Structured Review

ibidi a549 cells
Aspergillus fumigatus hyphae grow parallel to <t>A549</t> cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.
A549 Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Images

1) Product Images from "Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen"

Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2016.00438

Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.
Figure Legend Snippet: Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.

Techniques Used: Staining, Standard Deviation

Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.
Figure Legend Snippet: Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

Techniques Used:

Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.
Figure Legend Snippet: Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

Techniques Used:

Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.
Figure Legend Snippet: Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.

Techniques Used: Blocking Assay

2) Product Images from "Cell senescence is an antiviral defense mechanism"

Article Title: Cell senescence is an antiviral defense mechanism

Journal: Scientific Reports

doi: 10.1038/srep37007

Chemotherapy-induced senescence of human tumor cells restricts VSV infection. ( A ) Microscopy images of human tumor A549 cells showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (A549-NT, upper panels) and bleomycin-induced senescent (A549-B, bottom panels) A549 cells. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). ( B ) Western-blot analysis of senescence markers p53 and p21 in untreated A549 cells (A549-NT) or after bleomycin treatment of A549 cells (A549-B). GAPDH is shown as loading control. ( C ) Expression levels of CDKN1A (coding for p21) mRNA relative to GAPDH (x10 −3 ) as determined by qRT-PCR in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. ( D ) Viral titers (PFU/mL) determined in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at a MOI of 0.5 PFU/cell. ( E ) Western-blot analysis of VSV protein synthesis in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at MOIs of 0.05 PFU/cell (upper panel) or 10 PFU/cell (lower panel). Actin is shown as loading control. ( F ) Microscopy images of MEFs showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (MEFs-NT, upper panels) and bleomycin-induced senescent (MEFs-B, bottom panels) MEFs. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). ( G ) Viral titers (PFU/mL) determined in untreated (MEFs-NT) or bleomycin-treated (MEFs-B) MEFs after the indicated periods of infection at MOIs of 0.05 PFU/cell (left panel) or 10 PFU/cell (right panel). ( G ) Percentage of apoptotic cells measured after mock or VSV infection at MOI of 10 PFU/cell, in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. Data are mean values +/− SE from at least three different experiments. *p
Figure Legend Snippet: Chemotherapy-induced senescence of human tumor cells restricts VSV infection. ( A ) Microscopy images of human tumor A549 cells showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (A549-NT, upper panels) and bleomycin-induced senescent (A549-B, bottom panels) A549 cells. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). ( B ) Western-blot analysis of senescence markers p53 and p21 in untreated A549 cells (A549-NT) or after bleomycin treatment of A549 cells (A549-B). GAPDH is shown as loading control. ( C ) Expression levels of CDKN1A (coding for p21) mRNA relative to GAPDH (x10 −3 ) as determined by qRT-PCR in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. ( D ) Viral titers (PFU/mL) determined in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at a MOI of 0.5 PFU/cell. ( E ) Western-blot analysis of VSV protein synthesis in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells after the indicated periods of infection at MOIs of 0.05 PFU/cell (upper panel) or 10 PFU/cell (lower panel). Actin is shown as loading control. ( F ) Microscopy images of MEFs showing morphology (left panels) and SA-beta-gal staining (right panels) of untreated (MEFs-NT, upper panels) and bleomycin-induced senescent (MEFs-B, bottom panels) MEFs. Quantification of the SA-beta-gal positive cells is shown below (at least 200 cells were counted per condition). ( G ) Viral titers (PFU/mL) determined in untreated (MEFs-NT) or bleomycin-treated (MEFs-B) MEFs after the indicated periods of infection at MOIs of 0.05 PFU/cell (left panel) or 10 PFU/cell (right panel). ( G ) Percentage of apoptotic cells measured after mock or VSV infection at MOI of 10 PFU/cell, in untreated (A549-NT) or bleomycin-treated (A549-B) A549 cells. Data are mean values +/− SE from at least three different experiments. *p

Techniques Used: Infection, Microscopy, Staining, Western Blot, Expressing, Quantitative RT-PCR

The senescence-induced antiviral response is partially mediated by the SASP. ( A ) Characterization of the expression of different SASP factors by qRT-PCR relative to GAPDH (x10 −3 ) in control untreated (black bars) or senescent bleomycin-treated (white bars) A549 cells. ( B ) Cell viability of A549 cells cultured with conditioned medium (CM) from control untreated or senescent bleomycin-treated A549, after VSV infection at different MOIs. Viability of senescent bleomycin-treated A549 cells or A549 cells cultured in the presence of interferon are shown as controls. ( C ) Cell viability of MEFs cultures with CM from control early passage or senescent late passage MEFs, after VSV infection at MOIs of 0.01 or 10 PFU/cell relative to mock-infected cells. Data are mean values +/− SE from at least three different experiments. *p
Figure Legend Snippet: The senescence-induced antiviral response is partially mediated by the SASP. ( A ) Characterization of the expression of different SASP factors by qRT-PCR relative to GAPDH (x10 −3 ) in control untreated (black bars) or senescent bleomycin-treated (white bars) A549 cells. ( B ) Cell viability of A549 cells cultured with conditioned medium (CM) from control untreated or senescent bleomycin-treated A549, after VSV infection at different MOIs. Viability of senescent bleomycin-treated A549 cells or A549 cells cultured in the presence of interferon are shown as controls. ( C ) Cell viability of MEFs cultures with CM from control early passage or senescent late passage MEFs, after VSV infection at MOIs of 0.01 or 10 PFU/cell relative to mock-infected cells. Data are mean values +/− SE from at least three different experiments. *p

Techniques Used: Expressing, Quantitative RT-PCR, Cell Culture, Infection

Senescence reduces the cell infectivity of VSV. ( A ) Fluorescent microscopy images of control untreated (A549-NT, left panels) or senescent bleomycin-treated (A549-BLEO, right panels) human tumor A549 cells showing virus spread at different times after infection with recombinant VSV expressing GFP (rVSV-GFP). ( B ) Percentage of rVSV-GFP positive cells after 6 or 24 hours post infection in control untreated (black bars) or senescent bleomycin-treated (white bars) human tumor A549 cells. ( C ) Percentage of rVSV-GFP positive cells after infection with MOIs of 0.05 or 0.5 PFU/cell in control early passage (black bars) or senescent late passage (white bars) MEFs. ( D ) Representative images of control (A549-NT, left panels) or bleomycin-induced senescent (A549-BLEO, right panels) A549 cells infected with VSV-GFP (MOI of 10 PFU/cell) taken at the indicated times. ( E ) Quantification of the intensity of GFP associated fluorescence per GFP positive cell (arbitrary units, a.u.). Data are mean values +/− SE from at least three different experiments. *p
Figure Legend Snippet: Senescence reduces the cell infectivity of VSV. ( A ) Fluorescent microscopy images of control untreated (A549-NT, left panels) or senescent bleomycin-treated (A549-BLEO, right panels) human tumor A549 cells showing virus spread at different times after infection with recombinant VSV expressing GFP (rVSV-GFP). ( B ) Percentage of rVSV-GFP positive cells after 6 or 24 hours post infection in control untreated (black bars) or senescent bleomycin-treated (white bars) human tumor A549 cells. ( C ) Percentage of rVSV-GFP positive cells after infection with MOIs of 0.05 or 0.5 PFU/cell in control early passage (black bars) or senescent late passage (white bars) MEFs. ( D ) Representative images of control (A549-NT, left panels) or bleomycin-induced senescent (A549-BLEO, right panels) A549 cells infected with VSV-GFP (MOI of 10 PFU/cell) taken at the indicated times. ( E ) Quantification of the intensity of GFP associated fluorescence per GFP positive cell (arbitrary units, a.u.). Data are mean values +/− SE from at least three different experiments. *p

Techniques Used: Infection, Microscopy, Recombinant, Expressing, Fluorescence

3) Product Images from "Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway"

Article Title: Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005307

LAI-1-dependent redistribution of IQGAP1 does not require Cdc42. (A) A549 cells were treated with siRNA against Cdc42 for 2 days, then with LAI-1 (10 μM, 1.5 h) and protein production or the subcellular localization of the IQGAP1 scaffold protein (green) and the small GTPase Cdc42 (red) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). (B) Quantification of protein production in A549 cells after RNAi treatment (percent cells producing protein of interest; n = 50). Means and standard deviations of 3 independent experiments are shown (*** p
Figure Legend Snippet: LAI-1-dependent redistribution of IQGAP1 does not require Cdc42. (A) A549 cells were treated with siRNA against Cdc42 for 2 days, then with LAI-1 (10 μM, 1.5 h) and protein production or the subcellular localization of the IQGAP1 scaffold protein (green) and the small GTPase Cdc42 (red) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). (B) Quantification of protein production in A549 cells after RNAi treatment (percent cells producing protein of interest; n = 50). Means and standard deviations of 3 independent experiments are shown (*** p

Techniques Used: Confocal Microscopy, Staining

LAI-1 reverses Icm/Dot-dependent inhibition of migration by L . pneumophila . (A) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity ( S7 Fig )) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; * p
Figure Legend Snippet: LAI-1 reverses Icm/Dot-dependent inhibition of migration by L . pneumophila . (A) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity ( S7 Fig )) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; * p

Techniques Used: Inhibition, Migration, Infection, Mutagenesis, Staining, Fluorescence, Chemotaxis Assay, Software, Imaging

Migration inhibition by L . pneumophila is augmented in the absence of Cdc42. (A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (*** p
Figure Legend Snippet: Migration inhibition by L . pneumophila is augmented in the absence of Cdc42. (A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (*** p

Techniques Used: Migration, Inhibition, Infection, Mutagenesis, Imaging, Software

LAI-1-dependent inhibition of cell migration requires IQGAP1 and Cdc42. (A) Confluent cell layers of A549 epithelial cells were treated with siRNA against IQGAP1, Cdc42, RhoA or Rac1 for 2 days. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of triplicate samples are shown (** p
Figure Legend Snippet: LAI-1-dependent inhibition of cell migration requires IQGAP1 and Cdc42. (A) Confluent cell layers of A549 epithelial cells were treated with siRNA against IQGAP1, Cdc42, RhoA or Rac1 for 2 days. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of triplicate samples are shown (** p

Techniques Used: Inhibition, Migration, Imaging, Software

Effect of L . pneumophila lqs genes on host cell migration. D . discoideum strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT or Δ lqsA mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: *** p
Figure Legend Snippet: Effect of L . pneumophila lqs genes on host cell migration. D . discoideum strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT or Δ lqsA mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: *** p

Techniques Used: Migration, Infection, Mutagenesis, Fluorescence, Staining, Imaging, Software

LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9. (A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (*** p
Figure Legend Snippet: LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9. (A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (*** p

Techniques Used: Inhibition, Migration, Imaging, Software

LAI-1 promotes inactivation of Cdc42 and redistribution of IQGAP1 to the cell cortex. (A) A549 cells were treated with LAI-1 (10 μM, 1 h), and the activation state of Cdc42 was analyzed by Western blot using an antibody recognizing Cdc42(GTP/GDP) (left panel). Quantification by densitometry was performed using ImageJ (right panel). A549 cells were treated with LAI-1 (10 μM, 1 h), fixed, stained with antibodies against (B) IQGAP1 or (C) Cdc42 and analyzed by confocal microscopy (left panels; green, FITC; blue, DAPI). The graphs (right panels) are based on the relative fluorescence intensity along cell sections (n = 50, *** p
Figure Legend Snippet: LAI-1 promotes inactivation of Cdc42 and redistribution of IQGAP1 to the cell cortex. (A) A549 cells were treated with LAI-1 (10 μM, 1 h), and the activation state of Cdc42 was analyzed by Western blot using an antibody recognizing Cdc42(GTP/GDP) (left panel). Quantification by densitometry was performed using ImageJ (right panel). A549 cells were treated with LAI-1 (10 μM, 1 h), fixed, stained with antibodies against (B) IQGAP1 or (C) Cdc42 and analyzed by confocal microscopy (left panels; green, FITC; blue, DAPI). The graphs (right panels) are based on the relative fluorescence intensity along cell sections (n = 50, *** p

Techniques Used: Activation Assay, Western Blot, Staining, Confocal Microscopy, Fluorescence

4) Product Images from "Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells"

Article Title: Coming in and Finding Out: Blending Receptor-Targeted Delivery and Efficient Endosomal Escape in a Novel Bio-Responsive siRNA Delivery System for Gene Knockdown in Pulmonary T Cells

Journal: Advanced therapeutics

doi: 10.1002/adtp.201900047

A) Fluorescence microscopy images of A549 cells after staining with acridine orange and treatment with chloroquine (A2), PEI polyplexes (A3), Tf-PEI polyplexes (A4), Mel-PEI polyplexes (A5), and Tf-Mel-PEI polyplexes (A6). A1 represents untreated cells as blank. B) Confocal images after transfection of Jurkat cells and staining with DAPI (blue, depicting the cell nuclei) and LysoTracker Red DND-99 (red, representing the lysosomes). B1 shows the DAPI and Lysotracker only control, B2-B6 show cells transfected with free siRNA, PEI, Tf-PEI, Mel-PEI, and Tf-Mel-PEI polyplexes, respectively.
Figure Legend Snippet: A) Fluorescence microscopy images of A549 cells after staining with acridine orange and treatment with chloroquine (A2), PEI polyplexes (A3), Tf-PEI polyplexes (A4), Mel-PEI polyplexes (A5), and Tf-Mel-PEI polyplexes (A6). A1 represents untreated cells as blank. B) Confocal images after transfection of Jurkat cells and staining with DAPI (blue, depicting the cell nuclei) and LysoTracker Red DND-99 (red, representing the lysosomes). B1 shows the DAPI and Lysotracker only control, B2-B6 show cells transfected with free siRNA, PEI, Tf-PEI, Mel-PEI, and Tf-Mel-PEI polyplexes, respectively.

Techniques Used: Fluorescence, Microscopy, Staining, Transfection

5) Product Images from "Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549"

Article Title: Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549

Journal: Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry

doi: 10.3390/molecules22060929

Curcumin inhibits lung cancer cell migration and invasion by regulating miR-98 expression. ( A , B ) Curcumin increased miR-98 level in A549 cells in a dose- and time-dependent manner (100 μm curcumin), as determined by qRT-PCR. * p
Figure Legend Snippet: Curcumin inhibits lung cancer cell migration and invasion by regulating miR-98 expression. ( A , B ) Curcumin increased miR-98 level in A549 cells in a dose- and time-dependent manner (100 μm curcumin), as determined by qRT-PCR. * p

Techniques Used: Migration, Expressing, Quantitative RT-PCR

Curcumin inhibits tumor growth and MMP2/9 expression in a xenograft model. ( A , B ) Severe combined immunodeficiency mice were inoculated in the right flank with A549 cells. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted. Tumors were weighed at the end of the experiment; each plot shows mean ± SEM of six mice per group. * p
Figure Legend Snippet: Curcumin inhibits tumor growth and MMP2/9 expression in a xenograft model. ( A , B ) Severe combined immunodeficiency mice were inoculated in the right flank with A549 cells. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted. Tumors were weighed at the end of the experiment; each plot shows mean ± SEM of six mice per group. * p

Techniques Used: Expressing, Mouse Assay

Curcumin reduces LIN28A-induced MMP2/9 expression and blocks cancer metastasis. ( A ) Curcumin (100 μm) treatment decreased LIN28A expression. ( B ) Various curcumin (25, 50, 100 µm) treatment reduced LIN28A, MMP2/9 protein expression. ( C , D ) LIN28A silencing decreased LIN28A expression and MMP2/9 mRNA and protein expression in A549 cells, as determined by qRT-PCR and western blotting, respectively. LIN28A (30nM) knockdown significantly suppressed A549 cell migration ( E,F ) and invasion ( G ). Data represent mean ± SEM of three independent experiments. * p
Figure Legend Snippet: Curcumin reduces LIN28A-induced MMP2/9 expression and blocks cancer metastasis. ( A ) Curcumin (100 μm) treatment decreased LIN28A expression. ( B ) Various curcumin (25, 50, 100 µm) treatment reduced LIN28A, MMP2/9 protein expression. ( C , D ) LIN28A silencing decreased LIN28A expression and MMP2/9 mRNA and protein expression in A549 cells, as determined by qRT-PCR and western blotting, respectively. LIN28A (30nM) knockdown significantly suppressed A549 cell migration ( E,F ) and invasion ( G ). Data represent mean ± SEM of three independent experiments. * p

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

6) Product Images from "Tumor-Selective Cytotoxicity of Nitidine Results from Its Rapid Accumulation into Mitochondria"

Article Title: Tumor-Selective Cytotoxicity of Nitidine Results from Its Rapid Accumulation into Mitochondria

Journal: BioMed Research International

doi: 10.1155/2017/2130594

Inhibitory effect of continuous treatment with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) in A549 cells measured using the xCELLigence system. Cell growth was assessed with impedance using the xCELLigence system for A549 cells. Cells were treated with 5 μ M of each compound after 18 h preculture (0 h). Cell growth was then monitored continuously for 48 h.
Figure Legend Snippet: Inhibitory effect of continuous treatment with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) in A549 cells measured using the xCELLigence system. Cell growth was assessed with impedance using the xCELLigence system for A549 cells. Cells were treated with 5 μ M of each compound after 18 h preculture (0 h). Cell growth was then monitored continuously for 48 h.

Techniques Used: Cycling Probe Technology

Inhibitory effect of short-term treatment with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) in A549 cells measured using the xCELLigence system. Cell growth was assessed with impedance using the xCELLigence system for A549 cells. Cells were treated with 2 or 5 μ M of each compound after 18 h preculture (0 h). After 2 h of treatment, wells of the short-term group were washed and replaced with complete medium without compound. Cell growth was then monitored continuously for 118 h.
Figure Legend Snippet: Inhibitory effect of short-term treatment with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) in A549 cells measured using the xCELLigence system. Cell growth was assessed with impedance using the xCELLigence system for A549 cells. Cells were treated with 2 or 5 μ M of each compound after 18 h preculture (0 h). After 2 h of treatment, wells of the short-term group were washed and replaced with complete medium without compound. Cell growth was then monitored continuously for 118 h.

Techniques Used: Cycling Probe Technology

Time-dependent changes of the mitochondrial membrane potential. A549 cells were treated with JC-1 for 30 min. After washing with phosphate-buffered saline (PBS) (−), the cells were treated with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) (0 h). The fluorescence of JC-1 was observed by confocal fluorescent microscopy (0, 1, 2, 4, 8, and 21 h after treatment). (a) Orange fluorescence represents JC-1, which formed J-aggregates in mitochondria in a mitochondrial membrane potential-dependent manner. (b) The fluorescent intensity of the image was quantified. All values were quantified from three random areas. Data are presented as the related intensity corrected by the value of 0 h. ∗∗ p
Figure Legend Snippet: Time-dependent changes of the mitochondrial membrane potential. A549 cells were treated with JC-1 for 30 min. After washing with phosphate-buffered saline (PBS) (−), the cells were treated with nitidine (NTD), camptothecin (CPT), or topotecan (TPT) (0 h). The fluorescence of JC-1 was observed by confocal fluorescent microscopy (0, 1, 2, 4, 8, and 21 h after treatment). (a) Orange fluorescence represents JC-1, which formed J-aggregates in mitochondria in a mitochondrial membrane potential-dependent manner. (b) The fluorescent intensity of the image was quantified. All values were quantified from three random areas. Data are presented as the related intensity corrected by the value of 0 h. ∗∗ p

Techniques Used: Cycling Probe Technology, Fluorescence, Microscopy

Fluorescent images of nitidine (NTD) and organelle-specific fluorescent proteins. A549 cells were transfected with each organelle-specific recombinant protein supplied in the Organelle Lights™ kit (red fluorescent protein (RFP)). The fluorescent images of NTD were observed under ultraviolet (UV) light (358 ± 28 nm).
Figure Legend Snippet: Fluorescent images of nitidine (NTD) and organelle-specific fluorescent proteins. A549 cells were transfected with each organelle-specific recombinant protein supplied in the Organelle Lights™ kit (red fluorescent protein (RFP)). The fluorescent images of NTD were observed under ultraviolet (UV) light (358 ± 28 nm).

Techniques Used: Transfection, Recombinant

Inhibition of nitidine (NTD) accumulation into mitochondria by treatment of mitochondrial membrane depolarizer. A549 cells were treated with or without 50 μ M of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 h. Disappearance of mitochondrial membrane potential (MMP) was detected by JC-1 (2 μ M) fluorescence. Control cells show accumulation of JC-1 or NTD in mitochondria. CCCP-treated cells showed disappearance of MMP (ΔΨm). In the same condition, NTD fluorescence also disappeared.
Figure Legend Snippet: Inhibition of nitidine (NTD) accumulation into mitochondria by treatment of mitochondrial membrane depolarizer. A549 cells were treated with or without 50 μ M of carbonyl cyanide m-chlorophenyl hydrazone (CCCP) for 1 h. Disappearance of mitochondrial membrane potential (MMP) was detected by JC-1 (2 μ M) fluorescence. Control cells show accumulation of JC-1 or NTD in mitochondria. CCCP-treated cells showed disappearance of MMP (ΔΨm). In the same condition, NTD fluorescence also disappeared.

