutant. A549 cells were transfected with either vector or the SOCS3-KIR mutant for 48 h, and cell lysates were subjected to western blot using the indicated antibodies. Cells pretreated with β-lactacystin showed that PYK2 expression, Tyr402 and ERK1/2 phosphorylations were restored to some extent. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration inhibited by the SOCS3-KIR mutant was statistically significant, and β-lactacystin pretreatment restored cell migration to some degree. The values are means of three replicates.
    Figure Legend Snippet: Effects of SOCS3-KIR mutant on PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells . (a) Down-regulations of PYK2 expression, Tyr402 and ERK1/2 phosphorylations by SOCS3-KIR mutant. A549 cells were transfected with either vector or the SOCS3-KIR mutant for 48 h, and cell lysates were subjected to western blot using the indicated antibodies. Cells pretreated with β-lactacystin showed that PYK2 expression, Tyr402 and ERK1/2 phosphorylations were restored to some extent. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration inhibited by the SOCS3-KIR mutant was statistically significant, and β-lactacystin pretreatment restored cell migration to some degree. The values are means of three replicates.

    Techniques Used: Mutagenesis, Expressing, Migration, Transfection, Plasmid Preparation, Western Blot, Amplification, Polymerase Chain Reaction

    Interactions of exogenously expressed SOCS3 mutants with PYK2 in A549 cells . A549 cells were transfected with either vector or SOCS3 mutants with distinct functional domains deleted, and pretreated with β-lactacystin 4 h before transfection until the cell lysates were subjected to anti-myc tag immunoprecipitations and analysed by western blot using the anti-PYK2 antibody. The co-immunoprecipitation product was clearly observed in the SOCS-box mutant transfected cells. The results are representative of three individual experiments.
    Figure Legend Snippet: Interactions of exogenously expressed SOCS3 mutants with PYK2 in A549 cells . A549 cells were transfected with either vector or SOCS3 mutants with distinct functional domains deleted, and pretreated with β-lactacystin 4 h before transfection until the cell lysates were subjected to anti-myc tag immunoprecipitations and analysed by western blot using the anti-PYK2 antibody. The co-immunoprecipitation product was clearly observed in the SOCS-box mutant transfected cells. The results are representative of three individual experiments.

    Techniques Used: Transfection, Plasmid Preparation, Functional Assay, Western Blot, Immunoprecipitation, Mutagenesis

    PYK2 expression, Tyr402 and ERK1/2 phosphorylations, as well as cell migration in HBE and A549 cells. (a) PYK2 expression, Tyr402 and ERK1/2 phosphorylations in HBE and A549 cells by western blot. (b) Comparisons of migratory abilities between HBE and A549 cells by Transwell assay. The data are representative of three individual experiments.
    Figure Legend Snippet: PYK2 expression, Tyr402 and ERK1/2 phosphorylations, as well as cell migration in HBE and A549 cells. (a) PYK2 expression, Tyr402 and ERK1/2 phosphorylations in HBE and A549 cells by western blot. (b) Comparisons of migratory abilities between HBE and A549 cells by Transwell assay. The data are representative of three individual experiments.

    Techniques Used: Expressing, Migration, Western Blot, Transwell Assay

    Methylation status and expression of SOCS3 in HBE and A549 cells. (a) Methylation status of SOCS3 in HBE and A549 cells, using the primer set designed in the exon 2, and detection of SOCS3 expression by western blot. The observed bands in lane M are methylated 159 bp products with methylation-specific primers and that in lane U are unmethylated 159 bp products with unmethylation-specific primers. Methylation was found in A549 cells but not in HBE cells and HBE cells expressed higher level of SOCS3. (b) The visible band in lane U appeared in A549 cells and SOCS3 was reactivated by the treatment of 5-aza-2'-deoxycytidine. The results are representative of three independent experiments.
    Figure Legend Snippet: Methylation status and expression of SOCS3 in HBE and A549 cells. (a) Methylation status of SOCS3 in HBE and A549 cells, using the primer set designed in the exon 2, and detection of SOCS3 expression by western blot. The observed bands in lane M are methylated 159 bp products with methylation-specific primers and that in lane U are unmethylated 159 bp products with unmethylation-specific primers. Methylation was found in A549 cells but not in HBE cells and HBE cells expressed higher level of SOCS3. (b) The visible band in lane U appeared in A549 cells and SOCS3 was reactivated by the treatment of 5-aza-2'-deoxycytidine. The results are representative of three independent experiments.

    Techniques Used: Methylation, Expressing, Western Blot

    Up-regulation of SOCS3 by 5-aza-2'-deoxycytidine treatment inhibited cell migration and associated PYK2 expression, Tyr402 and ERK1/2 phosphorylations. (a) Decreased PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells were observed by SOCS3 up-regulation. A549 cells were treated with 5-aza-2'-deoxycytidine for 6 d, and cell lysates were analysed by western blot using the indicated antibodies. Elevated PYK2 expression, Tyr402 and ERK1/2 phosphorylations were found by the pretreatment of β-lactacystin. (b) PYK2 mRNA levels remained invariable regardless of treatment or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration was suppressed by SOCS3 restoration, and the β-lactacystin pretreatment facilitated cell migration to some extent. The values are means of three replicates.
    Figure Legend Snippet: Up-regulation of SOCS3 by 5-aza-2'-deoxycytidine treatment inhibited cell migration and associated PYK2 expression, Tyr402 and ERK1/2 phosphorylations. (a) Decreased PYK2 expression, Tyr402 and ERK1/2 activations and migration of A549 cells were observed by SOCS3 up-regulation. A549 cells were treated with 5-aza-2'-deoxycytidine for 6 d, and cell lysates were analysed by western blot using the indicated antibodies. Elevated PYK2 expression, Tyr402 and ERK1/2 phosphorylations were found by the pretreatment of β-lactacystin. (b) PYK2 mRNA levels remained invariable regardless of treatment or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration was suppressed by SOCS3 restoration, and the β-lactacystin pretreatment facilitated cell migration to some extent. The values are means of three replicates.

    Techniques Used: Migration, Expressing, Western Blot, Amplification, Polymerase Chain Reaction

    Effects of SOCS-box mutant on PYK2 expression, Tyr402 and ERK1/2 activations and cell migration . (a) Decreased PYK2 and ERK1/2 phosphorylations by SOCS-box mutant with the same PYK2 expression. A549 cells were vector- or the SOCS-box mutant-transfected for 48 h, and cell lysates were analysed by western blot using the indicated antibodies. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration suppressed by the SOCS-box mutant was not statistically significant. The values are means of three replicates.
    Figure Legend Snippet: Effects of SOCS-box mutant on PYK2 expression, Tyr402 and ERK1/2 activations and cell migration . (a) Decreased PYK2 and ERK1/2 phosphorylations by SOCS-box mutant with the same PYK2 expression. A549 cells were vector- or the SOCS-box mutant-transfected for 48 h, and cell lysates were analysed by western blot using the indicated antibodies. (b) PYK2 mRNA levels were unaffected regardless of transfection or not. The amplified PYK2 products were 629 bp in length. β-actin amplification demonstrated the consistency of PT-PCR. (c) Cell migration suppressed by the SOCS-box mutant was not statistically significant. The values are means of three replicates.

    Techniques Used: Mutagenesis, Expressing, Migration, Plasmid Preparation, Transfection, Western Blot, Amplification, Polymerase Chain Reaction

    33) Product Images from "Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation"

    Article Title: Pentabromopseudilin: a myosin V inhibitor suppresses TGF- β activity by recruiting the type II TGF- β receptor to lysosomal degradation

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    doi: 10.1080/14756366.2018.1465416

    PBrP treatment resulted in the internalisation and rapid degradation of T β RII. Serum-starved Mv1Lu (A) and A549 (B) cells were treated with increasing concentrations of PBrP (0.01–1 μ M) for 6 h and then stimulated with 20 or 100 pM of TGF- β for 30 min. Cells were analysed through Western blotting for T β RII protein levels. Similarly, Mv1Lu (C), A549 (D), HepG2 (E) and Clone 9 (F) cells growing in a low-serum medium were treated with 0.2 or 0.5 μ M of PBrP for 0, 1, 2, 4 and 6 h followed by 50 or 100 pM of TGF- β stimulation for 30 min. Cell lysates were analysed for T β RII, T β RI or β -actin protein expression through Western blotting. The show results are those of the same samples from Figure 2 (A to G) and share the same β -actin for load control. Panels (A to D) show the representative images and illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (% of untreated control); * p < .05, ** p < .01. Mv1Lu cells transiently expressing T β RII-flag were treated with 0.01–0.2 μ M of PBrP for 6 h (G) or 0.2 μ M of PBrP for 0, 1, 2, 4 and 6 h (H). Subsequently, the cells lysates were subjected to Western blot analysis using an antibody against the flag tag. The mock showed no signal, which suggested that the flag signal from the T β RII-flag was not an artefact. (I) PBrP reduces T β RII protein stability. Mv1Lu cells were treated with 0.5 μ M of PBrP or a relevant amount of DMSO for the indicated time in the presence or absence of CHX. The amounts of T β RII protein from the cell lysates were analysed through Western blotting using anti-T β RII and anti- β -actin (as a loading control) antibodies. The percentage of the control indicates the T β RII amount at each time point relative to the control. (J) PBrP reduced T β R-II abundance at the cell surface, as assessed by affinity purification of cell surface proteins using NeutrAvidin-conjugated beads and Western blot. Cells expressing T β R-II-flag were pretreated with 0.2 μ M PBrP for 0, 1, 2, 3 and 4 h in DMEM containing 0.2% of FBS at 37 °C followed by cell surface biotinylation. NeutrAvidin-precipitated proteins and total lysates were subjected to immunoblotting to detect T β R-II-flag. Changes of T β R-II abundance were represented as % of control (mean ± SD) from three independent experiments; * p < .05, ** p < .01. (K) HEK293 cells co-expressing T β RII-flag and Rab5-RFP were treated with 0.2 μ M of PBrP for 1 h, followed by fixation and permeabilisation. T β RII-flag localisation was visualised by immunofluorescence staining using the anti-flag antibody and Alexa Fluor 488-conjugated secondary antibodies. Bar: 10 μ m.
    Figure Legend Snippet: PBrP treatment resulted in the internalisation and rapid degradation of T β RII. Serum-starved Mv1Lu (A) and A549 (B) cells were treated with increasing concentrations of PBrP (0.01–1 μ M) for 6 h and then stimulated with 20 or 100 pM of TGF- β for 30 min. Cells were analysed through Western blotting for T β RII protein levels. Similarly, Mv1Lu (C), A549 (D), HepG2 (E) and Clone 9 (F) cells growing in a low-serum medium were treated with 0.2 or 0.5 μ M of PBrP for 0, 1, 2, 4 and 6 h followed by 50 or 100 pM of TGF- β stimulation for 30 min. Cell lysates were analysed for T β RII, T β RI or β -actin protein expression through Western blotting. The show results are those of the same samples from Figure 2 (A to G) and share the same β -actin for load control. Panels (A to D) show the representative images and illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (% of untreated control); * p < .05, ** p < .01. Mv1Lu cells transiently expressing T β RII-flag were treated with 0.01–0.2 μ M of PBrP for 6 h (G) or 0.2 μ M of PBrP for 0, 1, 2, 4 and 6 h (H). Subsequently, the cells lysates were subjected to Western blot analysis using an antibody against the flag tag. The mock showed no signal, which suggested that the flag signal from the T β RII-flag was not an artefact. (I) PBrP reduces T β RII protein stability. Mv1Lu cells were treated with 0.5 μ M of PBrP or a relevant amount of DMSO for the indicated time in the presence or absence of CHX. The amounts of T β RII protein from the cell lysates were analysed through Western blotting using anti-T β RII and anti- β -actin (as a loading control) antibodies. The percentage of the control indicates the T β RII amount at each time point relative to the control. (J) PBrP reduced T β R-II abundance at the cell surface, as assessed by affinity purification of cell surface proteins using NeutrAvidin-conjugated beads and Western blot. Cells expressing T β R-II-flag were pretreated with 0.2 μ M PBrP for 0, 1, 2, 3 and 4 h in DMEM containing 0.2% of FBS at 37 °C followed by cell surface biotinylation. NeutrAvidin-precipitated proteins and total lysates were subjected to immunoblotting to detect T β R-II-flag. Changes of T β R-II abundance were represented as % of control (mean ± SD) from three independent experiments; * p < .05, ** p < .01. (K) HEK293 cells co-expressing T β RII-flag and Rab5-RFP were treated with 0.2 μ M of PBrP for 1 h, followed by fixation and permeabilisation. T β RII-flag localisation was visualised by immunofluorescence staining using the anti-flag antibody and Alexa Fluor 488-conjugated secondary antibodies. Bar: 10 μ m.

    Techniques Used: Western Blot, Expressing, FLAG-tag, Affinity Purification, Immunofluorescence, Staining

    PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p
    Figure Legend Snippet: PBrP delays TGF- β -induced cell migration. Confluent monolayers of A549 cells in a 24-well cluster tissue culture plate were scratched and incubated at 37 °C for 20 h in DMEM containing 0.2% of FBS (Control; top). Cell motility was measured using a fully automatic microscope at 10× phase objective. Cell migration was observed by performing time-lapse microscopy and images of all four experimental conditions were captured simultaneously every 20 min. The red lines indicate the starting point of cell migration. Representative micrographs from three experiments are shown in panel (A). Right graphs illustrate of quantitative analyses of cell covering area (mean ± SD) from three independent experiments are shown (B) (% of TGF- β treatment alone); ** p

    Techniques Used: Migration, Incubation, Microscopy, Time-lapse Microscopy

    PBrP inhibits TGF- β -stimulated Smad2/3 phosphorylation and nuclear translocation. Mv1Lu (A), A549 (B) and HepG2 (C) cells were pretreated with increasing concentrations (0.01–1 μ M) of PBrP for 6 h followed by 30 min of TGF- β stimulation. Mv1Lu (D), A549 (E), HepG2 (F) and Clone 9 (G) cells were pretreated with 0.5 μ M of PBrP for 0.5, 1, 2, 4 and 6 h followed by 30 min of TGF- β stimulation. Cell lysates were subjected to Western blot analysis with monoclonal antibodies against p-Smad2/3 and Smad2/3. An equal amount of protein loading was verified by Smad2/3 and β -actin (images were duplicated from the experiments illustrated in ). (H) Nuclear translocation of Smad proteins was analysed by separating nuclear fractions after treating A549 cells with TGF- β or PBrP for 6 h. Smad2/3 and pSmad2/3 proteins were detected through Western blot analysis using antibodies against Smad2/3 and p-Smad2/3. Complete fractionation of nuclear proteins and equivalent loading were verified through Western blot analysis using antibodies against Lamin B. Panels (A to H) show the representative images and right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (compare with TGF- β treatment alone); * p < .05, ** p < .01. (I) Blockade of TGF- β -induced nuclear import of Smad2/3 by PBrP in A549 cells was determined through immunofluorescence. Cells were serum starved and pretreated with 0.5 μ M of PBrP for 6 h followed by 30 min of TGF- β stimulation, and processed for immunofluorescence using the anti-Smad2/3 antibody. Fluorescence was visualised using the Alexa Fluor 488-conjugated donkey anti-goat antibody and a ZeissObserver fluorescence microscope. DAPI was used as a counterstain. Each experiment was repeated three times. Bar: 20 μ m.
    Figure Legend Snippet: PBrP inhibits TGF- β -stimulated Smad2/3 phosphorylation and nuclear translocation. Mv1Lu (A), A549 (B) and HepG2 (C) cells were pretreated with increasing concentrations (0.01–1 μ M) of PBrP for 6 h followed by 30 min of TGF- β stimulation. Mv1Lu (D), A549 (E), HepG2 (F) and Clone 9 (G) cells were pretreated with 0.5 μ M of PBrP for 0.5, 1, 2, 4 and 6 h followed by 30 min of TGF- β stimulation. Cell lysates were subjected to Western blot analysis with monoclonal antibodies against p-Smad2/3 and Smad2/3. An equal amount of protein loading was verified by Smad2/3 and β -actin (images were duplicated from the experiments illustrated in ). (H) Nuclear translocation of Smad proteins was analysed by separating nuclear fractions after treating A549 cells with TGF- β or PBrP for 6 h. Smad2/3 and pSmad2/3 proteins were detected through Western blot analysis using antibodies against Smad2/3 and p-Smad2/3. Complete fractionation of nuclear proteins and equivalent loading were verified through Western blot analysis using antibodies against Lamin B. Panels (A to H) show the representative images and right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments (compare with TGF- β treatment alone); * p < .05, ** p < .01. (I) Blockade of TGF- β -induced nuclear import of Smad2/3 by PBrP in A549 cells was determined through immunofluorescence. Cells were serum starved and pretreated with 0.5 μ M of PBrP for 6 h followed by 30 min of TGF- β stimulation, and processed for immunofluorescence using the anti-Smad2/3 antibody. Fluorescence was visualised using the Alexa Fluor 488-conjugated donkey anti-goat antibody and a ZeissObserver fluorescence microscope. DAPI was used as a counterstain. Each experiment was repeated three times. Bar: 20 μ m.

    Techniques Used: Translocation Assay, Western Blot, Fractionation, Immunofluorescence, Fluorescence, Microscopy

    PBrP blocks TGF- β -induced transcriptional responses and EMT protein production. (A) Inhibition of PAI-1 gene promoter-luciferase (PAI-1-Luc) activity in MLECs through a dose response of PBrP and the T β RI kinase inhibitor SB-431542 (SB) in response to TGF- β . (B to E) Control A549 cells (top) or cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were fixed and permeabilised. (B) Cells were incubated with TRITC-phalloidin (red) and DAPI (blue) to visualise the actin cytoskeleton and the nuclei, respectively. To visualize ZO-1 (C), fibronectin (D) and N-cadherin (E), cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were stained with specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. Representative micrographs from three experiments are shown. Bar: 20 μ m. For Western blot analysis, A549 (F) and HepG2 (G) cells were treated with increasing doses of PBrP in DMEM containing 0.1% of FBS for 6 h and continually stimulated with or without 100 pM of TGF- β for 48 h. (H) A549 cells were pretreated with 0.5 μ M of PBrP for 6 h and continually stimulated with 0, 10 or 100 pM of TGF- β for 42 h. Cell lysates were then analysed through Western blotting with desired antibodies as indicated. The representative images (F to H) and right graphs illustrate of quantitative analyses of ECL (mean ± SD) from three independent experiments are shown (% of TGF- β treatment alone); * p < .05, ** p < .01.
    Figure Legend Snippet: PBrP blocks TGF- β -induced transcriptional responses and EMT protein production. (A) Inhibition of PAI-1 gene promoter-luciferase (PAI-1-Luc) activity in MLECs through a dose response of PBrP and the T β RI kinase inhibitor SB-431542 (SB) in response to TGF- β . (B to E) Control A549 cells (top) or cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were fixed and permeabilised. (B) Cells were incubated with TRITC-phalloidin (red) and DAPI (blue) to visualise the actin cytoskeleton and the nuclei, respectively. To visualize ZO-1 (C), fibronectin (D) and N-cadherin (E), cells pretreated with 0.5 μ M of PBrP for 6 h followed by 42 h of TGF- β stimulation were stained with specific antibodies and Alexa Fluor 488-conjugated secondary antibodies. Representative micrographs from three experiments are shown. Bar: 20 μ m. For Western blot analysis, A549 (F) and HepG2 (G) cells were treated with increasing doses of PBrP in DMEM containing 0.1% of FBS for 6 h and continually stimulated with or without 100 pM of TGF- β for 48 h. (H) A549 cells were pretreated with 0.5 μ M of PBrP for 6 h and continually stimulated with 0, 10 or 100 pM of TGF- β for 42 h. Cell lysates were then analysed through Western blotting with desired antibodies as indicated. The representative images (F to H) and right graphs illustrate of quantitative analyses of ECL (mean ± SD) from three independent experiments are shown (% of TGF- β treatment alone); * p < .05, ** p < .01.