Techniques Used: Inhibition, Fluorescence

7) Product Images from "Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA"

Article Title: Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA

Journal: Scientific Reports

doi: 10.1038/s41598-017-18468-7

The effect of PLY on mitochondrial calcium, caspase-3/7 activation, membrane potential and opening of mPTP. ( A ) Representative time-lapse images of A549 cells loaded with the mitochondrial calcium sensor Rhod2-AM (red) and stimulated with 1.0 µg/ml PLY in the presence of the caspase-3/7 sensor (green). Scale bar represents 25 µm. ( B ) Quantification of mitochondrial calcium changes (Rhod2 F / F 0 ratios) in cells stimulated with 1.0 µg/ml and 0.1 µg/ml PLY or left untreated (mean ± SD from n = 5 independent experiments). ( C ) Quantification of caspase-3/7 induction ( F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( D ) Quantification of changes in mitochondrial membrane potential (TMRE F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( E ) Opening of the mPTP ( F / F 0 ratios) in cells stimulated with the indicated amount of PLY or left untreated (mean ± SD from n = 3 independent experiments).
Figure Legend Snippet: The effect of PLY on mitochondrial calcium, caspase-3/7 activation, membrane potential and opening of mPTP. ( A ) Representative time-lapse images of A549 cells loaded with the mitochondrial calcium sensor Rhod2-AM (red) and stimulated with 1.0 µg/ml PLY in the presence of the caspase-3/7 sensor (green). Scale bar represents 25 µm. ( B ) Quantification of mitochondrial calcium changes (Rhod2 F / F 0 ratios) in cells stimulated with 1.0 µg/ml and 0.1 µg/ml PLY or left untreated (mean ± SD from n = 5 independent experiments). ( C ) Quantification of caspase-3/7 induction ( F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( D ) Quantification of changes in mitochondrial membrane potential (TMRE F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( E ) Opening of the mPTP ( F / F 0 ratios) in cells stimulated with the indicated amount of PLY or left untreated (mean ± SD from n = 3 independent experiments).

Techniques Used: Activation Assay

Real-time measurements of PLY induced release of mtDNA in A549 and primary human alveolar epithelial cells. ( A ) Representative confocal 2D plane of mitochondria in A549 cells stained with Syto82 (red) and MitoTrackerOrange (grey) left unstimulated or stimulated with 1.0 µg/ml PLY for 5 min. The scale bar represents 15 µm. ( B ) Quantification of mtDNA release (Syto 82 F / F 0 ratios) in cells stimulated as described above (mean ± SD from n = 5 independent experiments, *** P
Figure Legend Snippet: Real-time measurements of PLY induced release of mtDNA in A549 and primary human alveolar epithelial cells. ( A ) Representative confocal 2D plane of mitochondria in A549 cells stained with Syto82 (red) and MitoTrackerOrange (grey) left unstimulated or stimulated with 1.0 µg/ml PLY for 5 min. The scale bar represents 15 µm. ( B ) Quantification of mtDNA release (Syto 82 F / F 0 ratios) in cells stimulated as described above (mean ± SD from n = 5 independent experiments, *** P

Techniques Used: Staining

Pneumolysin induces mitochondrial fragmentation and disrupts mitochondrial motility in epithelial cells and human lung tissue. ( A ) A549 cells were labelled with MitoTrackerOrange and subsequently infected with S . pn . D39Δ cps or S . pn . D39Δ cps Δ ply for 5 hours, stimulated with 1.0 µg/ml PLY for 15 min or left untreated. Cells were fixed, stained with DAPI and mitochondrial morphology was analysed by structured illumination microscopy. Mitochondria were pseudocoloured using YellowHot LUT and a reconstructed widefield image of the nucleus is shown in blue. Scale bar represents 5 µm. ( B , C ) Quantification of mitochondrial morphology in control and PLY treated cells exemplified in ( A ). The mitochondrial network was examined and quantified in stochastically selected mitochondria-rich parts of the cells. Integrative network/shape analysis (number of branches per mitochondrion ( B ) and branch length per mitochondrion ( C )) was performed (mean ± SD from n = 3 independent experiments, * P
Figure Legend Snippet: Pneumolysin induces mitochondrial fragmentation and disrupts mitochondrial motility in epithelial cells and human lung tissue. ( A ) A549 cells were labelled with MitoTrackerOrange and subsequently infected with S . pn . D39Δ cps or S . pn . D39Δ cps Δ ply for 5 hours, stimulated with 1.0 µg/ml PLY for 15 min or left untreated. Cells were fixed, stained with DAPI and mitochondrial morphology was analysed by structured illumination microscopy. Mitochondria were pseudocoloured using YellowHot LUT and a reconstructed widefield image of the nucleus is shown in blue. Scale bar represents 5 µm. ( B , C ) Quantification of mitochondrial morphology in control and PLY treated cells exemplified in ( A ). The mitochondrial network was examined and quantified in stochastically selected mitochondria-rich parts of the cells. Integrative network/shape analysis (number of branches per mitochondrion ( B ) and branch length per mitochondrion ( C )) was performed (mean ± SD from n = 3 independent experiments, * P

Techniques Used: Infection, Staining, Microscopy

Pneumolysin induces extracellular release of mtDNA. ( A ) Relative quantification of mitochondrial DNA released from A549 cells in the supernatant after stimulation with the indicated amounts of PLY after three hours. Data are expressed as amount of mitochondrial DNA normalized to unstimulated cells (ctrl). Bars represent mean ± SD from n = 5 independent experiments, * P
Figure Legend Snippet: Pneumolysin induces extracellular release of mtDNA. ( A ) Relative quantification of mitochondrial DNA released from A549 cells in the supernatant after stimulation with the indicated amounts of PLY after three hours. Data are expressed as amount of mitochondrial DNA normalized to unstimulated cells (ctrl). Bars represent mean ± SD from n = 5 independent experiments, * P

Techniques Used:

Infection with PLY expressing pneumococci and stimulation with PLY reduces cytosolic and mitochondrial ATP levels. A549 cells were transfected with plasmids encoding FRET-ATP sensors targeted to the cytosol ( A ) and mitochondria ( B ), respectively. 48 hours post transfection, cells were left unstimulated or infected with S . pn . D39Δ cps , S . pn . D39Δ cps Δ ply for 4 and 6 h, respectively, or stimulated with 0.25 µg/ml PLY for 30 min. Cells stimulated with 10 µg/ml Oligomycin for 30 min served as positive control. The ATP content is expressed as normalized ratio of the YFP/CFP peak intensity at 530 nm (YFP) and 478 nm (CFP), respectively measured by live spectral-FRET microscopy. Bars represent mean ± SD from n = 3 independent experiments. * P
Figure Legend Snippet: Infection with PLY expressing pneumococci and stimulation with PLY reduces cytosolic and mitochondrial ATP levels. A549 cells were transfected with plasmids encoding FRET-ATP sensors targeted to the cytosol ( A ) and mitochondria ( B ), respectively. 48 hours post transfection, cells were left unstimulated or infected with S . pn . D39Δ cps , S . pn . D39Δ cps Δ ply for 4 and 6 h, respectively, or stimulated with 0.25 µg/ml PLY for 30 min. Cells stimulated with 10 µg/ml Oligomycin for 30 min served as positive control. The ATP content is expressed as normalized ratio of the YFP/CFP peak intensity at 530 nm (YFP) and 478 nm (CFP), respectively measured by live spectral-FRET microscopy. Bars represent mean ± SD from n = 3 independent experiments. * P

Techniques Used: Infection, Expressing, Transfection, Positive Control, Microscopy

8) Product Images from "Lipids as Tumoricidal Components of Human ?-Lactalbumin Made Lethal to Tumor Cells (HAMLET)"

Article Title: Lipids as Tumoricidal Components of Human ?-Lactalbumin Made Lethal to Tumor Cells (HAMLET)

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.468405

HAMLET and oleate-HAMLET trigger tumor cell death, morphological change, and ion fluxes. A , cytotoxic responses in A549 lung carcinoma cells were treated with HAMLET or oleate-HAMLET (35 μ m ) and quantified by trypan blue exclusion and ATP levels.
Figure Legend Snippet: HAMLET and oleate-HAMLET trigger tumor cell death, morphological change, and ion fluxes. A , cytotoxic responses in A549 lung carcinoma cells were treated with HAMLET or oleate-HAMLET (35 μ m ) and quantified by trypan blue exclusion and ATP levels.

Techniques Used:

Metabolic responses to HAMLET, oleate, or oleic acid. Metabolites were quantified by GC/MS in extracts of A549 cells treated with HAMLET (35 μ m ) or oleate (175 μ m ) for 60 min. A , shown is a heatmap of differentially abundant metabolites.
Figure Legend Snippet: Metabolic responses to HAMLET, oleate, or oleic acid. Metabolites were quantified by GC/MS in extracts of A549 cells treated with HAMLET (35 μ m ) or oleate (175 μ m ) for 60 min. A , shown is a heatmap of differentially abundant metabolites.

Techniques Used: Gas Chromatography-Mass Spectrometry

9) Product Images from "Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA"

Article Title: Pneumolysin induced mitochondrial dysfunction leads to release of mitochondrial DNA

Journal: Scientific Reports

doi: 10.1038/s41598-017-18468-7

The effect of PLY on mitochondrial calcium, caspase-3/7 activation, membrane potential and opening of mPTP. ( A ) Representative time-lapse images of A549 cells loaded with the mitochondrial calcium sensor Rhod2-AM (red) and stimulated with 1.0 µg/ml PLY in the presence of the caspase-3/7 sensor (green). Scale bar represents 25 µm. ( B ) Quantification of mitochondrial calcium changes (Rhod2 F / F 0 ratios) in cells stimulated with 1.0 µg/ml and 0.1 µg/ml PLY or left untreated (mean ± SD from n = 5 independent experiments). ( C ) Quantification of caspase-3/7 induction ( F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( D ) Quantification of changes in mitochondrial membrane potential (TMRE F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( E ) Opening of the mPTP ( F / F 0 ratios) in cells stimulated with the indicated amount of PLY or left untreated (mean ± SD from n = 3 independent experiments).
Figure Legend Snippet: The effect of PLY on mitochondrial calcium, caspase-3/7 activation, membrane potential and opening of mPTP. ( A ) Representative time-lapse images of A549 cells loaded with the mitochondrial calcium sensor Rhod2-AM (red) and stimulated with 1.0 µg/ml PLY in the presence of the caspase-3/7 sensor (green). Scale bar represents 25 µm. ( B ) Quantification of mitochondrial calcium changes (Rhod2 F / F 0 ratios) in cells stimulated with 1.0 µg/ml and 0.1 µg/ml PLY or left untreated (mean ± SD from n = 5 independent experiments). ( C ) Quantification of caspase-3/7 induction ( F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( D ) Quantification of changes in mitochondrial membrane potential (TMRE F / F 0 ratios) in cells stimulated with PLY as described above or left untreated (mean ± SD from n = 3 independent experiments). ( E ) Opening of the mPTP ( F / F 0 ratios) in cells stimulated with the indicated amount of PLY or left untreated (mean ± SD from n = 3 independent experiments).

Techniques Used: Activation Assay

Real-time measurements of PLY induced release of mtDNA in A549 and primary human alveolar epithelial cells. ( A ) Representative confocal 2D plane of mitochondria in A549 cells stained with Syto82 (red) and MitoTrackerOrange (grey) left unstimulated or stimulated with 1.0 µg/ml PLY for 5 min. The scale bar represents 15 µm. ( B ) Quantification of mtDNA release (Syto 82 F / F 0 ratios) in cells stimulated as described above (mean ± SD from n = 5 independent experiments, *** P
Figure Legend Snippet: Real-time measurements of PLY induced release of mtDNA in A549 and primary human alveolar epithelial cells. ( A ) Representative confocal 2D plane of mitochondria in A549 cells stained with Syto82 (red) and MitoTrackerOrange (grey) left unstimulated or stimulated with 1.0 µg/ml PLY for 5 min. The scale bar represents 15 µm. ( B ) Quantification of mtDNA release (Syto 82 F / F 0 ratios) in cells stimulated as described above (mean ± SD from n = 5 independent experiments, *** P

Techniques Used: Staining

Pneumolysin induces mitochondrial fragmentation and disrupts mitochondrial motility in epithelial cells and human lung tissue. ( A ) A549 cells were labelled with MitoTrackerOrange and subsequently infected with S . pn . D39Δ cps or S . pn . D39Δ cps Δ ply for 5 hours, stimulated with 1.0 µg/ml PLY for 15 min or left untreated. Cells were fixed, stained with DAPI and mitochondrial morphology was analysed by structured illumination microscopy. Mitochondria were pseudocoloured using YellowHot LUT and a reconstructed widefield image of the nucleus is shown in blue. Scale bar represents 5 µm. ( B , C ) Quantification of mitochondrial morphology in control and PLY treated cells exemplified in ( A ). The mitochondrial network was examined and quantified in stochastically selected mitochondria-rich parts of the cells. Integrative network/shape analysis (number of branches per mitochondrion ( B ) and branch length per mitochondrion ( C )) was performed (mean ± SD from n = 3 independent experiments, * P
Figure Legend Snippet: Pneumolysin induces mitochondrial fragmentation and disrupts mitochondrial motility in epithelial cells and human lung tissue. ( A ) A549 cells were labelled with MitoTrackerOrange and subsequently infected with S . pn . D39Δ cps or S . pn . D39Δ cps Δ ply for 5 hours, stimulated with 1.0 µg/ml PLY for 15 min or left untreated. Cells were fixed, stained with DAPI and mitochondrial morphology was analysed by structured illumination microscopy. Mitochondria were pseudocoloured using YellowHot LUT and a reconstructed widefield image of the nucleus is shown in blue. Scale bar represents 5 µm. ( B , C ) Quantification of mitochondrial morphology in control and PLY treated cells exemplified in ( A ). The mitochondrial network was examined and quantified in stochastically selected mitochondria-rich parts of the cells. Integrative network/shape analysis (number of branches per mitochondrion ( B ) and branch length per mitochondrion ( C )) was performed (mean ± SD from n = 3 independent experiments, * P

Techniques Used: Infection, Staining, Microscopy

Pneumolysin induces extracellular release of mtDNA. ( A ) Relative quantification of mitochondrial DNA released from A549 cells in the supernatant after stimulation with the indicated amounts of PLY after three hours. Data are expressed as amount of mitochondrial DNA normalized to unstimulated cells (ctrl). Bars represent mean ± SD from n = 5 independent experiments, * P
Figure Legend Snippet: Pneumolysin induces extracellular release of mtDNA. ( A ) Relative quantification of mitochondrial DNA released from A549 cells in the supernatant after stimulation with the indicated amounts of PLY after three hours. Data are expressed as amount of mitochondrial DNA normalized to unstimulated cells (ctrl). Bars represent mean ± SD from n = 5 independent experiments, * P

Techniques Used:

Infection with PLY expressing pneumococci and stimulation with PLY reduces cytosolic and mitochondrial ATP levels. A549 cells were transfected with plasmids encoding FRET-ATP sensors targeted to the cytosol ( A ) and mitochondria ( B ), respectively. 48 hours post transfection, cells were left unstimulated or infected with S . pn . D39Δ cps , S . pn . D39Δ cps Δ ply for 4 and 6 h, respectively, or stimulated with 0.25 µg/ml PLY for 30 min. Cells stimulated with 10 µg/ml Oligomycin for 30 min served as positive control. The ATP content is expressed as normalized ratio of the YFP/CFP peak intensity at 530 nm (YFP) and 478 nm (CFP), respectively measured by live spectral-FRET microscopy. Bars represent mean ± SD from n = 3 independent experiments. * P
Figure Legend Snippet: Infection with PLY expressing pneumococci and stimulation with PLY reduces cytosolic and mitochondrial ATP levels. A549 cells were transfected with plasmids encoding FRET-ATP sensors targeted to the cytosol ( A ) and mitochondria ( B ), respectively. 48 hours post transfection, cells were left unstimulated or infected with S . pn . D39Δ cps , S . pn . D39Δ cps Δ ply for 4 and 6 h, respectively, or stimulated with 0.25 µg/ml PLY for 30 min. Cells stimulated with 10 µg/ml Oligomycin for 30 min served as positive control. The ATP content is expressed as normalized ratio of the YFP/CFP peak intensity at 530 nm (YFP) and 478 nm (CFP), respectively measured by live spectral-FRET microscopy. Bars represent mean ± SD from n = 3 independent experiments. * P

Techniques Used: Infection, Expressing, Transfection, Positive Control, Microscopy

10) Product Images from "Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles"

Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

Journal: Oncotarget

doi: 10.18632/oncotarget.24772

Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.
Figure Legend Snippet: Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.

Techniques Used: Incubation, Fluorescence, Software, Staining, Microscopy

Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Negative Control, Staining, Fluorescence, Microscopy

Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).
Figure Legend Snippet: Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Software

Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Concentration Assay, Incubation, Staining, Confocal Microscopy, Microscopy

Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.
Figure Legend Snippet: Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Fluorescence

Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy, Software

Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Sequencing

11) Product Images from "Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection"

Article Title: Bunyavirus requirement for endosomal K+ reveals new roles of cellular ion channels during infection

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1006845

K + channel modulation can impede normal K + accumulation across the endocytic network. (A) A549 cells were treated for 30 min with 10 mM TEA or left untreated. AG4 (10 μM) was then added in the presence or absence of drug for 40 min. Dye was removed and TEA was re-added onto cells. Fluorescence intensities were quantified using IncuCyte ZOOM imaging and analysis software, and data normalised to untreated (unt) controls over three independent cell populations. NS–no significant difference between no-drug and TEA treated controls (p≥0.05). Scale bar = 10 μM. (B) (i) Cells were treated with 10 mM TEA (or left untreated) and AG4 (10 μm) added as in A , with the addition of Magic Red during the 40 min incubation with AG4. Representative images are shown (n≥60 cells). Scale bar = 10 μM. (ii) Total number of AG4 positive puncta were counted per cell ± TEA and the % of colocalised puncta presented. n≥60 cells, (* = p≤0.05). Scale bar = 10 μM. (C) (i) Cells were treated with 10 mM TEA or left untreated, and AG4 (10 μM) added as in A , with the addition of the pH indicator pHrodo red dextran (10 μg/ml) during the 40 min incubation with AG4. Representative images are shown (Scale bar = 10 μM) and the % of co-localised puncta presented in (ii) n≥60 cells (* = p≤0.05). D Fluorescence intensity of Magic Red was quantified using IncuCyte ZOOM imaging and analysis software and data normalised to untreated controls over three independent cell populations. NS–no significant difference between untreated and TEA treated cells (p≥0.05). Representative images are also shown.
Figure Legend Snippet: K + channel modulation can impede normal K + accumulation across the endocytic network. (A) A549 cells were treated for 30 min with 10 mM TEA or left untreated. AG4 (10 μM) was then added in the presence or absence of drug for 40 min. Dye was removed and TEA was re-added onto cells. Fluorescence intensities were quantified using IncuCyte ZOOM imaging and analysis software, and data normalised to untreated (unt) controls over three independent cell populations. NS–no significant difference between no-drug and TEA treated controls (p≥0.05). Scale bar = 10 μM. (B) (i) Cells were treated with 10 mM TEA (or left untreated) and AG4 (10 μm) added as in A , with the addition of Magic Red during the 40 min incubation with AG4. Representative images are shown (n≥60 cells). Scale bar = 10 μM. (ii) Total number of AG4 positive puncta were counted per cell ± TEA and the % of colocalised puncta presented. n≥60 cells, (* = p≤0.05). Scale bar = 10 μM. (C) (i) Cells were treated with 10 mM TEA or left untreated, and AG4 (10 μM) added as in A , with the addition of the pH indicator pHrodo red dextran (10 μg/ml) during the 40 min incubation with AG4. Representative images are shown (Scale bar = 10 μM) and the % of co-localised puncta presented in (ii) n≥60 cells (* = p≤0.05). D Fluorescence intensity of Magic Red was quantified using IncuCyte ZOOM imaging and analysis software and data normalised to untreated controls over three independent cell populations. NS–no significant difference between untreated and TEA treated cells (p≥0.05). Representative images are also shown.

Techniques Used: Fluorescence, Imaging, Software, Incubation

K + channel modulation arrests BUNV trafficking in endosomes. (A) Cells were treated with TEA (10 mM) for 30 min (or left untreated) and infected with SYTO82/DiD-BUNV for a further 4 hrs in the presence/absence of TEA. EGF-488 was added for the final 15 min of infection and cells were fixed 4 hpi. Confocal images were taken and the EGF-488 fluorescence channel removed in the representative images showing only SYTO82 and DiDvbt (n≥40). Scale bar = 10 μM. (B)(i) Cells treated with TEA (10 mM) or left untreated as in A , were infected with SYTO82/DiD-BUNV and fixed at 2, 4 or 8 hpi. EGF-488 (2 μg/ml) was added for 15 min prior to fixation as in A , with the representative images showing only SYTO82 and DiDvbt channels (n > 65 cells). Scale bar = 10 μM. (ii) As in (i) but cells were treated with Qd (200 μM) and fixed 8 hpi (n > 65 cells). (iii) The number of SYTO82/DiD-BUNV virions per cell were quantified using images from (i) and (ii) for n > 65 cells and normalised to the untreated (no-drug) control. (C) A549 cells were infected with SYTO82/DiD-BUNV for 1 hour at 4°C and treated with cytopainter to label lysosomes. Cells were warmed to 37°C for 1 hr, virus/dye removed by washing and cells incubated for up to 8 hpi. Representative live cell images are shown (≥80 cells). Scale bar = 10 μM. (ii) The number of SYTO82/DiD-BUNV virions co-localising with cytopainter positive puncta were calculated and the % of co-localised puncta presented in (ii) (* = p≤0.05).
Figure Legend Snippet: K + channel modulation arrests BUNV trafficking in endosomes. (A) Cells were treated with TEA (10 mM) for 30 min (or left untreated) and infected with SYTO82/DiD-BUNV for a further 4 hrs in the presence/absence of TEA. EGF-488 was added for the final 15 min of infection and cells were fixed 4 hpi. Confocal images were taken and the EGF-488 fluorescence channel removed in the representative images showing only SYTO82 and DiDvbt (n≥40). Scale bar = 10 μM. (B)(i) Cells treated with TEA (10 mM) or left untreated as in A , were infected with SYTO82/DiD-BUNV and fixed at 2, 4 or 8 hpi. EGF-488 (2 μg/ml) was added for 15 min prior to fixation as in A , with the representative images showing only SYTO82 and DiDvbt channels (n > 65 cells). Scale bar = 10 μM. (ii) As in (i) but cells were treated with Qd (200 μM) and fixed 8 hpi (n > 65 cells). (iii) The number of SYTO82/DiD-BUNV virions per cell were quantified using images from (i) and (ii) for n > 65 cells and normalised to the untreated (no-drug) control. (C) A549 cells were infected with SYTO82/DiD-BUNV for 1 hour at 4°C and treated with cytopainter to label lysosomes. Cells were warmed to 37°C for 1 hr, virus/dye removed by washing and cells incubated for up to 8 hpi. Representative live cell images are shown (≥80 cells). Scale bar = 10 μM. (ii) The number of SYTO82/DiD-BUNV virions co-localising with cytopainter positive puncta were calculated and the % of co-localised puncta presented in (ii) (* = p≤0.05).