    Techniques Used: Inhibition, Luciferase, Activity Assay, Incubation, Staining, Western Blot

    MyoVa depletion enhances T β RII turnover and mitigates TGF- β /Smad signalling. A549 cells were infected with the lentivirus carrying control (Ctr shRNA) or MyoVa shRNA constructs (MyoVa shRNA#1 and MyoVa shRNA#2). Three days post-infection, total protein was extracted and subjected to Western blot using anti-MyoVa, anti-T β RII or anti- β -actin antibodies. (A) Two MyoVa shRNAs abolish MyoVa protein production and reduced the T β RII protein levels. (B) Effects of MyoVa on TGF- β -induced Smad2/3 phosphorylation. A549 cells harbouring MyoVa shRNA#1 or control shRNA were stimulated with 5, 10, 20, 50 and 100 pM of TGF- β for 30 min. The amount of pSmad2/3 obtained from the lysates of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using anti-pSmad2/3, anti-Smad2/3 and anti- β -actin were used as loading control. (C) MyoVa depletion inhibits TGF- β -induced fibronectin, PAI-1, and N-cadherin expression. Control and MyoVa-silenced A549 cells were treated with 5, 10, 20, 50 and 100 pM of TGF- β for 48 h. The protein abundance of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using appropriate antibodies. Right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments; ** p
    Figure Legend Snippet: MyoVa depletion enhances T β RII turnover and mitigates TGF- β /Smad signalling. A549 cells were infected with the lentivirus carrying control (Ctr shRNA) or MyoVa shRNA constructs (MyoVa shRNA#1 and MyoVa shRNA#2). Three days post-infection, total protein was extracted and subjected to Western blot using anti-MyoVa, anti-T β RII or anti- β -actin antibodies. (A) Two MyoVa shRNAs abolish MyoVa protein production and reduced the T β RII protein levels. (B) Effects of MyoVa on TGF- β -induced Smad2/3 phosphorylation. A549 cells harbouring MyoVa shRNA#1 or control shRNA were stimulated with 5, 10, 20, 50 and 100 pM of TGF- β for 30 min. The amount of pSmad2/3 obtained from the lysates of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using anti-pSmad2/3, anti-Smad2/3 and anti- β -actin were used as loading control. (C) MyoVa depletion inhibits TGF- β -induced fibronectin, PAI-1, and N-cadherin expression. Control and MyoVa-silenced A549 cells were treated with 5, 10, 20, 50 and 100 pM of TGF- β for 48 h. The protein abundance of cells in the absence (Lanes 8–12) or presence (Lanes 1–7) of MyoVa was analysed through Western blotting using appropriate antibodies. Right graphs illustrate quantitative analyses of ECL (mean ± SD) from at least three independent experiments; ** p

    Techniques Used: Infection, shRNA, Construct, Western Blot, Expressing

    PBrP induces T β RII degradation in lipid rafts. A549 cells or shRNA-silenced A549 cells were left untreated or incubated with 0.5 μ section. Thirty microgram of protein from each fraction was subjected to SDS-PAGE and transferred onto PVDF membranes, and blotted with anti-T β RII, anti-T β RI and EGFR antibodies. Protein samples from various treatments in acrylamide gel strips were synchronously electrotransferred onto the same PVDF membrane and then simultaneously proceeded for Western blotting; therefore, the signals of distinct proteins from various treatments were mutually comparable. (A) PBrP treatment reduced T β RII predominately in raft fractions within 3 h. (B) MyoVa depletion reduced T β RII in raft and non-raft fractions without altering the protein abundance and subcellular compartmentation of T β RI, EGFR or raft marker flotillin-2.
    Figure Legend Snippet: PBrP induces T β RII degradation in lipid rafts. A549 cells or shRNA-silenced A549 cells were left untreated or incubated with 0.5 μ section. Thirty microgram of protein from each fraction was subjected to SDS-PAGE and transferred onto PVDF membranes, and blotted with anti-T β RII, anti-T β RI and EGFR antibodies. Protein samples from various treatments in acrylamide gel strips were synchronously electrotransferred onto the same PVDF membrane and then simultaneously proceeded for Western blotting; therefore, the signals of distinct proteins from various treatments were mutually comparable. (A) PBrP treatment reduced T β RII predominately in raft fractions within 3 h. (B) MyoVa depletion reduced T β RII in raft and non-raft fractions without altering the protein abundance and subcellular compartmentation of T β RI, EGFR or raft marker flotillin-2.

    Techniques Used: shRNA, Incubation, SDS Page, Acrylamide Gel Assay, Western Blot, Marker

    34) Product Images from "SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion"

    Article Title: SpyAD, a Moonlighting Protein of Group A Streptococcus Contributing to Bacterial Division and Host Cell Adhesion

    Journal: Infection and Immunity

    doi: 10.1128/IAI.00064-14

    Biochemical characterization of SpyAD binding to epithelial cells. (A) A549 cells were treated with either medium alone (black bar) or with the indicated concentrations of pronase (white bars) or trypsin (gray bars), prior to incubation with recombinant
    Figure Legend Snippet: Biochemical characterization of SpyAD binding to epithelial cells. (A) A549 cells were treated with either medium alone (black bar) or with the indicated concentrations of pronase (white bars) or trypsin (gray bars), prior to incubation with recombinant

    Techniques Used: Binding Assay, Incubation, Recombinant

    Recombinant SpyAD binding to human epithelial cells. (A) Saturation curve of SpyAD binding to A549 cells. Cells were incubated for 1 h at 4°C with increasing concentrations of recombinant SpyAD from 0.08 to 7.28 μM ( x axis). Binding was
    Figure Legend Snippet: Recombinant SpyAD binding to human epithelial cells. (A) Saturation curve of SpyAD binding to A549 cells. Cells were incubated for 1 h at 4°C with increasing concentrations of recombinant SpyAD from 0.08 to 7.28 μM ( x axis). Binding was

    Techniques Used: Recombinant, Binding Assay, Incubation

    35) Product Images from "Enhanced Anticancer Activity of Gemcitabine in Combination with Noscapine via Antiangiogenic and Apoptotic Pathway against Non-Small Cell Lung Cancer"

    Article Title: Enhanced Anticancer Activity of Gemcitabine in Combination with Noscapine via Antiangiogenic and Apoptotic Pathway against Non-Small Cell Lung Cancer

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0027394

    Tube formation assay with HUVEC cells after 6 h (A); quantification of branching points (B); micrographs of cells stained with TUNEL after 72 h (C) and quantitation of apoptotic H460 (D) and A549 cells from TUNEL assay (E). For tube formation assay, HUVEC cells were incubated with Nos (30 µM), Gem (0.4 µg/ml) and NGC on polymerized Matrigel at 37°C. After 6 h, tube formation by endothelial cells was photographed and the capillary tube branch point formation were quantified (n = 3). For TUNEL assay, H460 cells were treated with Gem 0.4 µg/ml, Nos 30 µM, and, NGC and A549 cells were treated with Gem 0.3 µg/ml, Nos 50 µM, and, NGC. Control cells were untreated. Micron bar = 10 µm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean + SD (n = 6).
    Figure Legend Snippet: Tube formation assay with HUVEC cells after 6 h (A); quantification of branching points (B); micrographs of cells stained with TUNEL after 72 h (C) and quantitation of apoptotic H460 (D) and A549 cells from TUNEL assay (E). For tube formation assay, HUVEC cells were incubated with Nos (30 µM), Gem (0.4 µg/ml) and NGC on polymerized Matrigel at 37°C. After 6 h, tube formation by endothelial cells was photographed and the capillary tube branch point formation were quantified (n = 3). For TUNEL assay, H460 cells were treated with Gem 0.4 µg/ml, Nos 30 µM, and, NGC and A549 cells were treated with Gem 0.3 µg/ml, Nos 50 µM, and, NGC. Control cells were untreated. Micron bar = 10 µm. Cells were quantitated by counting 100 cells from 6 random microscopic fields. Data are expressed as mean + SD (n = 6).

    Techniques Used: Tube Formation Assay, Staining, TUNEL Assay, Quantitation Assay, Incubation

    36) Product Images from "Long non-coding RNA SNHG20 promotes non-small cell lung cancer cell proliferation and migration by epigenetically silencing of P21 expression"

    Article Title: Long non-coding RNA SNHG20 promotes non-small cell lung cancer cell proliferation and migration by epigenetically silencing of P21 expression

    Journal: Cell Death & Disease

    doi: 10.1038/cddis.2017.484

    Effect of SNHG20 on NSCLC tumorigenesis in vivo . ( a and b ) Stable SNHG20-knockdown A549 cells were used for in vivo assays. Growth curves of tumors from two groups of mice injected with A549 cells stably transfected with sh-SNHG20 or empty vectors are shown. Tumor volumes were calculated every 3 days. ( c ) Tumor weights from the two groups are represented. ( d ) qRT-PCR was performed to detect the average expression of SNHG20 in xenograft tumors ( n =4). ( e ) Tumors developed from sh-SNHG20-transfected A549 cells showed lower Ki-67 protein levels compared with tumors developed from control cells. Upper: hematoxylin and eosin staining; lower: immunohistochemical staining. * P
    Figure Legend Snippet: Effect of SNHG20 on NSCLC tumorigenesis in vivo . ( a and b ) Stable SNHG20-knockdown A549 cells were used for in vivo assays. Growth curves of tumors from two groups of mice injected with A549 cells stably transfected with sh-SNHG20 or empty vectors are shown. Tumor volumes were calculated every 3 days. ( c ) Tumor weights from the two groups are represented. ( d ) qRT-PCR was performed to detect the average expression of SNHG20 in xenograft tumors ( n =4). ( e ) Tumors developed from sh-SNHG20-transfected A549 cells showed lower Ki-67 protein levels compared with tumors developed from control cells. Upper: hematoxylin and eosin staining; lower: immunohistochemical staining. * P

    Techniques Used: In Vivo, Mouse Assay, Injection, Stable Transfection, Transfection, Quantitative RT-PCR, Expressing, Staining, Immunohistochemistry

    SNHG20 interacted with EZH2, and regulated P21 expression in NSCLC cells. ( a ) SNHG20 expression levels in the cytoplasm or nucleus of SPC-A1 and A549 cells were detected by qRT-PCR. GAPDH was used as a cytosol marker and U1 was used as a nuclear marker. ( b ) RNA immunoprecipitation experiments were performed in SPC-A1 and A549 cells and the coprecipitated RNA was subjected to qRT-PCR for SNHG20. SNHG20 RNA expression levels are presented as fold enrichment in EZH2 and SUZ12 immunoprecipitates relative to that of IgG. ( c ) The expression of p15, p16, P21 and p27 was determined using qRT-PCR after knockdown of SNHG20. ( d ) Western blot analysis was conducted to detect the level of P21 protein in SPC-A1 and A549 cells transfected with si-SNHG20. ( e – g ) qRT-PCR and western blot assays were used to detect EZH2 and P21 mRNA and protein levels in SPC-A1 and A549 cells transfected with si-EZH2. ( h ) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding to the P21 promoter in SPC-A1 and A549 cells treated with si-SNHG20 (48 h) or si-NC; IgG as a negative control. ( i ) Analysis of the relationship between SNHG20 expression and P21 mRNA levels (DCt value) in NSCLC patient tissue from GEO. Values are shown as the mean (S.D.) from three independent experiments. * P
    Figure Legend Snippet: SNHG20 interacted with EZH2, and regulated P21 expression in NSCLC cells. ( a ) SNHG20 expression levels in the cytoplasm or nucleus of SPC-A1 and A549 cells were detected by qRT-PCR. GAPDH was used as a cytosol marker and U1 was used as a nuclear marker. ( b ) RNA immunoprecipitation experiments were performed in SPC-A1 and A549 cells and the coprecipitated RNA was subjected to qRT-PCR for SNHG20. SNHG20 RNA expression levels are presented as fold enrichment in EZH2 and SUZ12 immunoprecipitates relative to that of IgG. ( c ) The expression of p15, p16, P21 and p27 was determined using qRT-PCR after knockdown of SNHG20. ( d ) Western blot analysis was conducted to detect the level of P21 protein in SPC-A1 and A549 cells transfected with si-SNHG20. ( e – g ) qRT-PCR and western blot assays were used to detect EZH2 and P21 mRNA and protein levels in SPC-A1 and A549 cells transfected with si-EZH2. ( h ) ChIP-qRT-PCR of EZH2 occupancy and H3K27me3 binding to the P21 promoter in SPC-A1 and A549 cells treated with si-SNHG20 (48 h) or si-NC; IgG as a negative control. ( i ) Analysis of the relationship between SNHG20 expression and P21 mRNA levels (DCt value) in NSCLC patient tissue from GEO. Values are shown as the mean (S.D.) from three independent experiments. * P

    Techniques Used: Expressing, Quantitative RT-PCR, Marker, Immunoprecipitation, RNA Expression, Western Blot, Transfection, Chromatin Immunoprecipitation, Binding Assay, Negative Control

    Effects of SNHG20 on NSCLC cell proliferation in vitro . ( a ) qRT-PCR analysis of SNHG20 expression in normal human bronchial epithelial cells (16HBE) and NSCLC cells. ( b ) qRT-PCR analysis of SNHG20 expression in control (scrambled), si-SNHG20 1#, si-SNHG20 2# and si-SNHG20 3# treated NSCLC cells. ( c ) MTT assays were used to determine the viability of si-SNHG20-transfected SPC-A1 and A549 cells. ( d ) Colony formation assays were performed to determine the proliferation of si-SNHG20-transfected SPC-A1 and A549 cells. Colonies were counted and captured. ( e ) EdU staining assays were performed to determine the growth of si-SNHG20 transfected SPC-A1 and A549 cells. Representative images and data are based on three independent experiments. Data are presented as the mean±S.E.M. * P
    Figure Legend Snippet: Effects of SNHG20 on NSCLC cell proliferation in vitro . ( a ) qRT-PCR analysis of SNHG20 expression in normal human bronchial epithelial cells (16HBE) and NSCLC cells. ( b ) qRT-PCR analysis of SNHG20 expression in control (scrambled), si-SNHG20 1#, si-SNHG20 2# and si-SNHG20 3# treated NSCLC cells. ( c ) MTT assays were used to determine the viability of si-SNHG20-transfected SPC-A1 and A549 cells. ( d ) Colony formation assays were performed to determine the proliferation of si-SNHG20-transfected SPC-A1 and A549 cells. Colonies were counted and captured. ( e ) EdU staining assays were performed to determine the growth of si-SNHG20 transfected SPC-A1 and A549 cells. Representative images and data are based on three independent experiments. Data are presented as the mean±S.E.M. * P

    Techniques Used: In Vitro, Quantitative RT-PCR, Expressing, MTT Assay, Transfection, Staining

    Effect of SNHG20 on NSCLC cell apoptosis, cell cycle and migration in vitro . ( a and b ) SPC-A1 and A549 cells were stained and analyzed by flow cytometry. LR, early apoptotic cells; UR, terminal apoptotic cells. ( c and d ) Flow cytometry showing significant decreases or increases in the proportion of cells in the S or G1 phase, respectively, when SNHG20 was silenced in SPC-A1 and A549 cells. ( e ) Transwell assays were performed to investigate changes in migratory abilities of SPC-A1 and A549 cells. Data are presented as the mean±S.E.M. All experiments were performed in triplicate with three technical replicates. * P
    Figure Legend Snippet: Effect of SNHG20 on NSCLC cell apoptosis, cell cycle and migration in vitro . ( a and b ) SPC-A1 and A549 cells were stained and analyzed by flow cytometry. LR, early apoptotic cells; UR, terminal apoptotic cells. ( c and d ) Flow cytometry showing significant decreases or increases in the proportion of cells in the S or G1 phase, respectively, when SNHG20 was silenced in SPC-A1 and A549 cells. ( e ) Transwell assays were performed to investigate changes in migratory abilities of SPC-A1 and A549 cells. Data are presented as the mean±S.E.M. All experiments were performed in triplicate with three technical replicates. * P

    Techniques Used: Migration, In Vitro, Staining, Flow Cytometry, Cytometry

    Downregulation of P21 promotes A549 cell proliferation and SNHG20 negatively regulates expression of P21. ( a ) A549 cells were transfected with si-P21. ( b and c ) MTT and colony formation assays were used to determine the cell viability of si-P21-transfected A549 cells. Experiments were performed in triplicate. ( d ) Flow cytometry assays were performed to analyze cell-cycle progression of A549 cells transfected with si-P21. The bar chart represents the percentage of cells in G0–G1, S or G2–M phases, as indicated. ( e and f ) MTT and colony formation assays were used to determine the cell viability of si-SNHG20 and si-P21 co-transfected A549 cells. ( g ) P21 expression was analyzed by western blotting. Values are shown as the mean (S.D.) from three independent experiments. * P
    Figure Legend Snippet: Downregulation of P21 promotes A549 cell proliferation and SNHG20 negatively regulates expression of P21. ( a ) A549 cells were transfected with si-P21. ( b and c ) MTT and colony formation assays were used to determine the cell viability of si-P21-transfected A549 cells. Experiments were performed in triplicate. ( d ) Flow cytometry assays were performed to analyze cell-cycle progression of A549 cells transfected with si-P21. The bar chart represents the percentage of cells in G0–G1, S or G2–M phases, as indicated. ( e and f ) MTT and colony formation assays were used to determine the cell viability of si-SNHG20 and si-P21 co-transfected A549 cells. ( g ) P21 expression was analyzed by western blotting. Values are shown as the mean (S.D.) from three independent experiments. * P

    Techniques Used: Expressing, Transfection, MTT Assay, Flow Cytometry, Cytometry, Western Blot

    37) Product Images from "The Step Further to Understand the Role of Cytosolic Phospholipase A2 Alpha and Group X Secretory Phospholipase A2 in Allergic Inflammation: Pilot Study"

    Article Title: The Step Further to Understand the Role of Cytosolic Phospholipase A2 Alpha and Group X Secretory Phospholipase A2 in Allergic Inflammation: Pilot Study

    Journal: BioMed Research International

    doi: 10.1155/2014/670814

    Morphological changes of A549 cells caused by rDerp1.
    Figure Legend Snippet: Morphological changes of A549 cells caused by rDerp1.

    Techniques Used:

    Relative cPLA 2 α protein expression in A549 cells stimulated with rFel d1. A549 were stimulated with rFel d1 (5 μ g/mL) for 24 hours. Control represents cells treated with the vehicle. The immunoblot is representative of three independent experiments in A549 cells, each showing similar results. The bar graph shows the densitometry results. Data are presented as the fold change compared with the vehicle-treated cells. Data represent the mean ± SE from at least three independent experiments. ∗ P
    Figure Legend Snippet: Relative cPLA 2 α protein expression in A549 cells stimulated with rFel d1. A549 were stimulated with rFel d1 (5 μ g/mL) for 24 hours. Control represents cells treated with the vehicle. The immunoblot is representative of three independent experiments in A549 cells, each showing similar results. The bar graph shows the densitometry results. Data are presented as the fold change compared with the vehicle-treated cells. Data represent the mean ± SE from at least three independent experiments. ∗ P

    Techniques Used: Expressing

    38) Product Images from "MiR‐646 suppresses proliferation and metastasis of non‐small cell lung cancer by repressing FGF2 and CCND2. MiR‐646 suppresses proliferation and metastasis of non‐small cell lung cancer by repressing FGF2 and CCND2"

    Article Title: MiR‐646 suppresses proliferation and metastasis of non‐small cell lung cancer by repressing FGF2 and CCND2. MiR‐646 suppresses proliferation and metastasis of non‐small cell lung cancer by repressing FGF2 and CCND2

    Journal: Cancer Medicine

    doi: 10.1002/cam4.3062

    MiR‐646 is downregulated in NSCLC tissues and cell lines. (A) qRT‐PCR assay was performed to quantify miR‐646 expression in 49 pairs of NSCLC tissues and adjacent noncancerous lung tissues. (B) miR‐646 expression in metastatic and nonmetastatic NSCLCs. (C) miR‐646 expression in different clinical stages of NSCLCs. (D) The endogenous levels of miR‐646 in four NSCLC cell lines (A549, SPC‐A1, H1299, and PC‐9) and the normal human bronchial epithelial cell line (BEAS‐2B). (E) qRT‐PCR analysis of miR‐646 expression in A549 and SPC‐A1 cells transfected with miR‐646 mimic or NC mimic. (F) qRT‐PCR analysis of miR‐646 expression in A549 cells transfected with miR‐646 inhibitor or NC inhibitor. * P
    Figure Legend Snippet: MiR‐646 is downregulated in NSCLC tissues and cell lines. (A) qRT‐PCR assay was performed to quantify miR‐646 expression in 49 pairs of NSCLC tissues and adjacent noncancerous lung tissues. (B) miR‐646 expression in metastatic and nonmetastatic NSCLCs. (C) miR‐646 expression in different clinical stages of NSCLCs. (D) The endogenous levels of miR‐646 in four NSCLC cell lines (A549, SPC‐A1, H1299, and PC‐9) and the normal human bronchial epithelial cell line (BEAS‐2B). (E) qRT‐PCR analysis of miR‐646 expression in A549 and SPC‐A1 cells transfected with miR‐646 mimic or NC mimic. (F) qRT‐PCR analysis of miR‐646 expression in A549 cells transfected with miR‐646 inhibitor or NC inhibitor. * P