Techniques Used: Infection, Fluorescence, Incubation

Production of SYTO82/DiD-BUNV to monitor virus trafficking. (A) Time course of A549 cells infected with BUNV. Cells were lysed at 3 hr intervals post-infection. Western blot analysis of BUNV-N protein and GAPDH (loading control) are shown (n = 3). (B) A549 cells were infected with BUNV supernatants collected from infected A549 cells at the indicated 3 hr intervals. Infected cells were fixed at 18 hpi and BUNV-N protein was labelled using anti-BUNV-N antibodies alongside Alexa-Fluor 594 nm secondary antibodies. Widefield images taken on the IncuCyte Zoom are shown (n = 3). Scale bar = 200μm (C) Schematic representation of BUNV labelling. A549 cells were infected with BUNV for 18 hrs, then SYTO82 dye was added to label the viral RNA segments until virus supernatants were collected at 24 hrs. Virus supernatants were concentrated, the BUNV envelope labelled with DiDvbt and SYTO82/DiD-BUNV was purified on a 10–30% iodixanol gradient. 1 ml fractions were collected (n = 3). (D) Fractions from SYTO82/DiD-BUNV purification were used to infect A549 cells for 18 hrs. Western blot analysis for BUNV-N was performed to confirm virus infectivity. (E) Cells were infected as in D , fixed, and stained with anti-BUNV-N and Alexa Fluor-488 antibodies. Widefield images were taken on the IncuCyte Zoom (n = 3). Scale bar = 200 μM. (F) A549 cells were infected with SYTO82/DiD-BUNV for 2 hrs and fixed. SYTO82 (em. max 560 nm) and DiDvbt (em. max 665 nm) fluorescent signals were imaged alongside DAPI in fixed cells. (G) Cells were infected with SYTO82/DiD-BUNV for 1 hr at 4°C, then heated to 37°C and infection was allowed to proceed until fixing at 2 hrs, 4 hrs, 8 hrs or 12 hrs. Biotinylated EGF-488 (2 μg/ml) was added for 15 min at 37°C prior to fixing to act as a cell marker. Confocal images were taken for n > 80 cells for each time point and the EGF-488 fluorescence channel was removed in the representative images (Scale bar = 10 μM). (H) NH 4 Cl (10 μM) was added at the indicated timepoints post-BUNV infection and BUNV-N expression assessed by western blot analysis 24 hours post-infection. CTL = no drug included during the timecourse (n = 3).
Figure Legend Snippet: Production of SYTO82/DiD-BUNV to monitor virus trafficking. (A) Time course of A549 cells infected with BUNV. Cells were lysed at 3 hr intervals post-infection. Western blot analysis of BUNV-N protein and GAPDH (loading control) are shown (n = 3). (B) A549 cells were infected with BUNV supernatants collected from infected A549 cells at the indicated 3 hr intervals. Infected cells were fixed at 18 hpi and BUNV-N protein was labelled using anti-BUNV-N antibodies alongside Alexa-Fluor 594 nm secondary antibodies. Widefield images taken on the IncuCyte Zoom are shown (n = 3). Scale bar = 200μm (C) Schematic representation of BUNV labelling. A549 cells were infected with BUNV for 18 hrs, then SYTO82 dye was added to label the viral RNA segments until virus supernatants were collected at 24 hrs. Virus supernatants were concentrated, the BUNV envelope labelled with DiDvbt and SYTO82/DiD-BUNV was purified on a 10–30% iodixanol gradient. 1 ml fractions were collected (n = 3). (D) Fractions from SYTO82/DiD-BUNV purification were used to infect A549 cells for 18 hrs. Western blot analysis for BUNV-N was performed to confirm virus infectivity. (E) Cells were infected as in D , fixed, and stained with anti-BUNV-N and Alexa Fluor-488 antibodies. Widefield images were taken on the IncuCyte Zoom (n = 3). Scale bar = 200 μM. (F) A549 cells were infected with SYTO82/DiD-BUNV for 2 hrs and fixed. SYTO82 (em. max 560 nm) and DiDvbt (em. max 665 nm) fluorescent signals were imaged alongside DAPI in fixed cells. (G) Cells were infected with SYTO82/DiD-BUNV for 1 hr at 4°C, then heated to 37°C and infection was allowed to proceed until fixing at 2 hrs, 4 hrs, 8 hrs or 12 hrs. Biotinylated EGF-488 (2 μg/ml) was added for 15 min at 37°C prior to fixing to act as a cell marker. Confocal images were taken for n > 80 cells for each time point and the EGF-488 fluorescence channel was removed in the representative images (Scale bar = 10 μM). (H) NH 4 Cl (10 μM) was added at the indicated timepoints post-BUNV infection and BUNV-N expression assessed by western blot analysis 24 hours post-infection. CTL = no drug included during the timecourse (n = 3).

Techniques Used: Infection, Western Blot, Purification, Staining, Activated Clotting Time Assay, Marker, Fluorescence, Expressing, CTL Assay

BUNV traffics through endosomes containing K + ions. (A) AG4 (10 μM) was added to A549 cells for 40 min to allow endosomal uptake, alongside (B)(i) Texas Red labelled EGF (2 μg/ml) or Magic Red cathepsin B dye. Non-internalised dyes were subsequently removed and live cells were imaged. Representative images are shown (n≥100 cells). Scale bar = 10 μM. (ii) Total numbers of AG4 positive puncta were counted per cell and % of colocalised AG4 puncta with each marker calculated in ≥100 cells. (C) AG4 (10 μM) was added to A549 cells for 40 min at either 37°C or 4°C and live cells were imaged as in A. Scale bar = 10 μM. (D) Schematic representation of AG4 uptake into endocytic vesicles and increased fluorescence with passage through early endosomes (EE) into late endosomes (LE) identifying K + -rich regions, identifiable using Texas Red labelled EGF. AG4 fluorescence decreases with passage into lysosomes (L). (E) A549 cells were infected with labelled-BUNV in the presence of AG4 (10μM) to allow virus penetration into cells and live cells were imaged 2 hrs or 8 hrs post-infection. Images are representative of ≥ 50 cells. (F)(i) A549 cells were infected with SYTO82/DiD-BUNV and EGF-488 (2 μg/ml) for 1 hour at 4°C and cells warmed to 37°C for the indicated timepoints. Images were taken of live cells at the indicated time points post-warming and are representative of ≥60 cells. Scale bar = 10 μM. (ii) Cells were transfected with Rab7-GFP and infected as in F(i) 24 hours post transfection. Images are representative of ≥ 40 cells. Scale bar = 10 μM. G(i) as in F(i) but cells were infected in the presence of 488-labelled Tf (25 μg/mL) or (ii) cells transfected with Rab11-GFP.
Figure Legend Snippet: BUNV traffics through endosomes containing K + ions. (A) AG4 (10 μM) was added to A549 cells for 40 min to allow endosomal uptake, alongside (B)(i) Texas Red labelled EGF (2 μg/ml) or Magic Red cathepsin B dye. Non-internalised dyes were subsequently removed and live cells were imaged. Representative images are shown (n≥100 cells). Scale bar = 10 μM. (ii) Total numbers of AG4 positive puncta were counted per cell and % of colocalised AG4 puncta with each marker calculated in ≥100 cells. (C) AG4 (10 μM) was added to A549 cells for 40 min at either 37°C or 4°C and live cells were imaged as in A. Scale bar = 10 μM. (D) Schematic representation of AG4 uptake into endocytic vesicles and increased fluorescence with passage through early endosomes (EE) into late endosomes (LE) identifying K + -rich regions, identifiable using Texas Red labelled EGF. AG4 fluorescence decreases with passage into lysosomes (L). (E) A549 cells were infected with labelled-BUNV in the presence of AG4 (10μM) to allow virus penetration into cells and live cells were imaged 2 hrs or 8 hrs post-infection. Images are representative of ≥ 50 cells. (F)(i) A549 cells were infected with SYTO82/DiD-BUNV and EGF-488 (2 μg/ml) for 1 hour at 4°C and cells warmed to 37°C for the indicated timepoints. Images were taken of live cells at the indicated time points post-warming and are representative of ≥60 cells. Scale bar = 10 μM. (ii) Cells were transfected with Rab7-GFP and infected as in F(i) 24 hours post transfection. Images are representative of ≥ 40 cells. Scale bar = 10 μM. G(i) as in F(i) but cells were infected in the presence of 488-labelled Tf (25 μg/mL) or (ii) cells transfected with Rab11-GFP.

Techniques Used: Marker, Fluorescence, Infection, Transfection

K + ions at pH 6.3 expedite BUNV infection. (A) Schematic protocol of BUNV priming at 37°C with buffers of varying pH and salt concentrations for 2 hrs. Buffers were subsequently diluted out with media and A549 cells were infected with treated virions for 18 hrs prior to cell lysis. Western blot analysis was performed on cell lysates using BUNV-N protein as a marker of BUNV infection and GAPDH as a loading control. (B) Cells were infected with BUNV that had been pre-treated with buffers at pH 7.3, 6.3 or 5.3 (no salt). Cells were lysed and analysed by western blot, as in A (n = 3). (C) BUNV virions were treated with pH 7.3, 6.3 and 5.3 buffers with and without 140 mM (i) KCl or (ii) NaCl. Cells were infected with treated BUNV diluted in media and lysates were analysed by western blot, as in A (n = 3). (iii) Levels of BUNV-N at pH 6.3 under the indicated conditions were quantified by densitometry ( n = 3). All error bars indicate mean ± SEM. *Significant difference from control ( P
Figure Legend Snippet: K + ions at pH 6.3 expedite BUNV infection. (A) Schematic protocol of BUNV priming at 37°C with buffers of varying pH and salt concentrations for 2 hrs. Buffers were subsequently diluted out with media and A549 cells were infected with treated virions for 18 hrs prior to cell lysis. Western blot analysis was performed on cell lysates using BUNV-N protein as a marker of BUNV infection and GAPDH as a loading control. (B) Cells were infected with BUNV that had been pre-treated with buffers at pH 7.3, 6.3 or 5.3 (no salt). Cells were lysed and analysed by western blot, as in A (n = 3). (C) BUNV virions were treated with pH 7.3, 6.3 and 5.3 buffers with and without 140 mM (i) KCl or (ii) NaCl. Cells were infected with treated BUNV diluted in media and lysates were analysed by western blot, as in A (n = 3). (iii) Levels of BUNV-N at pH 6.3 under the indicated conditions were quantified by densitometry ( n = 3). All error bars indicate mean ± SEM. *Significant difference from control ( P

Techniques Used: Infection, Lysis, Western Blot, Marker

12) Product Images from "Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells"

Article Title: Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbalip.2017.11.005

Analysis of selected phospholipid species formed when A549 cells were treated with purified PA-LOX. A549 cells were incubated in the presence/absence of varying amounts of PA-LOX (50 μg, 150 μg, 500 μg/ml) for 12 h and 24 h. Lipid extracts were analyzed by reverse-phase LC/MS/MS, in negative mode, using Luna column on 6500 Q Trap. Single incubations were carried out for each PA-LOX concentration. Each sample was analyzed once by RP-HPLC and in triplicate LC-MS/MS.
Figure Legend Snippet: Analysis of selected phospholipid species formed when A549 cells were treated with purified PA-LOX. A549 cells were incubated in the presence/absence of varying amounts of PA-LOX (50 μg, 150 μg, 500 μg/ml) for 12 h and 24 h. Lipid extracts were analyzed by reverse-phase LC/MS/MS, in negative mode, using Luna column on 6500 Q Trap. Single incubations were carried out for each PA-LOX concentration. Each sample was analyzed once by RP-HPLC and in triplicate LC-MS/MS.

Techniques Used: Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Concentration Assay, High Performance Liquid Chromatography

Confocal light micrographs of cultured A549 cells. Pre-confluenet A549 cells were incubated in the absence (panel A) and presence (panel B) of 250 μg/ml pure recombinant PA-LOX for 16 h. Four different no-enzyme controls wells and four PA-LOX incubation wells were set up. Representative images are shown and mitotic cells are labeled by the asterix.
Figure Legend Snippet: Confocal light micrographs of cultured A549 cells. Pre-confluenet A549 cells were incubated in the absence (panel A) and presence (panel B) of 250 μg/ml pure recombinant PA-LOX for 16 h. Four different no-enzyme controls wells and four PA-LOX incubation wells were set up. Representative images are shown and mitotic cells are labeled by the asterix.

Techniques Used: Cell Culture, Incubation, Recombinant, Labeling

13) Product Images from "ToF-SIMS 3D imaging unveils important insights on the cellular microenvironment during biomineralization of gold nanostructures"

Article Title: ToF-SIMS 3D imaging unveils important insights on the cellular microenvironment during biomineralization of gold nanostructures

Journal: Scientific Reports

doi: 10.1038/s41598-019-57136-w

High resolution transmission electron microscopy (HR-TEM) and nanoparticle tracking analysis (NTA) of biomineralized AuNP materials. The Brownian motion and light scattering information from NTA gives the size of distribution and HRTEM and shape of nanostructures formed by A549 cells treated with ( A ) 0.5 mM; ( B ) 1.0 mM; ( C ) 2.0 mM; and ( D ) 2.0 mM HAuCl 4 with spherical Au-NPs as seeds. ( E , F ) NTA plots showing size distribution after 5 rounds of tracking of spherical NPs ( E ) corresponding to scheme ( A ) and nanoribbons ( F ) corresponding to ( C ) of right panel in Fig. 1 .
Figure Legend Snippet: High resolution transmission electron microscopy (HR-TEM) and nanoparticle tracking analysis (NTA) of biomineralized AuNP materials. The Brownian motion and light scattering information from NTA gives the size of distribution and HRTEM and shape of nanostructures formed by A549 cells treated with ( A ) 0.5 mM; ( B ) 1.0 mM; ( C ) 2.0 mM; and ( D ) 2.0 mM HAuCl 4 with spherical Au-NPs as seeds. ( E , F ) NTA plots showing size distribution after 5 rounds of tracking of spherical NPs ( E ) corresponding to scheme ( A ) and nanoribbons ( F ) corresponding to ( C ) of right panel in Fig. 1 .

Techniques Used: Transmission Assay, Electron Microscopy, Transmission Electron Microscopy

The ion image 3D reconstruction of Au + signals (orange), and arginine-Au(I)-imine (blue) in top ( A ) view of the Au nanoribbon clusters on A549 cell. ( B ) The SIMS signals in side view from Au + (orange) and arginine-Au(I)-imine (blue) showing that there is a change in chemical specificity of biocomplexation during Au mineralization supplemented with spherical NPs as ‘seed’ in cellular microenvironment. ( C ) The secondary ion signals from Au + (m/z: 196.97) and arginine-Au(I)-imine (m/z: 417.06) (lower panel) and their comparison with untreated A549 cells (upper panel).
Figure Legend Snippet: The ion image 3D reconstruction of Au + signals (orange), and arginine-Au(I)-imine (blue) in top ( A ) view of the Au nanoribbon clusters on A549 cell. ( B ) The SIMS signals in side view from Au + (orange) and arginine-Au(I)-imine (blue) showing that there is a change in chemical specificity of biocomplexation during Au mineralization supplemented with spherical NPs as ‘seed’ in cellular microenvironment. ( C ) The secondary ion signals from Au + (m/z: 196.97) and arginine-Au(I)-imine (m/z: 417.06) (lower panel) and their comparison with untreated A549 cells (upper panel).

Techniques Used:

Scanning electron microscopy and ToF-SIMS molecular 3D imaging of condition ( A ) in schematic Fig. 1 . Scanning electron microscopy ( A , B ) and X-ray spectroscopic ( C , D ) analysis of A549 cells treated with 0.5 mM HAuCl 4 confirms NP formation (scale bar 5 µm in A,C and 200 nm in B ). The ion signals from Au + ( E , untreated A549 cells; G , ionic gold) and threonine- O -3-phosphate aureate ( F , untreated A549 cells; H , A549 cells treated with low dose of ionic gold). ( I , J ) Topography of extracellular spherical Au nanostructures on top of A549 cells (square ROI 130 µ × 130 µ in I and 80 µ × 80 µ in J). ( K ) Reconstructed 3D depth profile of mineralized Au structures in A549 cell cultures. The Au + signal (red, translucent) indicates the spherical Au-NP, whilst the signal for threonine- O -3-phosphate aureate is shown in green. ( L ) Enlarged view from ( K , arrow) showing the combined distribution of threonine- O -3-phosphate (green) and Au-NPs signals (orange) with occasional overlap (ring).
Figure Legend Snippet: Scanning electron microscopy and ToF-SIMS molecular 3D imaging of condition ( A ) in schematic Fig. 1 . Scanning electron microscopy ( A , B ) and X-ray spectroscopic ( C , D ) analysis of A549 cells treated with 0.5 mM HAuCl 4 confirms NP formation (scale bar 5 µm in A,C and 200 nm in B ). The ion signals from Au + ( E , untreated A549 cells; G , ionic gold) and threonine- O -3-phosphate aureate ( F , untreated A549 cells; H , A549 cells treated with low dose of ionic gold). ( I , J ) Topography of extracellular spherical Au nanostructures on top of A549 cells (square ROI 130 µ × 130 µ in I and 80 µ × 80 µ in J). ( K ) Reconstructed 3D depth profile of mineralized Au structures in A549 cell cultures. The Au + signal (red, translucent) indicates the spherical Au-NP, whilst the signal for threonine- O -3-phosphate aureate is shown in green. ( L ) Enlarged view from ( K , arrow) showing the combined distribution of threonine- O -3-phosphate (green) and Au-NPs signals (orange) with occasional overlap (ring).

Techniques Used: Electron Microscopy, Imaging

The constructed ion image reveals quasi spherical Au-NPs with increasing Au ions to 1 mM Au ions. ( A , B ) The topography is displayed by total ion reconstruction image of the A549 cell surface. Inset show SEM image of quasi-spherical nanostructure. ( C ) 3D reconstructed ion image of a single cluster with the Au + signals (m/z 196.97) picked from biomineralized cell surface. ( D ) The zoomed in-depth 3D reconstruction overlay of cluster shown in ( C ) in side view of spatiotemporal Au + signals (orange) with threonine- O -3-phosphate aureate (green).
Figure Legend Snippet: The constructed ion image reveals quasi spherical Au-NPs with increasing Au ions to 1 mM Au ions. ( A , B ) The topography is displayed by total ion reconstruction image of the A549 cell surface. Inset show SEM image of quasi-spherical nanostructure. ( C ) 3D reconstructed ion image of a single cluster with the Au + signals (m/z 196.97) picked from biomineralized cell surface. ( D ) The zoomed in-depth 3D reconstruction overlay of cluster shown in ( C ) in side view of spatiotemporal Au + signals (orange) with threonine- O -3-phosphate aureate (green).

Techniques Used: Construct

The schematic overview of the workflow of the 3D biomolecular imaging of de novo biomineralization of ionic gold into anisotropic (0, 1 2D) nanostructures. Left panel show the collection of secondary ions by the detector after the primary ion beam impacts freeze dried A549 cells with embedded anisotropic gold nanostructres. The right panel shows that ToF-SIMS images can be reconstructed into 3D space to give molecular distributions of gold and reducing agent in three different culture environments resulting in 3 different nanostructures (spheres, irregular particles and nanoribbons).
Figure Legend Snippet: The schematic overview of the workflow of the 3D biomolecular imaging of de novo biomineralization of ionic gold into anisotropic (0, 1 2D) nanostructures. Left panel show the collection of secondary ions by the detector after the primary ion beam impacts freeze dried A549 cells with embedded anisotropic gold nanostructres. The right panel shows that ToF-SIMS images can be reconstructed into 3D space to give molecular distributions of gold and reducing agent in three different culture environments resulting in 3 different nanostructures (spheres, irregular particles and nanoribbons).