    Techniques Used: Quantitative RT-PCR, Expressing, Transfection

    MiR‐646 suppresses the proliferation and invasion of NSCLC cells in vitro. (A) A549 and SPC‐A1 cells were transfected with miR‐646 mimic or its inhibitor, and cell proliferation was evaluated by CCK‐8 assay. (B) Representative micrographs (left) and quantification (right) of BrdU‐incorporating cells transfected with miR‐646 mimic or its inhibitor. (C) Colony formation assay. Representative images (left) and quantification (right) of colony formation of the transfected cells. (D) Representative images (left) and quantitation (right) of the Transwell assay. * P
    Figure Legend Snippet: MiR‐646 suppresses the proliferation and invasion of NSCLC cells in vitro. (A) A549 and SPC‐A1 cells were transfected with miR‐646 mimic or its inhibitor, and cell proliferation was evaluated by CCK‐8 assay. (B) Representative micrographs (left) and quantification (right) of BrdU‐incorporating cells transfected with miR‐646 mimic or its inhibitor. (C) Colony formation assay. Representative images (left) and quantification (right) of colony formation of the transfected cells. (D) Representative images (left) and quantitation (right) of the Transwell assay. * P

    Techniques Used: In Vitro, Transfection, CCK-8 Assay, Colony Assay, Quantitation Assay, Transwell Assay

    Suppression of FGF2 and CCND2 is key to the tumor‐repressive function of miR‐646. (A) Western blotting analyses of FGF2, CCND2, E‐cadherin, and vimentin in A549 cells transfected with FGF2 siRNA (siFGF2) or CCND2 siRNA (siCCND2). (B) CCK‐8 was used to determine the cell proliferation in different groups. (C) Quantification of BrdU‐incorporating cells. (D) Quantification of invading cells by Transwell assay. (E) Western blotting analyses of FGF2, CCND2, E‐cadherin, and vimentin in A549/LV‐miR‐646 cells after overexpression of FGF2 or CCND2. CCK‐8 (F), BrdU incorporation (G) and Transwell invasion (H) assays were performed. * P
    Figure Legend Snippet: Suppression of FGF2 and CCND2 is key to the tumor‐repressive function of miR‐646. (A) Western blotting analyses of FGF2, CCND2, E‐cadherin, and vimentin in A549 cells transfected with FGF2 siRNA (siFGF2) or CCND2 siRNA (siCCND2). (B) CCK‐8 was used to determine the cell proliferation in different groups. (C) Quantification of BrdU‐incorporating cells. (D) Quantification of invading cells by Transwell assay. (E) Western blotting analyses of FGF2, CCND2, E‐cadherin, and vimentin in A549/LV‐miR‐646 cells after overexpression of FGF2 or CCND2. CCK‐8 (F), BrdU incorporation (G) and Transwell invasion (H) assays were performed. * P

    Techniques Used: Western Blot, Transfection, CCK-8 Assay, Transwell Assay, Over Expression, BrdU Incorporation Assay

    MiR‐646 suppresses EMT process in NSCLC. (A) Western blotting analysis of E‐cadherin and vimentin expression in A549 and SPC‐A1 cells with miR‐646 mimic or inhibitor transfection. (B) Immunofluorescence staining of E‐cadherin and vimentin in A549 and SPC‐A1 cells with miR‐646 mimic transfection. (C) Immunohistochemical staining for E‐cadherin and vimentin in subcutaneous tumors formed with A549 carrying LV‐miR‐646 or LV‐NC. * P
    Figure Legend Snippet: MiR‐646 suppresses EMT process in NSCLC. (A) Western blotting analysis of E‐cadherin and vimentin expression in A549 and SPC‐A1 cells with miR‐646 mimic or inhibitor transfection. (B) Immunofluorescence staining of E‐cadherin and vimentin in A549 and SPC‐A1 cells with miR‐646 mimic transfection. (C) Immunohistochemical staining for E‐cadherin and vimentin in subcutaneous tumors formed with A549 carrying LV‐miR‐646 or LV‐NC. * P

    Techniques Used: Western Blot, Expressing, Transfection, Immunofluorescence, Staining, Immunohistochemistry

    MiR‐646 attenuated tumor growth and metastasis in vivo. (A) qRT‐PCR analysis of miR‐646 expression in A549 cells infected with miR‐646 overexpression or control lentivirus. (B) Flank tumors were established in nude mice after injection with miR‐646 overexpression or control cells. The mice were killed 4 weeks after flank injection and representative tumors were shown. (C) Growth curves of xenograft tumors. (D) Tumor weight. (E) Representative images of lungs isolated from mice that received tail vein injection of miR‐646 overexpression or control cells. (F) Representative images of HE staining of lungs. (G) The numbers of pulmonary metastatic nodules in the lungs. * P
    Figure Legend Snippet: MiR‐646 attenuated tumor growth and metastasis in vivo. (A) qRT‐PCR analysis of miR‐646 expression in A549 cells infected with miR‐646 overexpression or control lentivirus. (B) Flank tumors were established in nude mice after injection with miR‐646 overexpression or control cells. The mice were killed 4 weeks after flank injection and representative tumors were shown. (C) Growth curves of xenograft tumors. (D) Tumor weight. (E) Representative images of lungs isolated from mice that received tail vein injection of miR‐646 overexpression or control cells. (F) Representative images of HE staining of lungs. (G) The numbers of pulmonary metastatic nodules in the lungs. * P

    Techniques Used: In Vivo, Quantitative RT-PCR, Expressing, Infection, Over Expression, Mouse Assay, Injection, Isolation, Staining

    FGF2 and CCND2 were direct targets through which miR‐646 regulated proliferation and invasion of NSCLC cells. (A) Predicted binding sites of miR‐646 in the 3′UTR of FGF2 and CCND2 . (B) The expression levels of FGF2 and CCND2 mRNA and protein were detected by qRT‐PCR and Western blotting in A549 and SPC‐A1 cells after transfection of miR‐646 mimic or NC mimic. (C) Sequence of miR‐646‐mut. (D) Luciferase activity was analyzed in A549 and SPC‐A1 cells co‐transfected with miR‐646 mimic or miR‐646‐mut mimic with FGF2‐3'UTR or CCND2‐3'UTR reporters. (E,F)The average expression level of FGF2 and CCND2 in NSCLC tissues and adjacent noncancerous lung tissues. (G,H) Pearson's correlation analyses between miR‐646 expression and the expression of FGF2 and CCND2 in NSCLC tissues. * P
    Figure Legend Snippet: FGF2 and CCND2 were direct targets through which miR‐646 regulated proliferation and invasion of NSCLC cells. (A) Predicted binding sites of miR‐646 in the 3′UTR of FGF2 and CCND2 . (B) The expression levels of FGF2 and CCND2 mRNA and protein were detected by qRT‐PCR and Western blotting in A549 and SPC‐A1 cells after transfection of miR‐646 mimic or NC mimic. (C) Sequence of miR‐646‐mut. (D) Luciferase activity was analyzed in A549 and SPC‐A1 cells co‐transfected with miR‐646 mimic or miR‐646‐mut mimic with FGF2‐3'UTR or CCND2‐3'UTR reporters. (E,F)The average expression level of FGF2 and CCND2 in NSCLC tissues and adjacent noncancerous lung tissues. (G,H) Pearson's correlation analyses between miR‐646 expression and the expression of FGF2 and CCND2 in NSCLC tissues. * P

    Techniques Used: Binding Assay, Expressing, Quantitative RT-PCR, Western Blot, Transfection, Sequencing, Luciferase, Activity Assay

    39) Product Images from "2-aminopurine suppresses the TGF-β1-induced epithelial–mesenchymal transition and attenuates bleomycin-induced pulmonary fibrosis"

    Article Title: 2-aminopurine suppresses the TGF-β1-induced epithelial–mesenchymal transition and attenuates bleomycin-induced pulmonary fibrosis

    Journal: Cell Death Discovery

    doi: 10.1038/s41420-017-0016-3

    Identification of compounds that regulate the TGF-β1-induced EMT. a A549 cells were incubated with TGF-β1 (5 ng/mL) for 48 h, and the mean fluorescence intensity was determined by cellomics analysis. ** P
    Figure Legend Snippet: Identification of compounds that regulate the TGF-β1-induced EMT. a A549 cells were incubated with TGF-β1 (5 ng/mL) for 48 h, and the mean fluorescence intensity was determined by cellomics analysis. ** P

    Techniques Used: Incubation, Fluorescence

    Effect of 2-AP on expression of the epithelial marker E-cadherin and the mesenchymal markers fibronectin and vimentin. a A549 cells were treated with 2-AP and TGF-β1 as Fig. 1c , and the mean fluorescence intensity of E-cadherin was determined. b A549 cells were treated with DMSO (control), 5 ng/mL of TGF-β1, 5 ng/mL of TGF-β1 + 0.5 µM of 2-AP, or 5 ng/mL of TGF-β1 + 1 µM of 2-AP for 24 h. Results of mRNA expression analysis showed between-group differences for E-cadherin, fibronectin, and vimentin. Experiments were repeated at least in triplicate ( n = 3). * P
    Figure Legend Snippet: Effect of 2-AP on expression of the epithelial marker E-cadherin and the mesenchymal markers fibronectin and vimentin. a A549 cells were treated with 2-AP and TGF-β1 as Fig. 1c , and the mean fluorescence intensity of E-cadherin was determined. b A549 cells were treated with DMSO (control), 5 ng/mL of TGF-β1, 5 ng/mL of TGF-β1 + 0.5 µM of 2-AP, or 5 ng/mL of TGF-β1 + 1 µM of 2-AP for 24 h. Results of mRNA expression analysis showed between-group differences for E-cadherin, fibronectin, and vimentin. Experiments were repeated at least in triplicate ( n = 3). * P

    Techniques Used: Expressing, Marker, Fluorescence

    40) Product Images from "Temporal activation of NF-?B regulates an interferon-independent innate antiviral response against cytoplasmic RNA viruses"

    Article Title: Temporal activation of NF-?B regulates an interferon-independent innate antiviral response against cytoplasmic RNA viruses

    Journal: Proceedings of the National Academy of Sciences of the United States of America

    doi: 10.1073/pnas.1832775100

    Effect of TNF-α and IL-1β pretreatment on NF-κB-dependent restriction of RSV replication and cytopathogenicity. ( A ) Plaque assay analysis of medium supernatants from A549 cells pretreated with 20 ng/ml TNF-α or IL-1β for 8–16 h before RSV infection. ( B ) Plaque assay analysis of culture supernatants from IκB-SR or GFP expressing A549 cells, pretreated with either TNF-α or IL-1β for 16 h before RSV infection. ( C ) Expression of transfected NF-κB-Luc in A549 cells treated with either TNF-α or IL-1β (20 ng/ml for 2 h) in the presence of IκB-SR or control GFP. ( D ) Plaque assay analysis of medium supernatants from A549 cells pretreated with 20 ng/ml TNF-α or IL-1β for 24 h before HPIV-3 infection.
    Figure Legend Snippet: Effect of TNF-α and IL-1β pretreatment on NF-κB-dependent restriction of RSV replication and cytopathogenicity. ( A ) Plaque assay analysis of medium supernatants from A549 cells pretreated with 20 ng/ml TNF-α or IL-1β for 8–16 h before RSV infection. ( B ) Plaque assay analysis of culture supernatants from IκB-SR or GFP expressing A549 cells, pretreated with either TNF-α or IL-1β for 16 h before RSV infection. ( C ) Expression of transfected NF-κB-Luc in A549 cells treated with either TNF-α or IL-1β (20 ng/ml for 2 h) in the presence of IκB-SR or control GFP. ( D ) Plaque assay analysis of medium supernatants from A549 cells pretreated with 20 ng/ml TNF-α or IL-1β for 24 h before HPIV-3 infection.

    Techniques Used: Plaque Assay, Infection, Expressing, Transfection

    The noninvolvement of IFN and/or soluble secretory factor(s) in mediating the NF-κB-dependent antiviral response elicited by TNF-α and IL-1β. Plaque assay analysis of medium supernatants from A549 cells infected with RSV after a 16-h pretreatment of A549 cells with medium obtained from TNF-α-( A ) or IL-1β-( B ) treated (8 or 16 h) A549 cells in the presence of control antibody or anti-TNF-α ( A ) or anti-IL-1β ( B ) neutralizing antibodies. ( C ) Plaque assay analysis using medium supernatants from WT or STAT-1 – / – cells untreated or pretreated with TNF-α (20 ng/ml for 16 h) before infection with RSV. The percent infection indicated was calculated based on a ratio of number of plaques obtained in the presence of TNF-α over the number obtained from untreated cells.
    Figure Legend Snippet: The noninvolvement of IFN and/or soluble secretory factor(s) in mediating the NF-κB-dependent antiviral response elicited by TNF-α and IL-1β. Plaque assay analysis of medium supernatants from A549 cells infected with RSV after a 16-h pretreatment of A549 cells with medium obtained from TNF-α-( A ) or IL-1β-( B ) treated (8 or 16 h) A549 cells in the presence of control antibody or anti-TNF-α ( A ) or anti-IL-1β ( B ) neutralizing antibodies. ( C ) Plaque assay analysis using medium supernatants from WT or STAT-1 – / – cells untreated or pretreated with TNF-α (20 ng/ml for 16 h) before infection with RSV. The percent infection indicated was calculated based on a ratio of number of plaques obtained in the presence of TNF-α over the number obtained from untreated cells.

    Techniques Used: Plaque Assay, Infection

    Differential requirement of MyD88 for NF-κB induction by HPIV-3 and RSV. ( A ) Expression of transfected NF-κB-Luc in A549 cells infected with HPIV-3 or RSV in the presence of DN-MyD88 or control GFP. ( B ) The indicated medium supernatants from A549 cells infected with HPIV-3 or RSV in the absence or presence of prior infection with Ads encoding the DN-MyD88 or GFP were subjected to plaque assay analysis. ( C ) Phase contrast microscopic picture of CV-1 cells incubated with same dilutions of medium supernatants obtained from A549 cells infected with HPIV-3 and Ad-GFP or Ad-DN-MyD88.
    Figure Legend Snippet: Differential requirement of MyD88 for NF-κB induction by HPIV-3 and RSV. ( A ) Expression of transfected NF-κB-Luc in A549 cells infected with HPIV-3 or RSV in the presence of DN-MyD88 or control GFP. ( B ) The indicated medium supernatants from A549 cells infected with HPIV-3 or RSV in the absence or presence of prior infection with Ads encoding the DN-MyD88 or GFP were subjected to plaque assay analysis. ( C ) Phase contrast microscopic picture of CV-1 cells incubated with same dilutions of medium supernatants obtained from A549 cells infected with HPIV-3 and Ad-GFP or Ad-DN-MyD88.

    Techniques Used: Expressing, Transfection, Infection, Plaque Assay, Incubation

    Inhibition of NF-κB activation increases HPIV-3, but not RSV, replication and cytopathogenicity in an IFN- and/or soluble factor(s)-independent manner. Plaque assay analysis using medium supernatants from A549 cells mock infected or infected with either HPIV-3 or RSV in the absence or presence of PDTC (50 μM) ( A ), or A549 cells infected with HPIV-3 or RSV in the absence or presence of prior infection with Ads encoding the IκB-SR or GFP( B ). ( C ) Expression of transfected NF-κB-Luc in A549 cells infected with HPIV-3 or RSV in the presence of IκB-SR or control GFP. ( D ) Plaque assay analysis using medium supernatants from IKKγ – / – or WT MEFs infected with either HPIV-3 or RSV. Phase contrast microscopic picture of CV-1 cells incubated with same dilutions of medium supernatants obtained from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR ( E ) and HPIV-3-infected WT or IKKγ – / – MEFs ( F ). ( G ) Plaque assay analysis using medium supernatants from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR and treated with IFN-β (2,000 units/ml). Similar analysis was performed with medium supernatants from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR in the presence or absence of either mock CM or HPIV-3 CM (cleared free of virus).
    Figure Legend Snippet: Inhibition of NF-κB activation increases HPIV-3, but not RSV, replication and cytopathogenicity in an IFN- and/or soluble factor(s)-independent manner. Plaque assay analysis using medium supernatants from A549 cells mock infected or infected with either HPIV-3 or RSV in the absence or presence of PDTC (50 μM) ( A ), or A549 cells infected with HPIV-3 or RSV in the absence or presence of prior infection with Ads encoding the IκB-SR or GFP( B ). ( C ) Expression of transfected NF-κB-Luc in A549 cells infected with HPIV-3 or RSV in the presence of IκB-SR or control GFP. ( D ) Plaque assay analysis using medium supernatants from IKKγ – / – or WT MEFs infected with either HPIV-3 or RSV. Phase contrast microscopic picture of CV-1 cells incubated with same dilutions of medium supernatants obtained from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR ( E ) and HPIV-3-infected WT or IKKγ – / – MEFs ( F ). ( G ) Plaque assay analysis using medium supernatants from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR and treated with IFN-β (2,000 units/ml). Similar analysis was performed with medium supernatants from A549 cells infected with HPIV-3 and Ad-GFP or Ad-IκB-SR in the presence or absence of either mock CM or HPIV-3 CM (cleared free of virus).

    Techniques Used: Inhibition, Activation Assay, Plaque Assay, Infection, Expressing, Transfection, Incubation

    Replication kinetics and NF-κB-induction profile of HPIV-3 and RSV in human lung epithelial A549 cells. ( A ) A single-step growth kinetics of HPIV-3 and RSV (0.1 moi) in A549 cells was determined by plaque assay analysis. ( B ) Cytopathic effect analysis (48 h postinfection) of HPIV-3 and RSV (0.1 moi) from infected A549 cells was determined after addition of same dilution of A549 medium supernatants to CV-1 cells for plaque assay analysis. ( C ) NF-κB EMSA using nuclear extracts from uninfected (–) and RSV-infected (1, 2, 6, and 16 h postinfection) A549 cells, and HPIV-3-infected (0.5–24 h postinfection) A549 cells in the absence or presence of NF-κB p65 antibody (Ab), preimmune serum, specific NF-κB unlabeled probe, or mutant NF-κB unlabeled probe (NS) as indicated.
    Figure Legend Snippet: Replication kinetics and NF-κB-induction profile of HPIV-3 and RSV in human lung epithelial A549 cells. ( A ) A single-step growth kinetics of HPIV-3 and RSV (0.1 moi) in A549 cells was determined by plaque assay analysis. ( B ) Cytopathic effect analysis (48 h postinfection) of HPIV-3 and RSV (0.1 moi) from infected A549 cells was determined after addition of same dilution of A549 medium supernatants to CV-1 cells for plaque assay analysis. ( C ) NF-κB EMSA using nuclear extracts from uninfected (–) and RSV-infected (1, 2, 6, and 16 h postinfection) A549 cells, and HPIV-3-infected (0.5–24 h postinfection) A549 cells in the absence or presence of NF-κB p65 antibody (Ab), preimmune serum, specific NF-κB unlabeled probe, or mutant NF-κB unlabeled probe (NS) as indicated.

    Techniques Used: Plaque Assay, Infection, Mutagenesis

    Related Articles

    Modification:

    Article Title: p53-independent structure-activity relationships of 3-ring mesogenic compounds’ activity as cytotoxic effects against human non-small cell lung cancer lines
    Article Snippet: .. The A549 cells were maintained in Dulbecco’s modified Eagle’s medium (Sigma–Aldrich) supplemented with 1 % penicillin/streptomycin (GIBCO®; Invitrogen, CA, USA) and 10 % heat-inactivated fetal bovine serum (FBS; Japan Bioserum Co., Ltd., Japan) at 37 °C in a humidified atmosphere containing 5 % CO2 . .. WI-38 and EBC-1 cells were cultured in Eagle’s minimum essential medium (Sigma–Aldrich) supplemented with 1 % penicillin/streptomycin and 10 % heat-inactivated FBS at 37 °C in a humidified atmosphere containing 5 % CO2 .

    Invasion Assay:

    Article Title: Nuclear control of lung cancer cells migration, invasion and bioenergetics by eukaryotic translation initiation factor 3F
    Article Snippet: .. Invasion assay Cell invasion properties were analyzed on A549 cells control and overexpressing EIF3F using fluorimetric and colorimetric methods: ECMatrix Cell Invasion Assay (Millipore) and Tumor Cell Transendothelial Assay (Millipore). .. Tumors cells engraftment and optical imaging using bioluminescent tumors A549, H460, and H1975 tumor cells were transduced with a lentiviral construct containing the Fluc gene driven by an internal CMV promoter.

    SDS Page:

    Article Title: Pathogen Specific, IRF3-Dependent Signaling and Innate Resistance to Human Kidney Infection
    Article Snippet: .. SDS-PAGE and immunoblotting In order to detect phosphorylated TRAM, A549 cells grown in 6-well plates were stimulated with 1U/mL SMase, 0.1 µg/mL LPS+sCD14 (10+1µg/ml), 15 µg/mL C6 (Sigma) or RPMI medium alone for 45 and 90 min. .. Cells were lysed in ice-cold buffer (10 mM HEPES-KOH, 5 mM EDTA, 0.5% Nonidet P-40 and 10 mM KCl, pH 7.9) containing a protease inhibitor mix (Complete, Roche Diagnostics GmbH, Mannheim, Germany) and 1mM Na3 VO4 .