Techniques Used: Imaging

SEM and ToF-SIMS analysis of de novo biomineralized nanostructures at 2 mM Au ions: delta and rhombus shaped particles. ( A – D ) SEM and EDX chemical mapping to confirm anisotropic rhombus/delta shape Au nanoplate formation (Scale bar 1 micron). ( E , F ) The SIMS signals from Au + ( E ) and threonine- O -3-phosphate aureate ( F ). ( G ) Reconstructed ToF-SIMS ion image from the A549 cell surface demonstrating spherical Au particles. ( H ) The 3D reconstruction and overlay of Au + signal (orange) and threonine- O -3-phosphate aureate (green) from red square ROI from ( G ).
Figure Legend Snippet: SEM and ToF-SIMS analysis of de novo biomineralized nanostructures at 2 mM Au ions: delta and rhombus shaped particles. ( A – D ) SEM and EDX chemical mapping to confirm anisotropic rhombus/delta shape Au nanoplate formation (Scale bar 1 micron). ( E , F ) The SIMS signals from Au + ( E ) and threonine- O -3-phosphate aureate ( F ). ( G ) Reconstructed ToF-SIMS ion image from the A549 cell surface demonstrating spherical Au particles. ( H ) The 3D reconstruction and overlay of Au + signal (orange) and threonine- O -3-phosphate aureate (green) from red square ROI from ( G ).

Techniques Used:

14) Product Images from "Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells"

Article Title: Specific oxygenation of plasma membrane phospholipids by Pseudomonas aeruginosa lipoxygenase induces structural and functional alterations in mammalian cells

Journal: Biochimica et Biophysica Acta

doi: 10.1016/j.bbalip.2017.11.005

Analysis of selected phospholipid species formed when A549 cells were treated with purified PA-LOX. A549 cells were incubated in the presence/absence of varying amounts of PA-LOX (50 μg, 150 μg, 500 μg/ml) for 12 h and 24 h. Lipid extracts were analyzed by reverse-phase LC/MS/MS, in negative mode, using Luna column on 6500 Q Trap. Single incubations were carried out for each PA-LOX concentration. Each sample was analyzed once by RP-HPLC and in triplicate LC-MS/MS.
Figure Legend Snippet: Analysis of selected phospholipid species formed when A549 cells were treated with purified PA-LOX. A549 cells were incubated in the presence/absence of varying amounts of PA-LOX (50 μg, 150 μg, 500 μg/ml) for 12 h and 24 h. Lipid extracts were analyzed by reverse-phase LC/MS/MS, in negative mode, using Luna column on 6500 Q Trap. Single incubations were carried out for each PA-LOX concentration. Each sample was analyzed once by RP-HPLC and in triplicate LC-MS/MS.

Techniques Used: Purification, Incubation, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Concentration Assay, High Performance Liquid Chromatography

15) Product Images from "Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro"

Article Title: Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro

Journal: Oncology Letters

doi: 10.3892/ol.2018.9462

HBFs stimulate the invasive behavior of A549 cells. (A) A549 cells and AS HBFs were seeded at a density of 10 4 and 10 3 cells/cm 2 , respectively, and were incubated for 48 h in Dulbecco's modified Eagle's medium supplemented with a 10% fetal calf serum. The motility of A549 cells was estimated with time-lapse videomicroscopy and (C) its parameters were quantified in comparison to control A549 cells. (B) A549/HBF co-cultures were established as in (A) and cell morphology was visualized with NIC microscopy. Scale bars, 100 µm. (D) The motility of A549 cells in co-cultures with NA HBFs estimated by time-lapse videomicroscopy as in (A). At least 50 cell trajectories were drawn for each condition and presented in correlative plots. Dot-plots and column charts present movement parameters at the single cell and population level, respectively. Data are representative of 3 independent experiments. *P
Figure Legend Snippet: HBFs stimulate the invasive behavior of A549 cells. (A) A549 cells and AS HBFs were seeded at a density of 10 4 and 10 3 cells/cm 2 , respectively, and were incubated for 48 h in Dulbecco's modified Eagle's medium supplemented with a 10% fetal calf serum. The motility of A549 cells was estimated with time-lapse videomicroscopy and (C) its parameters were quantified in comparison to control A549 cells. (B) A549/HBF co-cultures were established as in (A) and cell morphology was visualized with NIC microscopy. Scale bars, 100 µm. (D) The motility of A549 cells in co-cultures with NA HBFs estimated by time-lapse videomicroscopy as in (A). At least 50 cell trajectories were drawn for each condition and presented in correlative plots. Dot-plots and column charts present movement parameters at the single cell and population level, respectively. Data are representative of 3 independent experiments. *P

Techniques Used: Incubation, Modification, Microscopy

AS HBFs induce the motility of A549 cells via contact-modulated paracrine signaling. (A) A549 cells were cultivated in the media conditioned by AS2 HBFs (left), ‘separated’ (middle) and ‘confronted’ AS HBF/A549 co-cultures (1:1 v/v with fresh medium) for 48 h and (C) the parameters of their motility were analyzed by time-lapse videomicroscopy in comparison to A549 motility in control conditions and in ‘open’ AS HBF/A549 co-cultures. (B) A549 cells were cultivated in the media conditioned by ‘separated’ co-cultures of A549 with AS and NA HBFs (1:1 v/v with fresh medium) for 48 h. Intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA was visualized with immunofluorescence and cytofluorimetry, respectively (left axes/blue line: DNA; right axes/green line: Snail-1). Scale bar, 50 µm; magnification, ×400. (D) The motility of A549 cells in the presence of the media conditioned by NA HBFs (left), ‘separated’ (middle) and ‘confronted’ NA HBF/A549 co-cultures (1:1 v/v with fresh medium) was analyzed as in (A). Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P
Figure Legend Snippet: AS HBFs induce the motility of A549 cells via contact-modulated paracrine signaling. (A) A549 cells were cultivated in the media conditioned by AS2 HBFs (left), ‘separated’ (middle) and ‘confronted’ AS HBF/A549 co-cultures (1:1 v/v with fresh medium) for 48 h and (C) the parameters of their motility were analyzed by time-lapse videomicroscopy in comparison to A549 motility in control conditions and in ‘open’ AS HBF/A549 co-cultures. (B) A549 cells were cultivated in the media conditioned by ‘separated’ co-cultures of A549 with AS and NA HBFs (1:1 v/v with fresh medium) for 48 h. Intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA was visualized with immunofluorescence and cytofluorimetry, respectively (left axes/blue line: DNA; right axes/green line: Snail-1). Scale bar, 50 µm; magnification, ×400. (D) The motility of A549 cells in the presence of the media conditioned by NA HBFs (left), ‘separated’ (middle) and ‘confronted’ NA HBF/A549 co-cultures (1:1 v/v with fresh medium) was analyzed as in (A). Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P

Techniques Used: Immunofluorescence

AS HBFs selectively modulate the invasion of A549 cells. (A) A549 cells were grown to confluence with NA HBFs (left-hand panel) or AS HBFs (right-hand panel) in 2-well silicone inserts and co-incubated for 48 h following diaphragm removal prior to fixation and immunostaining for α-SMA. Expression of α-SMA was visualized by immunofluorescence and cytofluorimetry, respectively. Scale bar, 100 µm; magnification, ×200. Inserts present A549 cells within the HBF monolayer. (B) Cells were cultivated as in (A) and intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA were visualized with immunofluorescence and cytofluorimetry, respectively (left axes: DNA; right axes: Snail-1). Photomicrographs in (A) and (B) present panoramic till scans of the interfaces between HBF and A549 monolayers obtained by the composition of two rows of succeeding images (7 pictures in a row). Scale bars, 100 µm. (C) NA HBFs and AS HBFs were cultivated in the presence of the media conditioned by ‘separated’ NA HBF/A549 and AS HBF/A549 co-cultures, respectively, for 48 h and immunostained against α-SMA, Snail-1 and Cx43. Scale bars, 50 µm; magnification, ×630. (D) A549 cells were seeded onto microporous membrane, placed in the wells filled with the media conditioned by A549/HBF co-cultures, and allowed to transmigrate for 48 h. The number of the transmigrated cells were counted following 24 h. Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P
Figure Legend Snippet: AS HBFs selectively modulate the invasion of A549 cells. (A) A549 cells were grown to confluence with NA HBFs (left-hand panel) or AS HBFs (right-hand panel) in 2-well silicone inserts and co-incubated for 48 h following diaphragm removal prior to fixation and immunostaining for α-SMA. Expression of α-SMA was visualized by immunofluorescence and cytofluorimetry, respectively. Scale bar, 100 µm; magnification, ×200. Inserts present A549 cells within the HBF monolayer. (B) Cells were cultivated as in (A) and intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA were visualized with immunofluorescence and cytofluorimetry, respectively (left axes: DNA; right axes: Snail-1). Photomicrographs in (A) and (B) present panoramic till scans of the interfaces between HBF and A549 monolayers obtained by the composition of two rows of succeeding images (7 pictures in a row). Scale bars, 100 µm. (C) NA HBFs and AS HBFs were cultivated in the presence of the media conditioned by ‘separated’ NA HBF/A549 and AS HBF/A549 co-cultures, respectively, for 48 h and immunostained against α-SMA, Snail-1 and Cx43. Scale bars, 50 µm; magnification, ×630. (D) A549 cells were seeded onto microporous membrane, placed in the wells filled with the media conditioned by A549/HBF co-cultures, and allowed to transmigrate for 48 h. The number of the transmigrated cells were counted following 24 h. Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P

Techniques Used: Incubation, Immunostaining, Expressing, Immunofluorescence

16) Product Images from "Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles"

Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

Journal: Oncotarget

doi: 10.18632/oncotarget.24772

Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.
Figure Legend Snippet: Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.

Techniques Used: Incubation, Fluorescence, Software, Staining, Microscopy

Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Negative Control, Staining, Fluorescence, Microscopy

Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).
Figure Legend Snippet: Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Software

Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Concentration Assay, Incubation, Staining, Confocal Microscopy, Microscopy

Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.
Figure Legend Snippet: Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Fluorescence

Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy, Software

Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Sequencing

17) Product Images from "Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis"

Article Title: Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis

Journal: Frontiers in Pharmacology

doi: 10.3389/fphar.2018.00738

The EMT gene expression at baseline and in the presence of TGF-β (100 pM, 24 h incubation) was measured in the presence of vehicle (Veh, 0.1% DMSO) or over a range of concentrations of PF670462 (0.1 – 10 μM), Pirfenidone (1 –100 μM)) or nintedanib (10 –1000 nM) in. Data are presented as mean and SEM of n = 4 independent experiments and show the TGF-β induced fold increase in the expression of genes that change during EMT or in response to TGF-β in A549 cells, including N -cadherin ( N -Cad), Vimentin (Vim), E -Cadherin ( E -Cad), α-smooth muscle actin (a-SMA) plasminogen-activator inhibitor-1 (PAI-1). Data were analyzed by two-way ANOVA with repeated measures, followed by comparisons at individual concentrations using Bonferroni’s correction for multiple comparisons. ∗ P
Figure Legend Snippet: The EMT gene expression at baseline and in the presence of TGF-β (100 pM, 24 h incubation) was measured in the presence of vehicle (Veh, 0.1% DMSO) or over a range of concentrations of PF670462 (0.1 – 10 μM), Pirfenidone (1 –100 μM)) or nintedanib (10 –1000 nM) in. Data are presented as mean and SEM of n = 4 independent experiments and show the TGF-β induced fold increase in the expression of genes that change during EMT or in response to TGF-β in A549 cells, including N -cadherin ( N -Cad), Vimentin (Vim), E -Cadherin ( E -Cad), α-smooth muscle actin (a-SMA) plasminogen-activator inhibitor-1 (PAI-1). Data were analyzed by two-way ANOVA with repeated measures, followed by comparisons at individual concentrations using Bonferroni’s correction for multiple comparisons. ∗ P

Techniques Used: Expressing, Incubation

(A) Effect of PF670462 on EMT associated gene induction in A549 alveolar epithelial cells 4 and 24 h after TGF-β (100 pM) stimulation ( n = 4). (B) Immunofluorescence of membrane E -cadherin expression in A549 alveolar epithelial cells. Cells were treated with TGF-β (100 pM) for 48 h with PF670462 added 30 min prior to TGF-β. Nuclei were stained with DAPI. Quantification of n = 4 experiments is shown in (C) with > 50 fields analyzed per well. Data are presented as mean ± SEM from 4 independent experiments on A549 epithelial cells. ∗ P
Figure Legend Snippet: (A) Effect of PF670462 on EMT associated gene induction in A549 alveolar epithelial cells 4 and 24 h after TGF-β (100 pM) stimulation ( n = 4). (B) Immunofluorescence of membrane E -cadherin expression in A549 alveolar epithelial cells. Cells were treated with TGF-β (100 pM) for 48 h with PF670462 added 30 min prior to TGF-β. Nuclei were stained with DAPI. Quantification of n = 4 experiments is shown in (C) with > 50 fields analyzed per well. Data are presented as mean ± SEM from 4 independent experiments on A549 epithelial cells. ∗ P

Techniques Used: Immunofluorescence, Expressing, Staining

18) Product Images from "Potential Toxicity of Polymyxins in Human Lung Epithelial Cells"

Article Title: Potential Toxicity of Polymyxins in Human Lung Epithelial Cells

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.02690-16

(A) Sensitivity of A549 (black bars) and HK-2 (gray bars) cells to polymyxin B. ****, P
Figure Legend Snippet: (A) Sensitivity of A549 (black bars) and HK-2 (gray bars) cells to polymyxin B. ****, P

Techniques Used:

Concentration (A, B)- and time (C, D)-dependent activation of caspase 3 in A549 cells. Activation was measured with the caspase 3-specific fluorogenic substrate Red-DEVD-FMK. For the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. ***, P
Figure Legend Snippet: Concentration (A, B)- and time (C, D)-dependent activation of caspase 3 in A549 cells. Activation was measured with the caspase 3-specific fluorogenic substrate Red-DEVD-FMK. For the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. ***, P

Techniques Used: Concentration Assay, Activation Assay

Colistin- and CMS-induced mitochondrial oxidative stress in A549 cells. (A, B) Concentration-dependent (2.0, 4.0, and 6.0 mM) mitochondrial oxidative stress was detected with MitoSOX only in the colistin group at 24 h. Scale bar, 50 μm. Results are presented as the mean ± SD ( n = 3). **, P
Figure Legend Snippet: Colistin- and CMS-induced mitochondrial oxidative stress in A549 cells. (A, B) Concentration-dependent (2.0, 4.0, and 6.0 mM) mitochondrial oxidative stress was detected with MitoSOX only in the colistin group at 24 h. Scale bar, 50 μm. Results are presented as the mean ± SD ( n = 3). **, P

Techniques Used: Concentration Assay

(A, B) Concentration- and time-dependent FasL expression in A549 cells treated with 0.25, 1.0, or 2.0 mM polymyxin B for 24 h. FasL expression was detected by flow cytometry with anti-FasL antibody CD178 conjugated with Alexa Fluor 647. (C, D) Time-dependent FasL expression of A549 cells treated with 1.0 mM (black bars) or 2.0 mM (gray bars) polymyxin B. The profiles are representative of three independent experiments. The percentage of FasL-positive cells is presented as the mean ± SD ( n = 3). *, P
Figure Legend Snippet: (A, B) Concentration- and time-dependent FasL expression in A549 cells treated with 0.25, 1.0, or 2.0 mM polymyxin B for 24 h. FasL expression was detected by flow cytometry with anti-FasL antibody CD178 conjugated with Alexa Fluor 647. (C, D) Time-dependent FasL expression of A549 cells treated with 1.0 mM (black bars) or 2.0 mM (gray bars) polymyxin B. The profiles are representative of three independent experiments. The percentage of FasL-positive cells is presented as the mean ± SD ( n = 3). *, P

Techniques Used: Concentration Assay, Expressing, Flow Cytometry, Cytometry

Concentration (A, B)- and time (C, D)-dependent activation of caspase 8 in A549 cells measured with the caspase 8-specific fluorogenic substrate Red-IETD-FMK. In the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. ***, P
Figure Legend Snippet: Concentration (A, B)- and time (C, D)-dependent activation of caspase 8 in A549 cells measured with the caspase 8-specific fluorogenic substrate Red-IETD-FMK. In the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. ***, P

Techniques Used: Concentration Assay, Activation Assay

A549 cell death and apoptosis induced by polymyxins. (A) A549 cell death at 24 h as a function of polymyxin B concentration. Data are presented as the mean ± SD ( n = 3). The EC 50 values of colistin and CMS were not calculated, as colistin-induced cell death (red) did not reach a plateau and no significant cell death was observed with CMS treatment (green). (B) Time-dependent cell death induced by polymyxin B at 2.0 mM (mean ± SD; n = 3). Filled circles represent polymyxin B treatment, and filled squares represent the untreated control.
Figure Legend Snippet: A549 cell death and apoptosis induced by polymyxins. (A) A549 cell death at 24 h as a function of polymyxin B concentration. Data are presented as the mean ± SD ( n = 3). The EC 50 values of colistin and CMS were not calculated, as colistin-induced cell death (red) did not reach a plateau and no significant cell death was observed with CMS treatment (green). (B) Time-dependent cell death induced by polymyxin B at 2.0 mM (mean ± SD; n = 3). Filled circles represent polymyxin B treatment, and filled squares represent the untreated control.

Techniques Used: Concentration Assay

Polymyxin B-induced mitochondrial oxidative stress in A549 cells. (A, B) Concentration-dependent (0.25, 1.0, and 2.0 mM) polymyxin-induced mitochondrial oxidative stress was detected with MitoSOX. (C, D) Time-dependent mitochondrial oxidative stress at 1.0 mM (black bars) or 2.0 mM (gray bars) polymyxin B. Scale bars, 50 μm. Results are presented as the mean ± SD ( n = 3). **, P
Figure Legend Snippet: Polymyxin B-induced mitochondrial oxidative stress in A549 cells. (A, B) Concentration-dependent (0.25, 1.0, and 2.0 mM) polymyxin-induced mitochondrial oxidative stress was detected with MitoSOX. (C, D) Time-dependent mitochondrial oxidative stress at 1.0 mM (black bars) or 2.0 mM (gray bars) polymyxin B. Scale bars, 50 μm. Results are presented as the mean ± SD ( n = 3). **, P

Techniques Used: Concentration Assay

Concentration (A, B)- and time (C, D)-dependent activation of caspase 9 in A549 cells. Activation was measured with the caspase 9-specific fluorogenic substrate Red-LEHD-FMK. In the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. *, P
Figure Legend Snippet: Concentration (A, B)- and time (C, D)-dependent activation of caspase 9 in A549 cells. Activation was measured with the caspase 9-specific fluorogenic substrate Red-LEHD-FMK. In the time-dependent experiments, the black and gray bars represent 1.0 and 2.0 mM polymyxin B, respectively. Scale bars, 50 μm. Group results are presented as the mean ± SD; n = 3. *, P

Techniques Used: Concentration Assay, Activation Assay

19) Product Images from "Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET"

Article Title: Non-Agonistic Bivalent Antibodies That Promote c-MET Degradation and Inhibit Tumor Growth and Others Specific for Tumor Related c-MET

Journal: PLoS ONE

doi: 10.1371/journal.pone.0034658

Antagonist activity of single chain variable fragments. LMH 85 and LMH 87 were converted into scFv format and tested for their ability to block HGF/SF stimulation of the c-MET receptor. A549 cells were treated with either scFv 85 or scFv 87 and then HGF/SF added. A vehicle control without HGF/SF was included. Whole cell lysates were then probed for total c-MET ( upper panel ), phosphorylated c-MET (Tyr1234/1235; middle panel ) and pan-actin to control for loading ( bottom panel ). The phosphorylated c-MET blot shows that scFv 85, but not scFv 87, inhibited HGF/SF stimulation of c-MET.
Figure Legend Snippet: Antagonist activity of single chain variable fragments. LMH 85 and LMH 87 were converted into scFv format and tested for their ability to block HGF/SF stimulation of the c-MET receptor. A549 cells were treated with either scFv 85 or scFv 87 and then HGF/SF added. A vehicle control without HGF/SF was included. Whole cell lysates were then probed for total c-MET ( upper panel ), phosphorylated c-MET (Tyr1234/1235; middle panel ) and pan-actin to control for loading ( bottom panel ). The phosphorylated c-MET blot shows that scFv 85, but not scFv 87, inhibited HGF/SF stimulation of c-MET.

Techniques Used: Activity Assay, Blocking Assay

LMH 80, 81 and 82 bind specifically to cell surface p170 c-MET precursor. (A) LMH 80, 81 and 82 only precipitate the p170 c-MET precursor. c-MET was IPed from LoVo cells ( top panel ) or A549 cells ( bottom panel ) using LMH 80, 81, 82 and LMH 85 and probed for total c-MET. A single 170 kDa band was visible in the LMH 80, 81 and 82 lanes, but not the 145 kDa β-chain. The 145 kDa β-chain was clearly present following IP with LMH 85. (B) LMH 80 binds p170 c-MET on the cell surface but is not internalized. Alexa-Fluor 488-labeled LMH 80 was bound to LoVo ( left panels ) and A549 ( right panels ) cells at 4°C to prevent internalization. Warm medium was added to initiate internalization and cells analyzed by confocal microscopy; no significant internalization was observed. A labeled LMH 85 positive control and labeled irrelevant isotype control antibody were included at 0 min.
Figure Legend Snippet: LMH 80, 81 and 82 bind specifically to cell surface p170 c-MET precursor. (A) LMH 80, 81 and 82 only precipitate the p170 c-MET precursor. c-MET was IPed from LoVo cells ( top panel ) or A549 cells ( bottom panel ) using LMH 80, 81, 82 and LMH 85 and probed for total c-MET. A single 170 kDa band was visible in the LMH 80, 81 and 82 lanes, but not the 145 kDa β-chain. The 145 kDa β-chain was clearly present following IP with LMH 85. (B) LMH 80 binds p170 c-MET on the cell surface but is not internalized. Alexa-Fluor 488-labeled LMH 80 was bound to LoVo ( left panels ) and A549 ( right panels ) cells at 4°C to prevent internalization. Warm medium was added to initiate internalization and cells analyzed by confocal microscopy; no significant internalization was observed. A labeled LMH 85 positive control and labeled irrelevant isotype control antibody were included at 0 min.