    Cell Culture:

    Article Title: Cardiac mitochondrial function depends on BUD23 mediated ribosome programming
    Article Snippet: .. A549 cells were cultured in DMEM growth medium (Sigma, D6546), supplemented with 2 mM L-Glutamine (Sigma, G7513), 10% foetal calf serum (FCS) (Sigma, F96665). .. Depletion of BUD23 with siRNA A549 cells were plated at 1 × 10^6 cells in a 10 cm cell culture dish and transfected with BUD23 specific or non-targeting control siRNA (S41529, S41530, S41531, negative control 1, negative control 2, Ambion Silencer Select) using Dharmafect DF-1 according to the manufacturer’s guidelines.

    Article Title: Scoulerine affects microtubule structure, inhibits proliferation, arrests cell cycle and thus culminates in the apoptotic death of cancer cells
    Article Snippet: .. A549 cells were cultured in Minimum Essential Medium Eagle with L-glutamine and sodium bicarbonate (Sigma-Aldrich, St. Louis, MO, USA) in the presence of 10% foetal calf serum, 1 mM pyruvate, 10 mM HEPES, 50 µg/ml penicillin and 50 µg/ml streptomycin (all supplements from Life Technologies, Grand Island, NY, USA). .. SK-BR-3 cells were cultured in McCoy’s 5 A medium (Life Technologies, Grand Island, NY, USA) supplemented with 10% foetal bovine serum and 50 µg/ml penicillin/streptomycin (all reagents and supplements from Life Technologies, Grand Island, NY, USA).

    In Vitro:

    Article Title: Dual Acting Neuraminidase Inhibitors Open New Opportunities to Disrupt the Lethal Synergism between Streptococcus pneumoniae and Influenza Virus
    Article Snippet: .. Test medium used for virus propagation and in vitro studies with virus was serum-free but contained 0.25 μg/mL (A549 cells) or 2 μg/mL (MDCK cells) trypsin (Sigma–Aldrich GmbH) and 1.3% sodium bicarbonate (Lonza Group Ltd.). ..

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  • 91
    Millipore a549 bar cells
    Fzd-CRD binding characterization of B12 and scFv a, Binding specificity of the B12-Fzd-CRD interaction determined by yeast cell surface titration. B12 was displayed on yeast and binding of monomeric Fzd-CRDs fluorescently labeled with SAV-Alexa647 was detected by flow cytometry. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. b–c, Binding affinity of the B12-Fzd5/8-CRD interaction determined by surface plasmon resonance. Fzd5/8-CRDs were immobilized on a streptavidin chip, and B12 was flown through as analyte. d, Inhibition of XWnt8 induced signaling in <t>A549</t> cells as measured by <t>BAR</t> luciferase reporter assay. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. e–h, Binding affinity of the scFv-DKK1c – Fzd1, Fzd5, Fzd7, Fzd8-CRD interaction determined by surface plasmon resonance. Fzd-CRDs were immobilized on a streptavidin chip, and scFv-DKK1c was flown through as analyte.
    A549 Bar Cells, supplied by Millipore, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a549 bar cells/product/Millipore
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    84
    Millipore a549 cell line human
    Expression of an EV-associated protein in <t>A549</t> cell lysates. Dot blot analyses of CD81 in A549 cell lysates following LPS treatment (0.1 µg/mL, 1 µg/mL, and 10 µg/mL).
    A549 Cell Line Human, supplied by Millipore, used in various techniques. Bioz Stars score: 84/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Fzd-CRD binding characterization of B12 and scFv a, Binding specificity of the B12-Fzd-CRD interaction determined by yeast cell surface titration. B12 was displayed on yeast and binding of monomeric Fzd-CRDs fluorescently labeled with SAV-Alexa647 was detected by flow cytometry. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. b–c, Binding affinity of the B12-Fzd5/8-CRD interaction determined by surface plasmon resonance. Fzd5/8-CRDs were immobilized on a streptavidin chip, and B12 was flown through as analyte. d, Inhibition of XWnt8 induced signaling in A549 cells as measured by BAR luciferase reporter assay. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. e–h, Binding affinity of the scFv-DKK1c – Fzd1, Fzd5, Fzd7, Fzd8-CRD interaction determined by surface plasmon resonance. Fzd-CRDs were immobilized on a streptavidin chip, and scFv-DKK1c was flown through as analyte.

    Journal: Nature

    Article Title: Surrogate Wnt agonists that phenocopy canonical Wnt/β-catenin signaling

    doi: 10.1038/nature22306

    Figure Lengend Snippet: Fzd-CRD binding characterization of B12 and scFv a, Binding specificity of the B12-Fzd-CRD interaction determined by yeast cell surface titration. B12 was displayed on yeast and binding of monomeric Fzd-CRDs fluorescently labeled with SAV-Alexa647 was detected by flow cytometry. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. b–c, Binding affinity of the B12-Fzd5/8-CRD interaction determined by surface plasmon resonance. Fzd5/8-CRDs were immobilized on a streptavidin chip, and B12 was flown through as analyte. d, Inhibition of XWnt8 induced signaling in A549 cells as measured by BAR luciferase reporter assay. Error bars represent S.D. of n=3 technical replicates from one of 2 representative experiments. e–h, Binding affinity of the scFv-DKK1c – Fzd1, Fzd5, Fzd7, Fzd8-CRD interaction determined by surface plasmon resonance. Fzd-CRDs were immobilized on a streptavidin chip, and scFv-DKK1c was flown through as analyte.

    Article Snippet: A549 BAR cells were plated at a density of 5’000 cells/well in the presence of 2 μM IWP-2 (Calbiochem) to suppress endogenous Wnt secretion, and treatment was started after 48 hrs in fresh medium containing fresh IWP-2.

    Techniques: Binding Assay, Titration, Labeling, Flow Cytometry, Cytometry, SPR Assay, Chromatin Immunoprecipitation, Inhibition, Luciferase, Reporter Assay

    Fzd-specific activation of canonical Wnt signaling by Wnt surrogates a–d, Activation of Wnt pathway by decreasing concentration of scFv-DKK1c, B12-DKK1c, XWnt8 or negative control proteins B12, DKK1, IL-2, IL-4 and EPO assayed by the BAR and STF reporters in (a) L cells (50-3 nM) , (b) A375 cells (250-15 nM), (c) SH-SY5Y cells (250-15 nM), and (d) A549 cells (100–1 nM). Error bars represent S.D of n=3 technical replicates from one of 2 representative experiments. The relative quantities of human Fzd mRNA in the relative cell lines determined by qRT-PCR are shown as insets, error bars represent S.D. of n=3 technical replicates. e–f , Selective inhibition of B12-DKK1c and scFv-DKK1c activity in A549 cells by B12, DKK1, Fzd1-CRD-Fc and Fzd8-CRD-Fc assayed by the BAR reporter, correlates with binding specificity. Error bars represent S.D. of n=3 technical replicates. g, Immunoblot analysis of Lrp6 phosphorylation and β-catenin accumulation in A375 BAR cells treated with scFv-DKK1c and hSirpα (0.1, 10, 50 nM), Wnt3a-CM and mock CM (30, 50 %). h, Axin2 transcription relative to GAPDH in SH-SY5Y BAR and A375 BAR cells treated with 50 nM scFv-DKK1c, B12 (negative control), and 30 % Wnt3a-CM analyzed by qRT-PCR. Error bars represent the S.D. of biological triplicates performed in technical triplicates (n=9). i, Axin2 transcription relative to GAPDH in A549 BAR cells treated with 50 nM B12-DKK1c variants, and XWnt8 analyzed by qRT-PCR. Error bars represent S.D. of biological triplicates performed in technical triplicates (n=9). j , Activation of Wnt signaling with distinct amplitudes by XWnt8 and B12-DKK1c variants assayed by the BAR reporter in A549 cells. Error bars represent S.D. of n=3 technical replicates from one of 3 representative experiments.

    Journal: Nature

    Article Title: Surrogate Wnt agonists that phenocopy canonical Wnt/β-catenin signaling

    doi: 10.1038/nature22306

    Figure Lengend Snippet: Fzd-specific activation of canonical Wnt signaling by Wnt surrogates a–d, Activation of Wnt pathway by decreasing concentration of scFv-DKK1c, B12-DKK1c, XWnt8 or negative control proteins B12, DKK1, IL-2, IL-4 and EPO assayed by the BAR and STF reporters in (a) L cells (50-3 nM) , (b) A375 cells (250-15 nM), (c) SH-SY5Y cells (250-15 nM), and (d) A549 cells (100–1 nM). Error bars represent S.D of n=3 technical replicates from one of 2 representative experiments. The relative quantities of human Fzd mRNA in the relative cell lines determined by qRT-PCR are shown as insets, error bars represent S.D. of n=3 technical replicates. e–f , Selective inhibition of B12-DKK1c and scFv-DKK1c activity in A549 cells by B12, DKK1, Fzd1-CRD-Fc and Fzd8-CRD-Fc assayed by the BAR reporter, correlates with binding specificity. Error bars represent S.D. of n=3 technical replicates. g, Immunoblot analysis of Lrp6 phosphorylation and β-catenin accumulation in A375 BAR cells treated with scFv-DKK1c and hSirpα (0.1, 10, 50 nM), Wnt3a-CM and mock CM (30, 50 %). h, Axin2 transcription relative to GAPDH in SH-SY5Y BAR and A375 BAR cells treated with 50 nM scFv-DKK1c, B12 (negative control), and 30 % Wnt3a-CM analyzed by qRT-PCR. Error bars represent the S.D. of biological triplicates performed in technical triplicates (n=9). i, Axin2 transcription relative to GAPDH in A549 BAR cells treated with 50 nM B12-DKK1c variants, and XWnt8 analyzed by qRT-PCR. Error bars represent S.D. of biological triplicates performed in technical triplicates (n=9). j , Activation of Wnt signaling with distinct amplitudes by XWnt8 and B12-DKK1c variants assayed by the BAR reporter in A549 cells. Error bars represent S.D. of n=3 technical replicates from one of 3 representative experiments.

    Article Snippet: A549 BAR cells were plated at a density of 5’000 cells/well in the presence of 2 μM IWP-2 (Calbiochem) to suppress endogenous Wnt secretion, and treatment was started after 48 hrs in fresh medium containing fresh IWP-2.

    Techniques: Activation Assay, Concentration Assay, Negative Control, Quantitative RT-PCR, Inhibition, Activity Assay, Binding Assay

    Expression of an EV-associated protein in A549 cell lysates. Dot blot analyses of CD81 in A549 cell lysates following LPS treatment (0.1 µg/mL, 1 µg/mL, and 10 µg/mL).