Techniques Used: Labeling, Confocal Microscopy, Positive Control

Representative data showing characterization of selected LMH antibodies. (A) flow cytometry analysis showing that LMH 85 and LMH 87 bind surface c-MET expressed on A549 cells. (B) western blots of representative LMH antibodies. c-MET was IPed with LMH 85 and membranes probed with indicated antibodies. The commercial 25H2 recognized the 170 kDa c-MET precursor (p170 c-MET; contains both α and β-chain) and the c-MET β-chain. Using this technique, LMH 80 only weakly bound the β-chain, while LMH 82, 84 and 87 bound p170 c-MET and α-chain. (C) IP with selected LMH antibodies. Following IP with different LMH antibodies, membranes were blotted with mAb 25H2. Antibodies shown were all capable of IP but LMH 80, LMH 81 and LMH 82 appeared specific for p170 c-MET using this technique. (D) biochemical activity of selected LMH antibodies. A459 cells were treated with antibody alone or antibody in the presence of HGF/SF and WCL were probed for total c-MET ( upper panels ) or phosphorylated c-MET (Y1234/Y1235) ( lower panels ). LMH 85 stimulated c-MET phosphorylation, whilst LMH 86 and LMH 87 did not. LMH 86 was unable to block HGF/SF stimulated c-MET phosphorylation. (E) effect of selected LMH antibodies on cell migration. Antibodies were tested for their ability to induce cell migration in SK-OV-3 cells. LMH 85 stimulated cell migration while LMH 87 had no effect ( upper panel ). Differing concentrations of LMH 87 were mixed with 3×10 −10 M HGF/SF to determine if it inhibited the HGF/SF induced migration of SK-OV-3 cells. LMH 87 substantially inhibited the migratory activity stimulated by HGF/SF ( lower panel ). Data in both graphs are presented as percentage migration versus 3×10 −10 M HGF/SF ± SD. A full summary of all LMH antibodies is contained in Table 1 .
Figure Legend Snippet: Representative data showing characterization of selected LMH antibodies. (A) flow cytometry analysis showing that LMH 85 and LMH 87 bind surface c-MET expressed on A549 cells. (B) western blots of representative LMH antibodies. c-MET was IPed with LMH 85 and membranes probed with indicated antibodies. The commercial 25H2 recognized the 170 kDa c-MET precursor (p170 c-MET; contains both α and β-chain) and the c-MET β-chain. Using this technique, LMH 80 only weakly bound the β-chain, while LMH 82, 84 and 87 bound p170 c-MET and α-chain. (C) IP with selected LMH antibodies. Following IP with different LMH antibodies, membranes were blotted with mAb 25H2. Antibodies shown were all capable of IP but LMH 80, LMH 81 and LMH 82 appeared specific for p170 c-MET using this technique. (D) biochemical activity of selected LMH antibodies. A459 cells were treated with antibody alone or antibody in the presence of HGF/SF and WCL were probed for total c-MET ( upper panels ) or phosphorylated c-MET (Y1234/Y1235) ( lower panels ). LMH 85 stimulated c-MET phosphorylation, whilst LMH 86 and LMH 87 did not. LMH 86 was unable to block HGF/SF stimulated c-MET phosphorylation. (E) effect of selected LMH antibodies on cell migration. Antibodies were tested for their ability to induce cell migration in SK-OV-3 cells. LMH 85 stimulated cell migration while LMH 87 had no effect ( upper panel ). Differing concentrations of LMH 87 were mixed with 3×10 −10 M HGF/SF to determine if it inhibited the HGF/SF induced migration of SK-OV-3 cells. LMH 87 substantially inhibited the migratory activity stimulated by HGF/SF ( lower panel ). Data in both graphs are presented as percentage migration versus 3×10 −10 M HGF/SF ± SD. A full summary of all LMH antibodies is contained in Table 1 .

Techniques Used: Flow Cytometry, Cytometry, Western Blot, Activity Assay, Blocking Assay, Migration

LMH 87 down-regulates surface c-MET and inhibits tumor cell growth. (A) c-MET was IPed using anti-c-MET antibody coupled to agarose beads from A549 lung cancer cells treated with LMH 87 and then cell surface biotinylated. Cell surface c-MET ( upper blot ) and total c-MET ( lower blot ) levels were determined by IB as detailed in the Materials Methods. Bar graphs show quantification by densitometry ± SEM. IRR = irrelevant antibody. (B) total c-MET remaining in U87MG glioma cells following incubation with LMH 87. Bar graph shows quantification by densitometry ± SEM. Both (A) and (B) are representative blots of repeated experiments. (C) LMH 85 and LMH 87 are internalized following engagement of the c-MET receptor. A549 cells were bound with LMH 85 ( top panels ) or LMH 87 ( bottom panels ) followed by AF488- labeled secondary antibody at 4°C. Internalization was induced with addition of media at 37°C. Significant internalization of both antibodies was observed at 30 min and 60 min as indicated by relocation of the fluorescent signal from the membrane to diffuse cytoplasmic and perinuclear locations. Scale bar = 10 µm. (D) xCELLigence analysis of A549 cells treated with LMH 87. A549 cells were treated with vehicle or different concentrations of LMH 87. Data is presented as the cell index normalized to 21 h ± SEM, the time point when antibody was added. Both the 50 and 100 µg/ml treatments caused a significant decrease ( p
Figure Legend Snippet: LMH 87 down-regulates surface c-MET and inhibits tumor cell growth. (A) c-MET was IPed using anti-c-MET antibody coupled to agarose beads from A549 lung cancer cells treated with LMH 87 and then cell surface biotinylated. Cell surface c-MET ( upper blot ) and total c-MET ( lower blot ) levels were determined by IB as detailed in the Materials Methods. Bar graphs show quantification by densitometry ± SEM. IRR = irrelevant antibody. (B) total c-MET remaining in U87MG glioma cells following incubation with LMH 87. Bar graph shows quantification by densitometry ± SEM. Both (A) and (B) are representative blots of repeated experiments. (C) LMH 85 and LMH 87 are internalized following engagement of the c-MET receptor. A549 cells were bound with LMH 85 ( top panels ) or LMH 87 ( bottom panels ) followed by AF488- labeled secondary antibody at 4°C. Internalization was induced with addition of media at 37°C. Significant internalization of both antibodies was observed at 30 min and 60 min as indicated by relocation of the fluorescent signal from the membrane to diffuse cytoplasmic and perinuclear locations. Scale bar = 10 µm. (D) xCELLigence analysis of A549 cells treated with LMH 87. A549 cells were treated with vehicle or different concentrations of LMH 87. Data is presented as the cell index normalized to 21 h ± SEM, the time point when antibody was added. Both the 50 and 100 µg/ml treatments caused a significant decrease ( p

Techniques Used: Incubation, Labeling

20) Product Images from "MicroRNA-92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells"

Article Title: MicroRNA-92a promotes cell proliferation, migration and survival by directly targeting the tumor suppressor gene NF2 in colorectal and lung cancer cells

Journal: Oncology Reports

doi: 10.3892/or.2019.7020

Overexpression of miR-92a-3p enhances cell proliferation and inhibits apoptosis induction of HCT116 and A549 cells. Proliferation of (A) HCT116 and (B) A549 cells transfected with empty vector (i.e., pmR-ZsGreen1) or miR-92a expression construct in 0.5% serum. (C) Caspase-3/7 activity in HCT116 cells transfected with miR-92a expression construct and maintained in 2% serum, with or without the presence of sodium butyrate (NaB). (D) Caspase-3/7 activity in A549 cells transfected with miR-92a expression construct maintained in 10 or 0.5% serum. Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01.
Figure Legend Snippet: Overexpression of miR-92a-3p enhances cell proliferation and inhibits apoptosis induction of HCT116 and A549 cells. Proliferation of (A) HCT116 and (B) A549 cells transfected with empty vector (i.e., pmR-ZsGreen1) or miR-92a expression construct in 0.5% serum. (C) Caspase-3/7 activity in HCT116 cells transfected with miR-92a expression construct and maintained in 2% serum, with or without the presence of sodium butyrate (NaB). (D) Caspase-3/7 activity in A549 cells transfected with miR-92a expression construct maintained in 10 or 0.5% serum. Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01.

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Construct, Activity Assay, Standard Deviation

Overexpression of miR-92a-3p enhances the motility of HCT116 and A549 cells. Representative micrographs of (A) HCT116 and (B) A549 wound fields after scratching (0 h) the monolayer and at 24 h post-scratch. Scale bars, 100 µm. Percent open wound of the field view area occupied by cells at 24 and 48 h post-scratch vs. time-point 0 h is shown. (C) Semi-quantitative reverse transcription-polymerase chain reaction (semi-RT-PCR) and (D) western blotting detection of NF2 /Merlin and EMT marker (E-cadherin, N-cadherin and vimentin) expression levels in HCT116 cells transfected with vector or miR-92a expression construct. The lysates used for western blotting were from the same independent trial shown in Fig. 2C . Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. EMT, epithelial-mesenchymal transition.
Figure Legend Snippet: Overexpression of miR-92a-3p enhances the motility of HCT116 and A549 cells. Representative micrographs of (A) HCT116 and (B) A549 wound fields after scratching (0 h) the monolayer and at 24 h post-scratch. Scale bars, 100 µm. Percent open wound of the field view area occupied by cells at 24 and 48 h post-scratch vs. time-point 0 h is shown. (C) Semi-quantitative reverse transcription-polymerase chain reaction (semi-RT-PCR) and (D) western blotting detection of NF2 /Merlin and EMT marker (E-cadherin, N-cadherin and vimentin) expression levels in HCT116 cells transfected with vector or miR-92a expression construct. The lysates used for western blotting were from the same independent trial shown in Fig. 2C . Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. EMT, epithelial-mesenchymal transition.

Techniques Used: Over Expression, Reverse Transcription Polymerase Chain Reaction, Western Blot, Marker, Expressing, Transfection, Plasmid Preparation, Construct, Standard Deviation

NF2 is downregulated by miR-92a-3p via its 3′UTR. (A) Schematic representation of the full and cloned 3′UTR region of NF2 isoform I mRNA and the predicted miR-92a-3p MREs within its sequence. Dual-Luciferase assay of (B) HCT116 and (C) A549 cells transfected with empty vector (i.e., pmirGLO) or NF2 −3′UTR-wild-type. (D and E) The expression levels of mature miR-92a-3p were measured by QuantiGene miRNA assay in untransfected (UTC) or miR-92a construct-transfected (OE) HCT116 and A549 cells. Dual-Luciferase assay of (F) HCT116 and (G) A549 cells co-transfected with increasing plasmid ratios of NF2 −3′UTR-wild-type:miR-92a. (H) Secondary structure analysis of the RNA-RNA interaction (boxed region) between miR-92a-3p (red) and its MRE within wild-type vs. mutant NF2 −3′UTR (green). Dual-Luciferase assay of (I) HCT116 and (J) A549 cells co-transfected with wild-type or miR-92a-3p MRE mutant NF2 −3′UTR and miR-92a expression vectors. All experiments were performed in cells maintained in 0.5% serum. Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. NF2 , neurofibromin 2; UTR, untranslated region; MRE, microRNA response element.
Figure Legend Snippet: NF2 is downregulated by miR-92a-3p via its 3′UTR. (A) Schematic representation of the full and cloned 3′UTR region of NF2 isoform I mRNA and the predicted miR-92a-3p MREs within its sequence. Dual-Luciferase assay of (B) HCT116 and (C) A549 cells transfected with empty vector (i.e., pmirGLO) or NF2 −3′UTR-wild-type. (D and E) The expression levels of mature miR-92a-3p were measured by QuantiGene miRNA assay in untransfected (UTC) or miR-92a construct-transfected (OE) HCT116 and A549 cells. Dual-Luciferase assay of (F) HCT116 and (G) A549 cells co-transfected with increasing plasmid ratios of NF2 −3′UTR-wild-type:miR-92a. (H) Secondary structure analysis of the RNA-RNA interaction (boxed region) between miR-92a-3p (red) and its MRE within wild-type vs. mutant NF2 −3′UTR (green). Dual-Luciferase assay of (I) HCT116 and (J) A549 cells co-transfected with wild-type or miR-92a-3p MRE mutant NF2 −3′UTR and miR-92a expression vectors. All experiments were performed in cells maintained in 0.5% serum. Data presented are representative of three independent trials and expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. NF2 , neurofibromin 2; UTR, untranslated region; MRE, microRNA response element.

Techniques Used: Clone Assay, Sequencing, Luciferase, Transfection, Plasmid Preparation, Expressing, Construct, Mutagenesis, Standard Deviation

siRNA-mediated NF2 silencing phenocopies pro-oncogenic effects of miR-92a-3p overexpression. (A) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and (B) western blotting detection of NF2 /Merlin expression in HCT116 cells transfected with negative control siRNA (siNEG) or NF2 siRNA. Proliferation of (C) HCT116 and (D) A549 cells transfected with siNEG or NF2 siRNA. Caspase-3/7 activity of (E) HCT116 and (F) A549 cells transfected with siNEG or NF2 siRNA. Representative micrographs of (G) HCT116 and (H) A549 wound fields after scratching (0 h) the monolayer and at 24 h post-scratch. Scale bars, 100 µm. (I) Semi-RT-qPCR and (J) western blotting detection of NF2 /Merlin and EMT marker expression levels in HCT116 cells transfected with siNEG or NF2 siRNA. Data are expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. (K) Fluorescent images showing the F-actin cytoskeletal organization of A549 cells co-transfected with pmR-ZsGreen1 and siNEG or NF2 siRNA. Yellow arrows indicate cells with sparse transverse actin filaments with a high number of intercellular adhesions. White arrows indicate cells with dense and prominent transverse F-actin fibers and formation of multiple pseudopodia, characteristic of motile cells (phalloidin: F-actin; Hoechst: nuclei, ZsGreen1: cells positively transfected with pmR-ZsGreen1). Scale bar, 25 µm. NF2 , neurofibromin 2; EMT, epithelial-mesenchymal transition.
Figure Legend Snippet: siRNA-mediated NF2 silencing phenocopies pro-oncogenic effects of miR-92a-3p overexpression. (A) Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and (B) western blotting detection of NF2 /Merlin expression in HCT116 cells transfected with negative control siRNA (siNEG) or NF2 siRNA. Proliferation of (C) HCT116 and (D) A549 cells transfected with siNEG or NF2 siRNA. Caspase-3/7 activity of (E) HCT116 and (F) A549 cells transfected with siNEG or NF2 siRNA. Representative micrographs of (G) HCT116 and (H) A549 wound fields after scratching (0 h) the monolayer and at 24 h post-scratch. Scale bars, 100 µm. (I) Semi-RT-qPCR and (J) western blotting detection of NF2 /Merlin and EMT marker expression levels in HCT116 cells transfected with siNEG or NF2 siRNA. Data are expressed as the mean ± standard deviation (SD). *P≤0.05, **P≤0.01, ***P≤0.001. (K) Fluorescent images showing the F-actin cytoskeletal organization of A549 cells co-transfected with pmR-ZsGreen1 and siNEG or NF2 siRNA. Yellow arrows indicate cells with sparse transverse actin filaments with a high number of intercellular adhesions. White arrows indicate cells with dense and prominent transverse F-actin fibers and formation of multiple pseudopodia, characteristic of motile cells (phalloidin: F-actin; Hoechst: nuclei, ZsGreen1: cells positively transfected with pmR-ZsGreen1). Scale bar, 25 µm. NF2 , neurofibromin 2; EMT, epithelial-mesenchymal transition.

Techniques Used: Over Expression, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Western Blot, Expressing, Transfection, Negative Control, Activity Assay, Marker, Standard Deviation

Overexpression of miR-92a-3p alters the cytoskeletal organization of A549 cells. Fluorescent images showing the F-actin cytoskeletal organization of A549 cells co-transfected with empty vector (i.e., pmiR-ZsGreen1) or miR-92a expression construct and TPneg or TP92a. Yellow arrows indicate cells with sparse transverse actin filaments with a high number of intercellular adhesions. White arrows indicate cells with dense and prominent transverse F-actin fibers and formation of multiple pseudopodia, which is characteristic of motile cells (phalloidin: F-actin; Hoechst: nuclei, ZsGreen1: cells positively transfected with pmR-ZsGreen1). Scale bars, 25 µm.
Figure Legend Snippet: Overexpression of miR-92a-3p alters the cytoskeletal organization of A549 cells. Fluorescent images showing the F-actin cytoskeletal organization of A549 cells co-transfected with empty vector (i.e., pmiR-ZsGreen1) or miR-92a expression construct and TPneg or TP92a. Yellow arrows indicate cells with sparse transverse actin filaments with a high number of intercellular adhesions. White arrows indicate cells with dense and prominent transverse F-actin fibers and formation of multiple pseudopodia, which is characteristic of motile cells (phalloidin: F-actin; Hoechst: nuclei, ZsGreen1: cells positively transfected with pmR-ZsGreen1). Scale bars, 25 µm.

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Expressing, Construct

21) Product Images from "Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine"

Article Title: Streptococcus pneumoniae Cell-Wall-Localized Phosphoenolpyruvate Protein Phosphotransferase Can Function as an Adhesin: Identification of Its Host Target Molecules and Evaluation of Its Potential as a Vaccine

Journal: PLoS ONE

doi: 10.1371/journal.pone.0150320

SIM images of CFDA-stained bacteria with Int β4 on A549 cells. CFDA-stained S . pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde and stained with a rabbit anti Int β4. Secondary antibody used was Alexa Fluor 594 (red) conjugated AffiniPure Goat Anti-Rabbit IgG (H+L). 3D SIM images were obtained with the Elyra SIM imaging system with a 63x oil objective (NA = 1.4); actual magnification of the image is as indicated in the scale bar. A) S . pneumoniae (CFDA 488 nm—green) adhering to A549 epithelial cells are seen coated by Int β4 (red). B) Higher magnification of bacteria (green) enveloped with Int β4 (red). C) Intensity alignment profile. D) Pedestal-like structure formed at the site of adherence of S . pneumoniae (green) to A549 cells recruits Int β4 (red). E) Higher magnification demonstrates the recruitment of Int β4 (red) to the pedestal-like structure underneath the adhered S . pneumoniae (green). F) Intensity alignment profile.
Figure Legend Snippet: SIM images of CFDA-stained bacteria with Int β4 on A549 cells. CFDA-stained S . pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde and stained with a rabbit anti Int β4. Secondary antibody used was Alexa Fluor 594 (red) conjugated AffiniPure Goat Anti-Rabbit IgG (H+L). 3D SIM images were obtained with the Elyra SIM imaging system with a 63x oil objective (NA = 1.4); actual magnification of the image is as indicated in the scale bar. A) S . pneumoniae (CFDA 488 nm—green) adhering to A549 epithelial cells are seen coated by Int β4 (red). B) Higher magnification of bacteria (green) enveloped with Int β4 (red). C) Intensity alignment profile. D) Pedestal-like structure formed at the site of adherence of S . pneumoniae (green) to A549 cells recruits Int β4 (red). E) Higher magnification demonstrates the recruitment of Int β4 (red) to the pedestal-like structure underneath the adhered S . pneumoniae (green). F) Intensity alignment profile.

Techniques Used: Staining, Incubation, Imaging

Inhibition of adhesion of S . pneumoniae cells to A549 cells by target-derived peptides. S . pneumoniae cells were treated for 1 h with peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) BMPER, WU2 ( P
Figure Legend Snippet: Inhibition of adhesion of S . pneumoniae cells to A549 cells by target-derived peptides. S . pneumoniae cells were treated for 1 h with peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) BMPER, WU2 ( P

Techniques Used: Inhibition, Derivative Assay

Immunostaining of S . pneumoniae adhesion to A549 cells. CFDA-stained S . pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde, stained, and viewed with a FluoView FV1000 confocal system (Olympus, Japan). The following stains were used: A) mouse anti-Eps 1 antiserum and rabbit anti-integrin β4 antiserum; C) mouse anti-epsin 1 antiserum and rabbit anti-BMPER antiserum; E) mouse anti-Eps 1 antiserum and rabbit anti-MMRN1 antiserum; and G) mouse anti-Eps 1 antiserum and anti-PCDH19 antiserum. The same cells viewed by Nomarsky microscopy overlaid with the CFDA-stained bacteria are shown, respectively, in B), D), F) and H). Secondary antibodies used were Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) antiserum and Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L (antiserum, in accordance with the primary antiserum used.
Figure Legend Snippet: Immunostaining of S . pneumoniae adhesion to A549 cells. CFDA-stained S . pneumoniae strain WU2 cells were incubated with A549 cells for 1 h. Excess bacteria were removed, and the culture was fixed with 4% para-formaldehyde, stained, and viewed with a FluoView FV1000 confocal system (Olympus, Japan). The following stains were used: A) mouse anti-Eps 1 antiserum and rabbit anti-integrin β4 antiserum; C) mouse anti-epsin 1 antiserum and rabbit anti-BMPER antiserum; E) mouse anti-Eps 1 antiserum and rabbit anti-MMRN1 antiserum; and G) mouse anti-Eps 1 antiserum and anti-PCDH19 antiserum. The same cells viewed by Nomarsky microscopy overlaid with the CFDA-stained bacteria are shown, respectively, in B), D), F) and H). Secondary antibodies used were Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (H+L) antiserum and Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L (antiserum, in accordance with the primary antiserum used.

Techniques Used: Immunostaining, Staining, Incubation, Microscopy

PtsA mediates S . pneumoniae adhesion to target host cells. A–E. A549 cells were grown to confluence and then blocked with 0,5% gelatin for 1 h. Excess gelatin was removed, and the A549 cells were incubated for 1 h with rPtsA at the denoted concentrations. Excess protein was then removed. S . pneumoniae was added for 1 h to the cells, non-adherent bacteria were removed, and cells were detached with trypsin and plated onto blood agar plates for counting. rPtsA inhibited the adhesion to A549 cells of: A) strain WU2 (p
Figure Legend Snippet: PtsA mediates S . pneumoniae adhesion to target host cells. A–E. A549 cells were grown to confluence and then blocked with 0,5% gelatin for 1 h. Excess gelatin was removed, and the A549 cells were incubated for 1 h with rPtsA at the denoted concentrations. Excess protein was then removed. S . pneumoniae was added for 1 h to the cells, non-adherent bacteria were removed, and cells were detached with trypsin and plated onto blood agar plates for counting. rPtsA inhibited the adhesion to A549 cells of: A) strain WU2 (p

Techniques Used: Incubation

Putative target molecules in A549 cells. A549 cells were fixed with 4% para-formaldehyde and stained with a combination of a mouse anti Eps 1 and one of the following: rabbit anti Int β4, PCDH19, MMRN1, or BMPER. The secondary antibodies used were either Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (blue) or Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (red) in accordance with the primary antibody used. 3D SIM images were taken using the Elyra SIM imaging system.
Figure Legend Snippet: Putative target molecules in A549 cells. A549 cells were fixed with 4% para-formaldehyde and stained with a combination of a mouse anti Eps 1 and one of the following: rabbit anti Int β4, PCDH19, MMRN1, or BMPER. The secondary antibodies used were either Alexa Fluor 405-conjugated AffiniPure Goat Anti-Mouse IgG (blue) or Alexa Fluor 594-conjugated AffiniPure Goat Anti-Rabbit IgG (red) in accordance with the primary antibody used. 3D SIM images were taken using the Elyra SIM imaging system.

Techniques Used: Staining, Imaging

Inhibition of S . pneumoniae adhesion to A549 cells by Eps 1-derived peptide. S . pneumoniae cells were treated for 1 h with Eps 1-derived peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) Strain WU2 (p
Figure Legend Snippet: Inhibition of S . pneumoniae adhesion to A549 cells by Eps 1-derived peptide. S . pneumoniae cells were treated for 1 h with Eps 1-derived peptide and added to A549 cells for 1 h; non-adherent bacteria were removed; and cells were detached with trypsin and plated onto blood agar plates for counting. A) Strain WU2 (p

Techniques Used: Inhibition, Derivative Assay

22) Product Images from "Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions"

Article Title: Bright Fluorescent Streptococcus pneumoniae for Live-Cell Imaging of Host-Pathogen Interactions

Journal: Journal of Bacteriology

doi: 10.1128/JB.02221-14

Unencapsulated S. pneumoniae cells adhere more efficiently to lung epithelial cells than to encapsulated bacteria. Adhesion of S. pneumoniae reporters on a confluent monolayer of A549, a type II lung epithelial cell line, was imaged using wide-field epifluorescence microscopy. Imaging was performed at 2 h postcoincubation. (A) Schematic overview of the coincubation system, which involves an equimolar mixture of encapsulated and unencapsulated S. pneumoniae strains expressing different fluorescent proteins, on an epithelial A549 monolayer. (B) Classical adherence assay: enumeration of pneumococcal CFU that adhere to A549 2 h after coincubation. The unencapsulated reporter strain, MK128, showed a significantly ( P
Figure Legend Snippet: Unencapsulated S. pneumoniae cells adhere more efficiently to lung epithelial cells than to encapsulated bacteria. Adhesion of S. pneumoniae reporters on a confluent monolayer of A549, a type II lung epithelial cell line, was imaged using wide-field epifluorescence microscopy. Imaging was performed at 2 h postcoincubation. (A) Schematic overview of the coincubation system, which involves an equimolar mixture of encapsulated and unencapsulated S. pneumoniae strains expressing different fluorescent proteins, on an epithelial A549 monolayer. (B) Classical adherence assay: enumeration of pneumococcal CFU that adhere to A549 2 h after coincubation. The unencapsulated reporter strain, MK128, showed a significantly ( P

Techniques Used: Epifluorescence Microscopy, Imaging, Expressing

23) Product Images from "A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging"

Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Journal: Nature Communications

doi: 10.1038/s41467-018-03191-2

Visualization of endogenously expressed BC2-tagged actin labeled with bivBC2-Nb AF647 . a Wide-field images of chemically fixed wild-type A549 and HeLa (-wt; left panel), as well as chemically fixed A549- BC2T ACTB and HeLa- BC2T ACTB cells (right panel). Cells were either left untreated (0 h) or stimulated for 48 h with TGFβ (5 ng ml −1 ) followed by staining with phalloidin AF555 and bivBC2-Nb AF647 . Scale bars, 25 µm. b dSTORM image of a representative HeLa- BC2T
Figure Legend Snippet: Visualization of endogenously expressed BC2-tagged actin labeled with bivBC2-Nb AF647 . a Wide-field images of chemically fixed wild-type A549 and HeLa (-wt; left panel), as well as chemically fixed A549- BC2T ACTB and HeLa- BC2T ACTB cells (right panel). Cells were either left untreated (0 h) or stimulated for 48 h with TGFβ (5 ng ml −1 ) followed by staining with phalloidin AF555 and bivBC2-Nb AF647 . Scale bars, 25 µm. b dSTORM image of a representative HeLa- BC2T

Techniques Used: Labeling, Staining

24) Product Images from "RalB regulates contractility-driven cancer dissemination upon TGFβ stimulation via the RhoGEF GEF-H1"

Article Title: RalB regulates contractility-driven cancer dissemination upon TGFβ stimulation via the RhoGEF GEF-H1

Journal: Scientific Reports

doi: 10.1038/srep11759

TGFβ1-induced EMT enhances actomyosin contractility and matrix remodelling. ( A ) EMT promotes traction forces on the substratum. Representative phase contrast images and corresponding traction force maps of untreated and TGFβ-treated A549 cells as revealed by Traction Force Microscopy (TFM). Color scale bar denotes traction stress (Pa). Scale bar, 50 μ m. ( B ) Quantification of the strain energy. TGFβ-treated cells generate significantly higher strain energies compared to untreated cells. Number of untreated cells n = 51, TGFβ-treated cells n = 35, from two experiments. ( C ) EMT stimulates Rho activity. Visualization of RhoA activity in representative untreated and TGFβ-treated cells. Cells were transfected with a plasmid expressing Raichu-RhoA (KRasCter) biosensor and observed by FRET microscopy the day after. Note the higher level of RhoA at cell periphery. Scale bars, 20 μm. ( D ) Quantification of whole-cell RhoA activity. YFP and CFP images were acquired for each cell and mean intensities were measured for the entire cell surface. The YFP/CFP ratio is a measure of whole-cell FRET, i.e. of whole-cell RhoA activity. Number of untreated cells n = 54, TGFβ-treated cells n = 41, from two experiments. ( E ) EMT induces MLC2 phosphorylation. The levels of total and phosphorylated MLC2 were analyzed by western-blot. The quantification of band intensities (with respect to untreated condition) and the calculated P-MLC/MLC ratio are indicated. The vertical lanes indicate positions were gel images were cut in order to juxtapose non-adjacent lanes coming from the same gel. ( F ) EMT stimulates ROCK-dependent actomyosin contractility. A549 cells were grown with or without TGF-β1 for 7 days and embedded in collagen I gels for 4 days. Gel contraction was monitored. Where indicated, cells were pre-treated for 2 hrs with 10 μM Y27632 ROCK inhibitor and submitted to collagen contraction assay in presence of the inhibitor. Number of untreated gels n = 2, TGFβ-treated gels n = 2, TGFβ/Y27632-treated gels n = 1, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p
Figure Legend Snippet: TGFβ1-induced EMT enhances actomyosin contractility and matrix remodelling. ( A ) EMT promotes traction forces on the substratum. Representative phase contrast images and corresponding traction force maps of untreated and TGFβ-treated A549 cells as revealed by Traction Force Microscopy (TFM). Color scale bar denotes traction stress (Pa). Scale bar, 50 μ m. ( B ) Quantification of the strain energy. TGFβ-treated cells generate significantly higher strain energies compared to untreated cells. Number of untreated cells n = 51, TGFβ-treated cells n = 35, from two experiments. ( C ) EMT stimulates Rho activity. Visualization of RhoA activity in representative untreated and TGFβ-treated cells. Cells were transfected with a plasmid expressing Raichu-RhoA (KRasCter) biosensor and observed by FRET microscopy the day after. Note the higher level of RhoA at cell periphery. Scale bars, 20 μm. ( D ) Quantification of whole-cell RhoA activity. YFP and CFP images were acquired for each cell and mean intensities were measured for the entire cell surface. The YFP/CFP ratio is a measure of whole-cell FRET, i.e. of whole-cell RhoA activity. Number of untreated cells n = 54, TGFβ-treated cells n = 41, from two experiments. ( E ) EMT induces MLC2 phosphorylation. The levels of total and phosphorylated MLC2 were analyzed by western-blot. The quantification of band intensities (with respect to untreated condition) and the calculated P-MLC/MLC ratio are indicated. The vertical lanes indicate positions were gel images were cut in order to juxtapose non-adjacent lanes coming from the same gel. ( F ) EMT stimulates ROCK-dependent actomyosin contractility. A549 cells were grown with or without TGF-β1 for 7 days and embedded in collagen I gels for 4 days. Gel contraction was monitored. Where indicated, cells were pre-treated for 2 hrs with 10 μM Y27632 ROCK inhibitor and submitted to collagen contraction assay in presence of the inhibitor. Number of untreated gels n = 2, TGFβ-treated gels n = 2, TGFβ/Y27632-treated gels n = 1, from two experiments. Error bars represent SEM. p values come from two-tailed Student’s t test. *p

Techniques Used: Microscopy, Activity Assay, Transfection, Plasmid Preparation, Expressing, Western Blot, Contraction Assay, Two Tailed Test

Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent. ( A ) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. ( B ) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. ( C ) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. ( D ) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.( E ) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). ( F ) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. ( G ) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n > 30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p
Figure Legend Snippet: Dissemination of TGFβ-treated A549 cells in 2/3D is proteolysis independent. ( A ) Depiction of the 2/3D Circular Invasion Assay (CIA). Note that we kept the original name of the assay, but that the actual shape of the stopper we used is not circular but square. ( B ) EMT promotes cell dissemination. A549 cells were treated with 2 ng/mL TGFβ for 7 days, submitted to CIA and compared to untreated cells. Selected time points from a representative experiment are shown. See Supplementary Movie S1 for entire video sequence. Scale bar, 100 μm. ( C ) Quantification of cell dissemination. Individual cells were tracked using the ImageJ software. Number of cells n = 40 for untreated and n = 163 for TGFβ-treated conditions from at least four experiments per condition. ( D ) TGFβ greatly stimulates invadopodia formation. Cells were stimulated with TGFβ for 7 days and invadopodia were visualized as dots positive for matrix degradation (black), for cortactin staining (green) and for F-actin staining (red). Scale bar, 20 μm.( E ) Quantification of cells positive for at least one invadopodia. Counting was performed on three independent experiments (n≥100 cells/condition per experiment). ( F ) TGFβ induces strong secretion of MMP2 and MMP9 metalloproteinases. Conditioned media from untreated and TGFβ-treated cells were collected and processed for gelatin zymogram assay. A representative zymogram image is shown. ( G ) MMP-dependent proteolysis is dispensable for TGFβ-induced dissemination in 2/3D. TGFβ-treated cells were incubated with 25 μM GM6001 MMPs inhibitor (2 hrs before CIA and during CIA) or depleted of MMP2 and MMP9, and submitted to CIA. Number of cells n > 30 per condition from three experiments with GM6001 and one experiment with siRNAs. Error bars represent SEM. p values come from two-tailed Student’s t test. *p

Techniques Used: Invasion Assay, Sequencing, Software, Staining, Incubation, Two Tailed Test

Model outlining the role of RalB/Exocyst pathway in the dissemination of TGFβ-treated A549 cells. TGFβ-treated cells generate traction forces which are required to remodel the extracellular matrix and to disseminate in 2/3D environment. This requires the activation of the Rho/ROCK pathway and actomyosin function. The Ral pathway controls the dissemination of TGFβ-treated cells by modulating RhoA-dependent actomyosin contractility via the interaction between the Sec5 subunit of Exocyst and the RhoGEF GEF-H1.
Figure Legend Snippet: Model outlining the role of RalB/Exocyst pathway in the dissemination of TGFβ-treated A549 cells. TGFβ-treated cells generate traction forces which are required to remodel the extracellular matrix and to disseminate in 2/3D environment. This requires the activation of the Rho/ROCK pathway and actomyosin function. The Ral pathway controls the dissemination of TGFβ-treated cells by modulating RhoA-dependent actomyosin contractility via the interaction between the Sec5 subunit of Exocyst and the RhoGEF GEF-H1.

Techniques Used: Activation Assay

25) Product Images from "Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles"

Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

Journal: Oncotarget

doi: 10.18632/oncotarget.24772

Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.
Figure Legend Snippet: Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.

Techniques Used: Incubation, Fluorescence, Software, Staining, Microscopy

Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Negative Control, Staining, Fluorescence, Microscopy

Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).
Figure Legend Snippet: Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Software

Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Concentration Assay, Incubation, Staining, Confocal Microscopy, Microscopy

Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.
Figure Legend Snippet: Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Fluorescence

Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy, Software

Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Sequencing

26) Product Images from "Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro"

Article Title: Invasive bronchial fibroblasts derived from asthmatic patients activate lung cancer A549 cells in vitro

Journal: Oncology Letters

doi: 10.3892/ol.2018.9462

AS HBFs induce the motility of A549 cells via contact-modulated paracrine signaling. (A) A549 cells were cultivated in the media conditioned by AS2 HBFs (left), ‘separated’ (middle) and ‘confronted’ AS HBF/A549 co-cultures (1:1 v/v with fresh medium) for 48 h and (C) the parameters of their motility were analyzed by time-lapse videomicroscopy in comparison to A549 motility in control conditions and in ‘open’ AS HBF/A549 co-cultures. (B) A549 cells were cultivated in the media conditioned by ‘separated’ co-cultures of A549 with AS and NA HBFs (1:1 v/v with fresh medium) for 48 h. Intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA was visualized with immunofluorescence and cytofluorimetry, respectively (left axes/blue line: DNA; right axes/green line: Snail-1). Scale bar, 50 µm; magnification, ×400. (D) The motility of A549 cells in the presence of the media conditioned by NA HBFs (left), ‘separated’ (middle) and ‘confronted’ NA HBF/A549 co-cultures (1:1 v/v with fresh medium) was analyzed as in (A). Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P
Figure Legend Snippet: AS HBFs induce the motility of A549 cells via contact-modulated paracrine signaling. (A) A549 cells were cultivated in the media conditioned by AS2 HBFs (left), ‘separated’ (middle) and ‘confronted’ AS HBF/A549 co-cultures (1:1 v/v with fresh medium) for 48 h and (C) the parameters of their motility were analyzed by time-lapse videomicroscopy in comparison to A549 motility in control conditions and in ‘open’ AS HBF/A549 co-cultures. (B) A549 cells were cultivated in the media conditioned by ‘separated’ co-cultures of A549 with AS and NA HBFs (1:1 v/v with fresh medium) for 48 h. Intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA was visualized with immunofluorescence and cytofluorimetry, respectively (left axes/blue line: DNA; right axes/green line: Snail-1). Scale bar, 50 µm; magnification, ×400. (D) The motility of A549 cells in the presence of the media conditioned by NA HBFs (left), ‘separated’ (middle) and ‘confronted’ NA HBF/A549 co-cultures (1:1 v/v with fresh medium) was analyzed as in (A). Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P

Techniques Used: Immunofluorescence

AS HBFs selectively modulate the invasion of A549 cells. (A) A549 cells were grown to confluence with NA HBFs (left-hand panel) or AS HBFs (right-hand panel) in 2-well silicone inserts and co-incubated for 48 h following diaphragm removal prior to fixation and immunostaining for α-SMA. Expression of α-SMA was visualized by immunofluorescence and cytofluorimetry, respectively. Scale bar, 100 µm; magnification, ×200. Inserts present A549 cells within the HBF monolayer. (B) Cells were cultivated as in (A) and intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA were visualized with immunofluorescence and cytofluorimetry, respectively (left axes: DNA; right axes: Snail-1). Photomicrographs in (A) and (B) present panoramic till scans of the interfaces between HBF and A549 monolayers obtained by the composition of two rows of succeeding images (7 pictures in a row). Scale bars, 100 µm. (C) NA HBFs and AS HBFs were cultivated in the presence of the media conditioned by ‘separated’ NA HBF/A549 and AS HBF/A549 co-cultures, respectively, for 48 h and immunostained against α-SMA, Snail-1 and Cx43. Scale bars, 50 µm; magnification, ×630. (D) A549 cells were seeded onto microporous membrane, placed in the wells filled with the media conditioned by A549/HBF co-cultures, and allowed to transmigrate for 48 h. The number of the transmigrated cells were counted following 24 h. Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P
Figure Legend Snippet: AS HBFs selectively modulate the invasion of A549 cells. (A) A549 cells were grown to confluence with NA HBFs (left-hand panel) or AS HBFs (right-hand panel) in 2-well silicone inserts and co-incubated for 48 h following diaphragm removal prior to fixation and immunostaining for α-SMA. Expression of α-SMA was visualized by immunofluorescence and cytofluorimetry, respectively. Scale bar, 100 µm; magnification, ×200. Inserts present A549 cells within the HBF monolayer. (B) Cells were cultivated as in (A) and intracellular localization of Snail-1/Cx43 and co-localization of Snail-1/DNA were visualized with immunofluorescence and cytofluorimetry, respectively (left axes: DNA; right axes: Snail-1). Photomicrographs in (A) and (B) present panoramic till scans of the interfaces between HBF and A549 monolayers obtained by the composition of two rows of succeeding images (7 pictures in a row). Scale bars, 100 µm. (C) NA HBFs and AS HBFs were cultivated in the presence of the media conditioned by ‘separated’ NA HBF/A549 and AS HBF/A549 co-cultures, respectively, for 48 h and immunostained against α-SMA, Snail-1 and Cx43. Scale bars, 50 µm; magnification, ×630. (D) A549 cells were seeded onto microporous membrane, placed in the wells filled with the media conditioned by A549/HBF co-cultures, and allowed to transmigrate for 48 h. The number of the transmigrated cells were counted following 24 h. Data are presented as the mean ± standard error of the mean of 3 independent experiments. *P

Techniques Used: Incubation, Immunostaining, Expressing, Immunofluorescence

27) Product Images from "Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway"

Article Title: Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway

Journal: PLoS Pathogens

doi: 10.1371/journal.ppat.1005307

LAI-1-dependent redistribution of IQGAP1 does not require Cdc42. (A) A549 cells were treated with siRNA against Cdc42 for 2 days, then with LAI-1 (10 μM, 1.5 h) and protein production or the subcellular localization of the IQGAP1 scaffold protein (green) and the small GTPase Cdc42 (red) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). (B) Quantification of protein production in A549 cells after RNAi treatment (percent cells producing protein of interest; n = 50). Means and standard deviations of 3 independent experiments are shown (*** p
Figure Legend Snippet: LAI-1-dependent redistribution of IQGAP1 does not require Cdc42. (A) A549 cells were treated with siRNA against Cdc42 for 2 days, then with LAI-1 (10 μM, 1.5 h) and protein production or the subcellular localization of the IQGAP1 scaffold protein (green) and the small GTPase Cdc42 (red) was analyzed by confocal microscopy using antibodies against IQGAP1 or Cdc42. Nuclei were stained with DAPI (blue). (B) Quantification of protein production in A549 cells after RNAi treatment (percent cells producing protein of interest; n = 50). Means and standard deviations of 3 independent experiments are shown (*** p

Techniques Used: Confocal Microscopy, Staining

LAI-1 reverses Icm/Dot-dependent inhibition of migration by L . pneumophila . (A) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity ( S7 Fig )) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; * p
Figure Legend Snippet: LAI-1 reverses Icm/Dot-dependent inhibition of migration by L . pneumophila . (A) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (B) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with different concentrations of LAI-1 (1, 5 and 10 μM) or not. The effect of LAI-1 on migration of amoebae towards folate (1 mM) or macrophages towards CCL5 (100 ng/ml) was monitored in under-agarose assays for 4 hours. Macrophages were stained with Cell Tracker Green BODIPY. Graphs depict the per cent fluorescence intensity versus migration distance. (C) D . discoideum Ax3 amoebae harboring pSW102 (GFP) or (D) RAW 264.7 macrophages were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria and treated with LAI-1 (10 μM, 1 h) or not. Single cell migration towards folate (1 mM) or CCL5 (100 ng/ml) was tracked in an under-agarose assay for 15 min or 1 h, respectively. Motility parameters (forward migration index, FMI, and velocity ( S7 Fig )) were analyzed using the ImageJ manual tracker and Ibidi chemotaxis software. (E) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, treated with LAI-1 (10 μM) or not, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (F) The scratch area was quantified at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (C, D, F; means and standard deviations; * p