    Journal: bioRxiv

    Article Title: Lipopolysaccharide administration alters extracellular vesicles in human lung-cancer cells and mice

    doi: 10.1101/2020.04.17.046367

    Figure Lengend Snippet: Expression of an EV-associated protein in A549 cell lysates. Dot blot analyses of CD81 in A549 cell lysates following LPS treatment (0.1 µg/mL, 1 µg/mL, and 10 µg/mL).

    Article Snippet: A549 cells were seeded independently in 96-well tissue culture plates (10,000 cells/well) and maintained in culture for 24 hrs prior to treatment.

    Techniques: Expressing, Dot Blot

    LPS treatment alters EVs from A549 cells. (A) Mean sizes and (B) particle concentrations (per mL) were determined for A549-derived EVs following LPS treatment using Nanosight Tracking Analysis. ELISAs of A549-derived EVs were used to examine expressions of (C) LAMP-1, and (D) RRP44/DIS3 proteins. Mean fold change ± SEM data are from a total of five experiments. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

    Journal: bioRxiv

    Article Title: Lipopolysaccharide administration alters extracellular vesicles in human lung-cancer cells and mice

    doi: 10.1101/2020.04.17.046367

    Figure Lengend Snippet: LPS treatment alters EVs from A549 cells. (A) Mean sizes and (B) particle concentrations (per mL) were determined for A549-derived EVs following LPS treatment using Nanosight Tracking Analysis. ELISAs of A549-derived EVs were used to examine expressions of (C) LAMP-1, and (D) RRP44/DIS3 proteins. Mean fold change ± SEM data are from a total of five experiments. *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

    Article Snippet: A549 cells were seeded independently in 96-well tissue culture plates (10,000 cells/well) and maintained in culture for 24 hrs prior to treatment.

    Techniques: Derivative Assay

    Transmission of SARS-CoV-2 genome into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) through EVs. A , Schematic depiction of study design. Nsp1 indicates non-structural protein 1; Nsp12, non-structural protein 12; E, envelope protein; N, nucleocapsid protein. B , Expression of SARS-Cov-2 genes in A549 lung epithelial cells. A549 cells were infected with indicated lentiviral particles for 48 hours and mRNA levels were measured by qRT-PCR (left) (n=3, mean± S.D) and semiquantitative PCR (right). *P

    Journal: bioRxiv

    Article Title: Detection of Synthetic Viral RNA Fragments in Human iPSC-Cardiomyocytes following Treatment with Precipitated Extracellular Vesicles from SARS-CoV-2 Coding-Sequence-Overexpressing Lung Epithelial Cells

    doi: 10.1101/2020.05.14.093583

    Figure Lengend Snippet: Transmission of SARS-CoV-2 genome into human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) through EVs. A , Schematic depiction of study design. Nsp1 indicates non-structural protein 1; Nsp12, non-structural protein 12; E, envelope protein; N, nucleocapsid protein. B , Expression of SARS-Cov-2 genes in A549 lung epithelial cells. A549 cells were infected with indicated lentiviral particles for 48 hours and mRNA levels were measured by qRT-PCR (left) (n=3, mean± S.D) and semiquantitative PCR (right). *P

    Article Snippet: Isolation of extracellular vesicles Supernatant of A549 cells was collected at 48 hours after transduction with lentivirus for isolation of EVs.

    Techniques: Transmission Assay, Derivative Assay, Expressing, Infection, Quantitative RT-PCR, Polymerase Chain Reaction

    Effect of curcumin and/or LPS on cell viability and apoptosis of A549 cells. A549 cells were treated with curcumin (10 µM) and/or LPS (1 µg/ml) for different time. (A) Cell viability was monitored by CellTiter 96® AQueous One Solution Cell Proliferation Assay. Data is presented as mean ± Standard error of mean of six data points. (B) Cells were fixed and then stained with propidium iodide (50 µg/ml) and Annexin V-FITC. The DNA contents were analyzed by flow cytometry. Representative result from three independent experiments is presented. x- and y-axis present DNA content and cell number, respectively. (C) Cells (in %) undergoing early- apoptotic or late-apoptotic phase were calculated using Flowing software.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: Effect of curcumin and/or LPS on cell viability and apoptosis of A549 cells. A549 cells were treated with curcumin (10 µM) and/or LPS (1 µg/ml) for different time. (A) Cell viability was monitored by CellTiter 96® AQueous One Solution Cell Proliferation Assay. Data is presented as mean ± Standard error of mean of six data points. (B) Cells were fixed and then stained with propidium iodide (50 µg/ml) and Annexin V-FITC. The DNA contents were analyzed by flow cytometry. Representative result from three independent experiments is presented. x- and y-axis present DNA content and cell number, respectively. (C) Cells (in %) undergoing early- apoptotic or late-apoptotic phase were calculated using Flowing software.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Proliferation Assay, Staining, Flow Cytometry, Software

    Global A549 proteome and common proteins identified in curcumin and/or LPS treated cells. (A) Venn diagram representing the total number of proteins identified in CURI, LPSI and LPSCUR experiments. (B) Subnetwork of 778 common genes (696 were mapped to the database with 65 linker genes) to all 3 experiments (CURI, LPSI and LPSCUR). (C) Venn diagram representing the proteins identified in CURI, LPSI and LPSCUR experiments with similar abundance in expression. (D) Subnetwork of 427 common genes (385 genes were mapped to database with 47 linker genes) with similar abundance in all the 3 experiments are shown. Color coded modules in which the interaction of the genes are highest based on the modularity. The functions of the genes are predicted by Reactome FI plugin in Cytoscape 3.4.0. The circled maps of the subnetworks involve different biological processes. Linker genes are shown by diamond shaped nodes.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: Global A549 proteome and common proteins identified in curcumin and/or LPS treated cells. (A) Venn diagram representing the total number of proteins identified in CURI, LPSI and LPSCUR experiments. (B) Subnetwork of 778 common genes (696 were mapped to the database with 65 linker genes) to all 3 experiments (CURI, LPSI and LPSCUR). (C) Venn diagram representing the proteins identified in CURI, LPSI and LPSCUR experiments with similar abundance in expression. (D) Subnetwork of 427 common genes (385 genes were mapped to database with 47 linker genes) with similar abundance in all the 3 experiments are shown. Color coded modules in which the interaction of the genes are highest based on the modularity. The functions of the genes are predicted by Reactome FI plugin in Cytoscape 3.4.0. The circled maps of the subnetworks involve different biological processes. Linker genes are shown by diamond shaped nodes.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Expressing

    Schematic diagram showing probable mechanism of curcumin action on LPS treated A549 cells. Curcumin (C) inhibits activation of several proteins and the ones marked with dark red oval are identified in this study.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: Schematic diagram showing probable mechanism of curcumin action on LPS treated A549 cells. Curcumin (C) inhibits activation of several proteins and the ones marked with dark red oval are identified in this study.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Activation Assay

    Deregulated global proteome in the A549 cells treated with curcumin and/or LPS. (A) Treatment of curcumin (10 µM) or LPS (1 µg/ml) for 24 hours alters proteome levels in A549 cell lines. Subnetwork in (B) Functional categorization and distribution of deregulated proteins in CURI (305) and LPSI (346) experiments. (C) Mitochondrial proteins showing inverse relationship in both CURI and LPSI experiments. (D) Simultaneous treatment of curcumin (10 µM) and LPS (1 µg/ml) for 48 hours induced proteome change in A549 cell lines. (E) Functional categorization and the distribution of LPSCUR (372) differentially expressed proteins to extract the information of their known functions.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: Deregulated global proteome in the A549 cells treated with curcumin and/or LPS. (A) Treatment of curcumin (10 µM) or LPS (1 µg/ml) for 24 hours alters proteome levels in A549 cell lines. Subnetwork in (B) Functional categorization and distribution of deregulated proteins in CURI (305) and LPSI (346) experiments. (C) Mitochondrial proteins showing inverse relationship in both CURI and LPSI experiments. (D) Simultaneous treatment of curcumin (10 µM) and LPS (1 µg/ml) for 48 hours induced proteome change in A549 cell lines. (E) Functional categorization and the distribution of LPSCUR (372) differentially expressed proteins to extract the information of their known functions.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Functional Assay

    Experimental strategy adopted in this study to understand the anti-inflammatory role of curcumin in A549 cells treated with LPS. Experimental design used to analyze altered proteome in A549 cells treated with curcumin (10 μM) and LPS (1 μg/ml) in SILAC experiments and other function assays used in this study including Mycobacteria infection.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: Experimental strategy adopted in this study to understand the anti-inflammatory role of curcumin in A549 cells treated with LPS. Experimental design used to analyze altered proteome in A549 cells treated with curcumin (10 μM) and LPS (1 μg/ml) in SILAC experiments and other function assays used in this study including Mycobacteria infection.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Infection

    H37Rv-GFP infected A549 cells, treated with curcumin and/or LPS, for 24 hours at multiple of infectivity (1:5) showed successful Mtb internalization and varied bacterial load. (A) Representative confocal microscopy images of cells stained with DAPI (blue, 1 µg/ml) to stain nucleus. Scale: 20 µm and calculation from average of 5 different focal points in triplicates for each experiment. Additional images are available in Figure S9A, S9B and S9C. (B) Colony forming unit (CFU) counts at different conditions. * at 95 % confidence and ** at 99 % confidence. (C) Expression of BID and AIFM1 proteins as probed using Western blot analysis. GAPDH was used as loading control and relative band intensity are presented.

    Journal: bioRxiv

    Article Title: Anti-inflammatory role of curcumin in Lipopolysaccharide treated A549 cells at global proteome level and on mycobacterial infection

    doi: 10.1101/721100

    Figure Lengend Snippet: H37Rv-GFP infected A549 cells, treated with curcumin and/or LPS, for 24 hours at multiple of infectivity (1:5) showed successful Mtb internalization and varied bacterial load. (A) Representative confocal microscopy images of cells stained with DAPI (blue, 1 µg/ml) to stain nucleus. Scale: 20 µm and calculation from average of 5 different focal points in triplicates for each experiment. Additional images are available in Figure S9A, S9B and S9C. (B) Colony forming unit (CFU) counts at different conditions. * at 95 % confidence and ** at 99 % confidence. (C) Expression of BID and AIFM1 proteins as probed using Western blot analysis. GAPDH was used as loading control and relative band intensity are presented.

    Article Snippet: Monitoring Mitochondrial distribution in treated A549 cells using Confocal Microscopy A549 cells were grown overnight on 18 mm coverslip in twelve well plates at 37 °C with 5% CO2 and incubated with curcumin/LPS/both for appropriate durations.

    Techniques: Infection, Confocal Microscopy, Staining, Expressing, Western Blot