Techniques Used: Inhibition, Migration, Infection, Mutagenesis, Staining, Fluorescence, Chemotaxis Assay, Software, Imaging

Migration inhibition by L . pneumophila is augmented in the absence of Cdc42. (A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (*** p
Figure Legend Snippet: Migration inhibition by L . pneumophila is augmented in the absence of Cdc42. (A) Confluent cell layers of A549 cells were treated with (A) scrambled siRNA or siRNA against (B) Cdc42 or (C) Rac1 for 2 days, left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type or Δ icmT mutant bacteria, scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified after 24 h at 7 different positions per condition using ImageJ software. Means and standard deviations of triplicate samples per condition are shown, which are representative of 3 independent experiments (*** p

Techniques Used: Migration, Inhibition, Infection, Mutagenesis, Imaging, Software

LAI-1-dependent inhibition of cell migration requires IQGAP1 and Cdc42. (A) Confluent cell layers of A549 epithelial cells were treated with siRNA against IQGAP1, Cdc42, RhoA or Rac1 for 2 days. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of triplicate samples are shown (** p
Figure Legend Snippet: LAI-1-dependent inhibition of cell migration requires IQGAP1 and Cdc42. (A) Confluent cell layers of A549 epithelial cells were treated with siRNA against IQGAP1, Cdc42, RhoA or Rac1 for 2 days. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of triplicate samples are shown (** p

Techniques Used: Inhibition, Migration, Imaging, Software

Effect of L . pneumophila lqs genes on host cell migration. D . discoideum strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT or Δ lqsA mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: *** p
Figure Legend Snippet: Effect of L . pneumophila lqs genes on host cell migration. D . discoideum strain Ax3 producing GFP (pSW102) was infected (MOI 10, 1 h) with (A) L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains harboring pSW001 (DsRed), or with (D) the strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA). An under-agarose assay was used to monitor the migration towards folate (1 mM) for another 4 h. The white lines represent the edge of the sample wells. (B, E) Graphs of the data from (A, D) plotted as per cent GFP fluorescence intensity versus migration distance. (C) Murine RAWs 264.7 macrophages were infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT , Δ lqsS , Δ lqsT , ΔlqsS-lqsT , Δ lqsR or Δ lqsA mutant strains. Cells were stained with Cell Tracker Green BODIPY and let migrate towards CCL5 (100 ng/ml) in an under-agarose assay for another 4 h. Graphs show the per cent fluorescence intensity versus migration distance. (F) Confluent cell layers of A549 epithelial cells were left uninfected or infected (MOI 10, 1 h) with L . pneumophila wild-type, Δ icmT or Δ lqsA mutant strains harboring pNT28 (GFP) or pNT36 (GFP, LqsA), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (G) The scratch area was quantified using ImageJ software at 7 different positions per condition in triplicate samples. Means and standard deviations of the triplicate samples are shown (pNT28 vs. pNT36: *** p

Techniques Used: Migration, Infection, Mutagenesis, Fluorescence, Staining, Imaging, Software

LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9. (A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (*** p
Figure Legend Snippet: LAI-1-dependent inhibition of cell migration requires the Cdc42 GEF ARHGEF9. (A) Confluent cell layers of A549 cells were treated for 2 days with siRNA against the different Cdc42 GEFs or GAPs indicated. The cells were then treated or not with LAI-1 (10 μM, 1.5 h), scratched and let migrate for 24 h. Prior to imaging (0, 24 h), the detached cells were washed off. (B) The scratch area was quantified at 6 different positions per condition using ImageJ software. Means and standard deviations of 3 samples are shown, which are representative of 3 independent experiments (*** p

Techniques Used: Inhibition, Migration, Imaging, Software

LAI-1 promotes inactivation of Cdc42 and redistribution of IQGAP1 to the cell cortex. (A) A549 cells were treated with LAI-1 (10 μM, 1 h), and the activation state of Cdc42 was analyzed by Western blot using an antibody recognizing Cdc42(GTP/GDP) (left panel). Quantification by densitometry was performed using ImageJ (right panel). A549 cells were treated with LAI-1 (10 μM, 1 h), fixed, stained with antibodies against (B) IQGAP1 or (C) Cdc42 and analyzed by confocal microscopy (left panels; green, FITC; blue, DAPI). The graphs (right panels) are based on the relative fluorescence intensity along cell sections (n = 50, *** p
Figure Legend Snippet: LAI-1 promotes inactivation of Cdc42 and redistribution of IQGAP1 to the cell cortex. (A) A549 cells were treated with LAI-1 (10 μM, 1 h), and the activation state of Cdc42 was analyzed by Western blot using an antibody recognizing Cdc42(GTP/GDP) (left panel). Quantification by densitometry was performed using ImageJ (right panel). A549 cells were treated with LAI-1 (10 μM, 1 h), fixed, stained with antibodies against (B) IQGAP1 or (C) Cdc42 and analyzed by confocal microscopy (left panels; green, FITC; blue, DAPI). The graphs (right panels) are based on the relative fluorescence intensity along cell sections (n = 50, *** p

Techniques Used: Activation Assay, Western Blot, Staining, Confocal Microscopy, Fluorescence

28) Product Images from "Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles"

Article Title: Cancer cell-selective, clathrin-mediated endocytosis of aptamer decorated nanoparticles

Journal: Oncotarget

doi: 10.18632/oncotarget.24772

Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.
Figure Legend Snippet: Impact of metabolic energy deprivation on S15-APT internalization in A549 cells Left: ( A ) Control A549 cells incubated with S15-APT QDs (100 nM) for 1 h in inhibitor-free growth medium; ( B ) A549 cells were pre-incubated for 30 min with 5 mM sodium azide and incubated for 1 h with 100 nM S15-APT QDs, along with 5 (μg/ml) FCCP and 5 mM sodium azide, which disrupt mitochondrial ATP synthesis; Right: Mean fluorescence values of panels A and B were determined using IMARIS software. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10–100 for all presented images.

Techniques Used: Incubation, Fluorescence, Software, Staining, Microscopy

Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Pitstop-2 confirms that the predominant mechanism of S15-APT internalization is clathrin-mediated endocytosis ( A ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, an established CME inhibitor, followed by incubation for 40 min with 25 μM Pitstop-2 and 100 nM S15-APT QDs; ( B ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2 negative control (which does not inhibit endocytosis) and incubated for 40 min with 25 μM Pitstop-2 negative control along with 100 nM S15-APT QDs; ( C ) A549 cells were pre-incubated for 10 min with 25 μM Pitstop-2, then incubated for 50 min in growth medium containing 10% FCS (the medium was refreshed once) to restore the ability of cells to undergo CME. Cells were then incubated with 100 nM S15-APT QDs for 40 min. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Negative Control, Staining, Fluorescence, Microscopy

Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).
Figure Legend Snippet: Kinetic study of cellular internalization of S15-APT QDs into A549 cells A549 cells were incubated with 50 nM S15-APT QDs for 10 min, 0.5 h, 1 h, 2 h, 4 h, and 6 h at 37° C. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Quantification of the average number of endolysosomes per cell was determined using Imaris Software. The red fluorescence channel was defined between 10-100 for all presented images. Fitting the dependence of the average number of fluoresent endolysosomes/cell (Y(t)) to the incubation time (t) was performed by nonlinear curve fitting (Eq. 1).

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Software

Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Determination of the dissociation constant of S15-APT from A549 target cells and demonstration of selective binding of S15-APTs to A549 cells ( A ) The equilibrium dissociation constant (K d ) of the S15-APT-cell interaction was evaluated by flow cytometry. The K d was obtained by fitting the results of mean red fluorescence intensity of specific binding vs. the concentration of the aptamers to a Langmuir model equation (Eq. 2). A549 cells were incubated for 50 min on ice (to prevent internalization) with increasing concentrations of S15-APT QDs from 0.78 to 200 nM. ( B ) A549 cells were pre-incubated with a 100-fold molar excess of free APT for 15 min on ice, followed by incubation with 50 nM S15-APT QDs along with 5 μM free APT on ice; ( C ) A549 cells were incubated with 50 nM S15-APT QDs for 50 min on ice; Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Binding Assay, Flow Cytometry, Cytometry, Fluorescence, Concentration Assay, Incubation, Staining, Confocal Microscopy, Microscopy

Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.
Figure Legend Snippet: Characterization of the active transport of S15-APT The impact of temperature on cellular accumulation of S15-APT QDs (100 nM) in A549 cells was studied at 4° C vs. 37° C using flow cytometry. A549 cells were incubated for 2 h in growth medium in the absence of S15-APT QDs or in the presence of 100 nM S15-APT QDs at 4° C, or at 37° C. Trypsin treatment was applied to remove the putative target cell surface protein to which S15-APT QDs presumably bind. Cellular fluorescence was determined using flow cytometry. Left panel: schematic diagram of the experimental principle; Right panel: Mean fluorescence intensity.

Techniques Used: Flow Cytometry, Cytometry, Incubation, Fluorescence

Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Disruption of clathrin-mediated endocytosis of S15-APTs with different inhibitors Right: A549 cells were pre-incubated with: ( A ) 5 μM cytochalasin D for 30 min to block endocytosis of S15-APT QDs; ( B ) 80 μM Dynasore for 30 min; ( C ) 1 mM Amiloride for 10 min; ( D ) 1 μg/ml Filipin for 30 min; and ( E ) drug-free medium; followed by a further incubation of 2 h with 100 nM S15-APT QDs. Nuclei were stained with Hoechst 33342. Fluorescence microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. Left: Mean fluorescence intensity (M.F.I) values of S15-APT QDs in A549 cells incubated with different inhibitors were determined with IMARIS software for analysis of image data. The red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Blocking Assay, Staining, Fluorescence, Microscopy, Software

Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.
Figure Legend Snippet: Selective internalization of S15 APTs into human non-small cell lung A549 target cells Cells were incubated with 50 nM S15-APT QDs for 2 h. Nuclei were stained with 2 μg/ml Hoechst 33342. Fluorescence confocal microscopy was performed using an inverted confocal microscope (Zeiss LSM 710) at ×630 magnification. ( A ) Human A549 non-small cell lung carcinoma cells; ( B ) Normal human bronchial epithelial BEAS2B cells which served as normal non-target cells, ( C ) Human colon adenocarcinoma CaCo-2 cells; ( D ) Human cervical carcinoma HeLa cells; ( E ) A549 cells were incubated with 50 nM S15-APT QDs along with 5 μM free APT; ( F ) A549 cells were incubated with 50 nM random sequence APT-QDs; ( G ) A549 cells incubated with no S15-APT QDs; H) A549 cells incubated with 50 nM Qdot ® 655; ( I ) ABCG2-overexpressing MDR subline A549/K1.5 cells incubated with 50 nM S15-APT QDs; the red fluorescence channel was defined between 10-100 for all presented images.

Techniques Used: Incubation, Staining, Fluorescence, Confocal Microscopy, Microscopy, Sequencing

29) Product Images from "MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1"

Article Title: MicroRNA-200c inhibits epithelial-mesenchymal transition, invasion, and migration of lung cancer by targeting HMGB1

Journal: PLoS ONE

doi: 10.1371/journal.pone.0180844

miR-200c suppresses the expression of EMT-associated proteins in A549 cells. A miR-200c mimic or inhibitor was applied to A459 cells for 24h and then the mRNA and protein expression levels of α-SMA, vimentin, and β-catenin were measured by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p
Figure Legend Snippet: miR-200c suppresses the expression of EMT-associated proteins in A549 cells. A miR-200c mimic or inhibitor was applied to A459 cells for 24h and then the mRNA and protein expression levels of α-SMA, vimentin, and β-catenin were measured by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p

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

HMGB1 induces the expression of EMT-associated proteins in A549 cells. Overexpression or silencing of HMGB1 in A549 cells regulates α-SMA, vimentin, and β-catenin mRNA and protein expression as determined by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p
Figure Legend Snippet: HMGB1 induces the expression of EMT-associated proteins in A549 cells. Overexpression or silencing of HMGB1 in A549 cells regulates α-SMA, vimentin, and β-catenin mRNA and protein expression as determined by real-time PCR (A), western blotting (B), and immunocytochemical staining (C). *p

Techniques Used: Expressing, Over Expression, Real-time Polymerase Chain Reaction, Western Blot, Staining

Overexpression of HMGB1 in NSCLC xenografts model. A549 cells and LV-HMGB1 cells were inoculated into the right flank of severe combined immunodeficiency mice. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted (A and B). *p
Figure Legend Snippet: Overexpression of HMGB1 in NSCLC xenografts model. A549 cells and LV-HMGB1 cells were inoculated into the right flank of severe combined immunodeficiency mice. Tumor volume was measured every 3 days with slide calipers starting from day 7, and a growth curve was plotted (A and B). *p

Techniques Used: Over Expression, Mouse Assay

30) Product Images from "A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging"

Article Title: A peptide tag-specific nanobody enables high-quality labeling for dSTORM imaging

Journal: Nature Communications

doi: 10.1038/s41467-018-03191-2

Visualization of endogenously expressed BC2-tagged actin labeled with bivBC2-Nb AF647 . a Wide-field images of chemically fixed wild-type A549 and HeLa (-wt; left panel), as well as chemically fixed A549- BC2T ACTB and HeLa- BC2T ACTB cells (right panel). Cells were either left untreated (0 h) or stimulated for 48 h with TGFβ (5 ng ml −1 ) followed by staining with phalloidin AF555 and bivBC2-Nb AF647 . Scale bars, 25 µm. b dSTORM image of a representative HeLa- BC2T ACTB cell. Scale bars, image 5 µm, insets 1 µm. Image reconstruction details are given in Methods section. Imaging sequence taken from raw data acquisition can be found in Supplementary Movie 5 , assessment of AF647 photophysics under dSTORM imaging conditions can be found in Supplementary Fig. 15
Figure Legend Snippet: Visualization of endogenously expressed BC2-tagged actin labeled with bivBC2-Nb AF647 . a Wide-field images of chemically fixed wild-type A549 and HeLa (-wt; left panel), as well as chemically fixed A549- BC2T ACTB and HeLa- BC2T ACTB cells (right panel). Cells were either left untreated (0 h) or stimulated for 48 h with TGFβ (5 ng ml −1 ) followed by staining with phalloidin AF555 and bivBC2-Nb AF647 . Scale bars, 25 µm. b dSTORM image of a representative HeLa- BC2T ACTB cell. Scale bars, image 5 µm, insets 1 µm. Image reconstruction details are given in Methods section. Imaging sequence taken from raw data acquisition can be found in Supplementary Movie 5 , assessment of AF647 photophysics under dSTORM imaging conditions can be found in Supplementary Fig. 15

Techniques Used: Labeling, Staining, Imaging, Sequencing

31) Product Images from "The cytochrome P450 slow metabolizers CYP2C9*2 and CYP2C9*3 directly regulate tumorigenesis via reduced epoxyeicosatrienoic acid production"

Article Title: The cytochrome P450 slow metabolizers CYP2C9*2 and CYP2C9*3 directly regulate tumorigenesis via reduced epoxyeicosatrienoic acid production

Journal: Cancer research

doi: 10.1158/0008-5472.CAN-17-3977

Analysis of EET production in A549 cells expressing CYP2C9* variant proteins. A, B, ). Circles indicate individual cell plates, and bars and errors show mean values and SD. The values in A and B are reported as ng/mg total cell protein or ng/ml total medium of EET + DHET (n=2 experiments). C, Levels of individual 8,9-, 11,12-, and 14,15- EET regioisomers and their DHET metabolites detected in A549 infected and analyzed as indicated above. Values are reported as ng/mg total protein (n=2 experiments).
Figure Legend Snippet: Analysis of EET production in A549 cells expressing CYP2C9* variant proteins. A, B, ). Circles indicate individual cell plates, and bars and errors show mean values and SD. The values in A and B are reported as ng/mg total cell protein or ng/ml total medium of EET + DHET (n=2 experiments). C, Levels of individual 8,9-, 11,12-, and 14,15- EET regioisomers and their DHET metabolites detected in A549 infected and analyzed as indicated above. Values are reported as ng/mg total protein (n=2 experiments).

Techniques Used: Expressing, Variant Assay, Infection

Effects of CYP2C9* variants on EC proliferation and migration. A, B, Cell lysates from HUVEC (A) or Cyp2c44KO (B) ECs infected with empty adenovirus or adenovirus carrying CYP2C9*1, CYP2C9*2, or CYP2C9*3 cDNAs were analyzed by Western blotting for expression of CYP2C9 proteins and FAK. C-F, HUVEC or Cyp2c44KO ECs infected as described above were plated in 96-well plates in complete medium. After 12 hrs, the cells were incubated with serum-free medium with 3 H-thymidine with or without AA (1 uM) ( C ), EET-A (1.25–5 μM) ( D ), 14,15-EEZE (1.25–5 μM) ( E ), or TEMPOL (4 μM) alone or in combination with EET-A (5 μM) ( F ). Values represent the mean ± SD of two experiments performed at least in triplicate. G, Schematic representation of the modified Boyden chamber assay used to evaluate migration of HUVEC or Cyp2c44KO ECs. The bottom well contains A549 cells infected with vector or the various CYP2C9* variants incubated with serum free medium that contained or not AA (1 uM) or 14,15-EEZE (5 uM). H, HUVEC or Cyp2c44KO ECs, added to the upper wells of Boyden chamber as described in G, were allowed to migrate for 4 hrs. The number of migrated cells was counted and expressed as the number of cells per microscopic field. Values are the mean ± SD of two experiments with at least eight microscopic fields evaluated.
Figure Legend Snippet: Effects of CYP2C9* variants on EC proliferation and migration. A, B, Cell lysates from HUVEC (A) or Cyp2c44KO (B) ECs infected with empty adenovirus or adenovirus carrying CYP2C9*1, CYP2C9*2, or CYP2C9*3 cDNAs were analyzed by Western blotting for expression of CYP2C9 proteins and FAK. C-F, HUVEC or Cyp2c44KO ECs infected as described above were plated in 96-well plates in complete medium. After 12 hrs, the cells were incubated with serum-free medium with 3 H-thymidine with or without AA (1 uM) ( C ), EET-A (1.25–5 μM) ( D ), 14,15-EEZE (1.25–5 μM) ( E ), or TEMPOL (4 μM) alone or in combination with EET-A (5 μM) ( F ). Values represent the mean ± SD of two experiments performed at least in triplicate. G, Schematic representation of the modified Boyden chamber assay used to evaluate migration of HUVEC or Cyp2c44KO ECs. The bottom well contains A549 cells infected with vector or the various CYP2C9* variants incubated with serum free medium that contained or not AA (1 uM) or 14,15-EEZE (5 uM). H, HUVEC or Cyp2c44KO ECs, added to the upper wells of Boyden chamber as described in G, were allowed to migrate for 4 hrs. The number of migrated cells was counted and expressed as the number of cells per microscopic field. Values are the mean ± SD of two experiments with at least eight microscopic fields evaluated.

Techniques Used: Migration, Infection, Western Blot, Expressing, Incubation, Modification, Boyden Chamber Assay, Plasmid Preparation

CYP2C9*1 promotes tumorigenesis. A, Cell lysates from A549 cells infected with empty adenovirus (Vector), or adenovirus carrying CYP2C9*1 , CYP2C9*2 , or CYP2C9*3 cDNAs were analyzed by Western blotting for expression of CYP2C9 and focal adhesion kinase (FAK, used as loading control) (n=3 infections). B, Images of tumors isolated from mice 14 days after receiving two s.c. injections of A549-Vector, −2C9*1, −2C9*2, or −2C9*3 cells. C, Tumor uptake was evaluated by counting the number of tumors in each mouse. The mice were divided into three groups: no tumors (0% uptake), one tumor (50% uptake), and two tumors (100% uptake). D, Tumor volume was evaluated with a caliper. Circles show individual tumors, while bars and errors show mean values and SD E, F, Tumor frozen sections were stained with anti-mouse CD31 antibody and vascularization was quantified as described in the Methods. Circles show individual mice, while bars and errors show mean values and SD.
Figure Legend Snippet: CYP2C9*1 promotes tumorigenesis. A, Cell lysates from A549 cells infected with empty adenovirus (Vector), or adenovirus carrying CYP2C9*1 , CYP2C9*2 , or CYP2C9*3 cDNAs were analyzed by Western blotting for expression of CYP2C9 and focal adhesion kinase (FAK, used as loading control) (n=3 infections). B, Images of tumors isolated from mice 14 days after receiving two s.c. injections of A549-Vector, −2C9*1, −2C9*2, or −2C9*3 cells. C, Tumor uptake was evaluated by counting the number of tumors in each mouse. The mice were divided into three groups: no tumors (0% uptake), one tumor (50% uptake), and two tumors (100% uptake). D, Tumor volume was evaluated with a caliper. Circles show individual tumors, while bars and errors show mean values and SD E, F, Tumor frozen sections were stained with anti-mouse CD31 antibody and vascularization was quantified as described in the Methods. Circles show individual mice, while bars and errors show mean values and SD.

Techniques Used: Infection, Plasmid Preparation, Western Blot, Expressing, Isolation, Mouse Assay, Staining

32) Product Images from "MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner"

Article Title: MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-018-0594-4

The CM derived from miR-16-transfected fibroblasts displays reduced pro-tumorigenic properties. a Serum-starved A549 cells were stimulated for the indicated periods with the CM collected from CAF154-hTERT fibroblasts and diluted 1:2 in medium without serum. Western blot was performed to detect the activation of cMet, AKT, and ERK pathways. Actin is shown as a loading control. b A549 (upper panel) and LT73 (bottom panel) cells were stimulated with CM derived from CAF154-hTERT fibroblasts transfected with control miR-C or miR-16, and cell proliferation was measured 72 h later by CTG (** p = 0.0066, n = 5; * p = 0.0386). c A549 cells stimulated as in b were employed in wound-healing experiments. d A549 cells were stimulated with CM collected from CAF154-hTERT fibroblasts transfected with a siRNA specific for HGF. e HGF concentration in the CM of a panel of primary patient-derived fibroblasts. Arrows indicate CM media used in f and i (black arrows for high-concentration and green arrows for low-concentration of HGF). f Wound-healing experiments performed as in c with A549 cells stimulated with CM shown in e , with o r without an HGF-neutralizing antibody (HGFi). g Results of migration experiments ( f ) after 24 h of migration (* p = 0.0355; *** p = 0.0004). h Time necessary to close half of the gap (T1/2) was plotted together with the concentration of HGF in the CM ( R 2 = 0.5647, slope = − 269.0 ± 83.51). i Migration experiments were performed with Calu-1 cells stimulated with a fibroblast-derived CM containing high levels of HGF (CAF206) and one with low levels of the cytokine (CAF190). j Results of migration experiments ( i ) after 12 h of migration in three independent experiments (* p = 0.0241)
Figure Legend Snippet: The CM derived from miR-16-transfected fibroblasts displays reduced pro-tumorigenic properties. a Serum-starved A549 cells were stimulated for the indicated periods with the CM collected from CAF154-hTERT fibroblasts and diluted 1:2 in medium without serum. Western blot was performed to detect the activation of cMet, AKT, and ERK pathways. Actin is shown as a loading control. b A549 (upper panel) and LT73 (bottom panel) cells were stimulated with CM derived from CAF154-hTERT fibroblasts transfected with control miR-C or miR-16, and cell proliferation was measured 72 h later by CTG (** p = 0.0066, n = 5; * p = 0.0386). c A549 cells stimulated as in b were employed in wound-healing experiments. d A549 cells were stimulated with CM collected from CAF154-hTERT fibroblasts transfected with a siRNA specific for HGF. e HGF concentration in the CM of a panel of primary patient-derived fibroblasts. Arrows indicate CM media used in f and i (black arrows for high-concentration and green arrows for low-concentration of HGF). f Wound-healing experiments performed as in c with A549 cells stimulated with CM shown in e , with o r without an HGF-neutralizing antibody (HGFi). g Results of migration experiments ( f ) after 24 h of migration (* p = 0.0355; *** p = 0.0004). h Time necessary to close half of the gap (T1/2) was plotted together with the concentration of HGF in the CM ( R 2 = 0.5647, slope = − 269.0 ± 83.51). i Migration experiments were performed with Calu-1 cells stimulated with a fibroblast-derived CM containing high levels of HGF (CAF206) and one with low levels of the cytokine (CAF190). j Results of migration experiments ( i ) after 12 h of migration in three independent experiments (* p = 0.0241)

Techniques Used: Derivative Assay, Transfection, Western Blot, Activation Assay, CTG Assay, Concentration Assay, Migration

MiR-16 affects the pro-tumorigenic properties of the fibroblasts in vivo. a HGF levels in the CM medium employed and b miR-16 expression in the CAF154-hTERT fibroblasts were evaluated by ELISA and real-time PCR, respectively. c A549 cells (5 × 10 5 cells) were injected in the flanks of immunosuppressed nude mice after 24 h culturing in CM (1:2) collected from CAF154-hTERT fibroblasts transfected with mir-C, with or without a neutralizing anti HGF antibody (HGFi), and miR-16 ( n = 6). Mice were considered engrafted when tumor volume reached 100 mm 3 (Gehan-Breslow-Wilcoxon test: miR-C vs miR-16 p = 0.0090, miR-C vs HGFi p = 0.0014). d A549 cancer cells were subcutaneously injected in nude mice together with the fibroblasts described in c (ratio cancer cells/fibroblasts 1:3; Gehan-Breslow-Wilcoxon test: p = 0.0437). e , f Lungs collected from mice described in c and b , respectively, were collected and analyzed by FACS for the presence of metastatic human cells ( e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735)
Figure Legend Snippet: MiR-16 affects the pro-tumorigenic properties of the fibroblasts in vivo. a HGF levels in the CM medium employed and b miR-16 expression in the CAF154-hTERT fibroblasts were evaluated by ELISA and real-time PCR, respectively. c A549 cells (5 × 10 5 cells) were injected in the flanks of immunosuppressed nude mice after 24 h culturing in CM (1:2) collected from CAF154-hTERT fibroblasts transfected with mir-C, with or without a neutralizing anti HGF antibody (HGFi), and miR-16 ( n = 6). Mice were considered engrafted when tumor volume reached 100 mm 3 (Gehan-Breslow-Wilcoxon test: miR-C vs miR-16 p = 0.0090, miR-C vs HGFi p = 0.0014). d A549 cancer cells were subcutaneously injected in nude mice together with the fibroblasts described in c (ratio cancer cells/fibroblasts 1:3; Gehan-Breslow-Wilcoxon test: p = 0.0437). e , f Lungs collected from mice described in c and b , respectively, were collected and analyzed by FACS for the presence of metastatic human cells ( e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735)

Techniques Used: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Injection, Mouse Assay, Transfection, FACS

High-throughput screening to identify miRNAs that modulate the pro-tumorigenic potential of cancer-associated fibroblasts. a Schematic of the HTS: 8000 CAFs/well were seeded in 96-well plates and reverse transfected with a library of human miRNA mimics composed of 988 mature miRNAs (875 unique sequences). After 48 h, A549-GFP lung cancer cells were added (3500 cells/well) and further cultured for 48 h. Nuclei were then stained with Hoechst 33342, and automated fluorescence microscopy analysis was performed to quantify the total number of fibroblasts (GFP-negative) and A549 (GFP-positive) cells. Two independent screenings were performed; plates were normalized for the median of the samples of each plate and miRNAs were scored for their capacity to increase or decrease A549 cell growth. b Representative images of the screening showing cells transfected with a miRNA inhibiting the growth of the A549 cells (upper panel), a control miRNA (middle panel), and a miRNA stimulating the proliferation of A549 cells (lower panel). c Summary of the screening results, showing the distribution of A549 cell number after transfection with the miRNA library. d Effect of miRNAs on CAF154-hTERT fibroblasts and co-cultured A549 cell number (Spearman r = 0.35). Not all the miRNAs displayed the same effect on A549 and CAF154-hTERT cells. For example, miR-20a, miR-20b, miR-519b-3p, and miR-519c-3p inhibited the proliferation of CAF154-hTERT cells (
Figure Legend Snippet: High-throughput screening to identify miRNAs that modulate the pro-tumorigenic potential of cancer-associated fibroblasts. a Schematic of the HTS: 8000 CAFs/well were seeded in 96-well plates and reverse transfected with a library of human miRNA mimics composed of 988 mature miRNAs (875 unique sequences). After 48 h, A549-GFP lung cancer cells were added (3500 cells/well) and further cultured for 48 h. Nuclei were then stained with Hoechst 33342, and automated fluorescence microscopy analysis was performed to quantify the total number of fibroblasts (GFP-negative) and A549 (GFP-positive) cells. Two independent screenings were performed; plates were normalized for the median of the samples of each plate and miRNAs were scored for their capacity to increase or decrease A549 cell growth. b Representative images of the screening showing cells transfected with a miRNA inhibiting the growth of the A549 cells (upper panel), a control miRNA (middle panel), and a miRNA stimulating the proliferation of A549 cells (lower panel). c Summary of the screening results, showing the distribution of A549 cell number after transfection with the miRNA library. d Effect of miRNAs on CAF154-hTERT fibroblasts and co-cultured A549 cell number (Spearman r = 0.35). Not all the miRNAs displayed the same effect on A549 and CAF154-hTERT cells. For example, miR-20a, miR-20b, miR-519b-3p, and miR-519c-3p inhibited the proliferation of CAF154-hTERT cells (

Techniques Used: High Throughput Screening Assay, Transfection, Cell Culture, Staining, Fluorescence, Microscopy

The FGFR-1 receptor regulates HGF secretion, and it is targeted by miR-16. a CAF154-hTERT fibroblasts (3 × 10 5 cells/well) were serum-starved for 48 h and then stimulated with increasing doses of FGF-2. CM was collected after 24 h and analyzed by ELISA to evaluate the levels of secreted HGF. b CAF154-hTERT fibroblasts were transfected with the non-targeting miR-C, miR-16, a control siRNA, or a siRNA targeting FGFR-1 or HGF. After 72 h, the CM was collected and, together with a non-conditioned medium, used to stimulate the A549 cells. Western blot was performed on transfected CAF154-hTERT fibroblasts and on stimulated A549 cells to detect the activated state of cMet and its total levels, the levels of FGFR-1, and the activated form of ERK1/2. Actin is shown as a loading control. c Luciferase assay performed as in Fig. 2 e, by using 293 T cells transfected with the pMirTarget FGFR-1 3′UTR and a Renilla -expressing plasmid. The FGFR-1 3′UTR displays two putative miR-16 binding sites (Additional file 3 : Figure S3). d Levels of HGF in CM of CAF154-hTERT fibroblasts transfected with the control miR-C and miR-16 or a control siRNA and siRNA specific for FGFR-1 or HGF. Non-conditioned medium is shown as a negative control. e CAF154-hTERT fibroblasts were treated with 10 μM FGFR-1 inhibitor SU5402 and CM collected at the indicated times to evaluate HGF concentration by ELISA. The graph is representative of two independent experiments. f Correlation between the expression of HGF and FGFR-1 in primary fibroblasts derived from lung cancer patients and expressing high levels of HGF ( R 2 = 0.3607, slope = 0.3797 ± 0.1054). g CAF154-hTERT fibroblasts were transduced with lentiviral particles to stably express FGFR-1 and transfected with control miR-C and miR-16. Western blot was performed 72 h after transfection to detect MEK1/2 and FGFR-1 levels and the activated form of ERK1/2. Actin is shown as a loading control. h ELISA performed to evaluate the levels of HGF in the CM of cells transfected as described in g
Figure Legend Snippet: The FGFR-1 receptor regulates HGF secretion, and it is targeted by miR-16. a CAF154-hTERT fibroblasts (3 × 10 5 cells/well) were serum-starved for 48 h and then stimulated with increasing doses of FGF-2. CM was collected after 24 h and analyzed by ELISA to evaluate the levels of secreted HGF. b CAF154-hTERT fibroblasts were transfected with the non-targeting miR-C, miR-16, a control siRNA, or a siRNA targeting FGFR-1 or HGF. After 72 h, the CM was collected and, together with a non-conditioned medium, used to stimulate the A549 cells. Western blot was performed on transfected CAF154-hTERT fibroblasts and on stimulated A549 cells to detect the activated state of cMet and its total levels, the levels of FGFR-1, and the activated form of ERK1/2. Actin is shown as a loading control. c Luciferase assay performed as in Fig. 2 e, by using 293 T cells transfected with the pMirTarget FGFR-1 3′UTR and a Renilla -expressing plasmid. The FGFR-1 3′UTR displays two putative miR-16 binding sites (Additional file 3 : Figure S3). d Levels of HGF in CM of CAF154-hTERT fibroblasts transfected with the control miR-C and miR-16 or a control siRNA and siRNA specific for FGFR-1 or HGF. Non-conditioned medium is shown as a negative control. e CAF154-hTERT fibroblasts were treated with 10 μM FGFR-1 inhibitor SU5402 and CM collected at the indicated times to evaluate HGF concentration by ELISA. The graph is representative of two independent experiments. f Correlation between the expression of HGF and FGFR-1 in primary fibroblasts derived from lung cancer patients and expressing high levels of HGF ( R 2 = 0.3607, slope = 0.3797 ± 0.1054). g CAF154-hTERT fibroblasts were transduced with lentiviral particles to stably express FGFR-1 and transfected with control miR-C and miR-16. Western blot was performed 72 h after transfection to detect MEK1/2 and FGFR-1 levels and the activated form of ERK1/2. Actin is shown as a loading control. h ELISA performed to evaluate the levels of HGF in the CM of cells transfected as described in g

Techniques Used: Enzyme-linked Immunosorbent Assay, Transfection, Western Blot, Luciferase, Expressing, Plasmid Preparation, Binding Assay, Negative Control, Concentration Assay, Derivative Assay, Transduction, Stable Transfection

33) Product Images from "MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner"

Article Title: MiR-16 regulates the pro-tumorigenic potential of lung fibroblasts through the inhibition of HGF production in an FGFR-1- and MEK1-dependent manner

Journal: Journal of Hematology & Oncology

doi: 10.1186/s13045-018-0594-4

The CM derived from miR-16-transfected fibroblasts displays reduced pro-tumorigenic properties. a Serum-starved A549 cells were stimulated for the indicated periods with the CM collected from CAF154-hTERT fibroblasts and diluted 1:2 in medium without serum. Western blot was performed to detect the activation of cMet, AKT, and ERK pathways. Actin is shown as a loading control. b A549 (upper panel) and LT73 (bottom panel) cells were stimulated with CM derived from CAF154-hTERT fibroblasts transfected with control miR-C or miR-16, and cell proliferation was measured 72 h later by CTG (** p = 0.0066, n = 5; * p = 0.0386). c A549 cells stimulated as in b were employed in wound-healing experiments. d A549 cells were stimulated with CM collected from CAF154-hTERT fibroblasts transfected with a siRNA specific for HGF. e HGF concentration in the CM of a panel of primary patient-derived fibroblasts. Arrows indicate CM media used in f and i (black arrows for high-concentration and green arrows for low-concentration of HGF). f Wound-healing experiments performed as in c with A549 cells stimulated with CM shown in e , with or without an HGF-neutralizing antibody (HGFi). g Results of migration experiments ( f ) after 24 h of migration (* p = 0.0355; *** p = 0.0004). h Time necessary to close half of the gap (T1/2) was plotted together with the concentration of HGF in the CM ( R 2 = 0.5647, slope = − 269.0 ± 83.51). i Migration experiments were performed with Calu-1 cells stimulated with a fibroblast-derived CM containing high levels of HGF (CAF206) and one with low levels of the cytokine (CAF190). j Results of migration experiments ( i ) after 12 h of migration in three independent experiments (* p = 0.0241)
Figure Legend Snippet: The CM derived from miR-16-transfected fibroblasts displays reduced pro-tumorigenic properties. a Serum-starved A549 cells were stimulated for the indicated periods with the CM collected from CAF154-hTERT fibroblasts and diluted 1:2 in medium without serum. Western blot was performed to detect the activation of cMet, AKT, and ERK pathways. Actin is shown as a loading control. b A549 (upper panel) and LT73 (bottom panel) cells were stimulated with CM derived from CAF154-hTERT fibroblasts transfected with control miR-C or miR-16, and cell proliferation was measured 72 h later by CTG (** p = 0.0066, n = 5; * p = 0.0386). c A549 cells stimulated as in b were employed in wound-healing experiments. d A549 cells were stimulated with CM collected from CAF154-hTERT fibroblasts transfected with a siRNA specific for HGF. e HGF concentration in the CM of a panel of primary patient-derived fibroblasts. Arrows indicate CM media used in f and i (black arrows for high-concentration and green arrows for low-concentration of HGF). f Wound-healing experiments performed as in c with A549 cells stimulated with CM shown in e , with or without an HGF-neutralizing antibody (HGFi). g Results of migration experiments ( f ) after 24 h of migration (* p = 0.0355; *** p = 0.0004). h Time necessary to close half of the gap (T1/2) was plotted together with the concentration of HGF in the CM ( R 2 = 0.5647, slope = − 269.0 ± 83.51). i Migration experiments were performed with Calu-1 cells stimulated with a fibroblast-derived CM containing high levels of HGF (CAF206) and one with low levels of the cytokine (CAF190). j Results of migration experiments ( i ) after 12 h of migration in three independent experiments (* p = 0.0241)

Techniques Used: Derivative Assay, Transfection, Western Blot, Activation Assay, CTG Assay, Concentration Assay, Migration

MiR-16 affects the pro-tumorigenic properties of the fibroblasts in vivo. a HGF levels in the CM medium employed and b miR-16 expression in the CAF154-hTERT fibroblasts were evaluated by ELISA and real-time PCR, respectively. c A549 cells (5 × 10 5 cells) were injected in the flanks of immunosuppressed nude mice after 24 h culturing in CM (1:2) collected from CAF154-hTERT fibroblasts transfected with mir-C, with or without a neutralizing anti HGF antibody (HGFi), and miR-16 ( n = 6). Mice were considered engrafted when tumor volume reached 100 mm 3 (Gehan-Breslow-Wilcoxon test: miR-C vs miR-16 p = 0.0090, miR-C vs HGFi p = 0.0014). d A549 cancer cells were subcutaneously injected in nude mice together with the fibroblasts described in c (ratio cancer cells/fibroblasts 1:3; Gehan-Breslow-Wilcoxon test: p = 0.0437). e , f Lungs collected from mice described in c and b , respectively, were collected and analyzed by FACS for the presence of metastatic human cells ( e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735)
Figure Legend Snippet: MiR-16 affects the pro-tumorigenic properties of the fibroblasts in vivo. a HGF levels in the CM medium employed and b miR-16 expression in the CAF154-hTERT fibroblasts were evaluated by ELISA and real-time PCR, respectively. c A549 cells (5 × 10 5 cells) were injected in the flanks of immunosuppressed nude mice after 24 h culturing in CM (1:2) collected from CAF154-hTERT fibroblasts transfected with mir-C, with or without a neutralizing anti HGF antibody (HGFi), and miR-16 ( n = 6). Mice were considered engrafted when tumor volume reached 100 mm 3 (Gehan-Breslow-Wilcoxon test: miR-C vs miR-16 p = 0.0090, miR-C vs HGFi p = 0.0014). d A549 cancer cells were subcutaneously injected in nude mice together with the fibroblasts described in c (ratio cancer cells/fibroblasts 1:3; Gehan-Breslow-Wilcoxon test: p = 0.0437). e , f Lungs collected from mice described in c and b , respectively, were collected and analyzed by FACS for the presence of metastatic human cells ( e miR-C vs miR-16, p = 0.2997; miR-C vs HGFi * p = 0.0312; f miR-C vs miR-16, p = 0.0735)

Techniques Used: In Vivo, Expressing, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Injection, Mouse Assay, Transfection, FACS

34) Product Images from "Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity"

Article Title: Low Resolution Solution Structure of HAMLET and the Importance of Its Alpha-Domains in Tumoricidal Activity

Journal: PLoS ONE

doi: 10.1371/journal.pone.0053051

Internalization of small peptides and induction of ion fluxes. (A) Internalization of biotinylated peptides (red) by A549 lung carcinoma cells counterstained with WGA (green) and Hoechst (blue) and examined by confocal microscopy. Peptides 1,10 and 11 were internalized in the absence of oleate and peptides 10 and 11 also in the presence of oleate. (B) K + and (C) Ca 2+ fluxes in tumor cells triggered by petides 1, 10 and 11 were measured by fluorescence spectrometry. Peptides 10 and 11 triggered Ca 2+ fluxes, in the presence and absence of oleate and peptide 6 a weaker Ca 2+ flux with oleate. (D) Insignificant Na + fluxes.
Figure Legend Snippet: Internalization of small peptides and induction of ion fluxes. (A) Internalization of biotinylated peptides (red) by A549 lung carcinoma cells counterstained with WGA (green) and Hoechst (blue) and examined by confocal microscopy. Peptides 1,10 and 11 were internalized in the absence of oleate and peptides 10 and 11 also in the presence of oleate. (B) K + and (C) Ca 2+ fluxes in tumor cells triggered by petides 1, 10 and 11 were measured by fluorescence spectrometry. Peptides 10 and 11 triggered Ca 2+ fluxes, in the presence and absence of oleate and peptide 6 a weaker Ca 2+ flux with oleate. (D) Insignificant Na + fluxes.

Techniques Used: Whole Genome Amplification, Confocal Microscopy, Fluorescence

Internalization of peptides into tumor cells and changes in tumor cell morphology. (A) Internalization of peptides. A549 lung carcinoma cells cultured on glass slides, were incubated with peptide-oleate mixtures for 1 hour, fixed and stained with AlexaFluor568-streptavidin, counterstained with WGA and examined by confocal microscopy. Alpha1 and alpha2 peptides, mixed with oleate, were internalized as shown by the red fluorescence. The beta peptide was not internalized. Scale bar 20 µm. (B) Morphological changes in A549 lung carcinoma cells treated with HAMLET, alpha1 peptide+oleate, alpha2 peptide+oleate and beta peptide+oleate recorded by holography imaging. Cells treated with HAMLET started to round up after 30 minutes and after 60 minutes, many cells had detached. Alpha1 peptide+oleate mixture triggers similar morphological changes as that by HAMLET. Alpha2 peptide+oleate mixture triggers similar morphological changes. Beta peptide+oleate mixture did not change cell morphology.
Figure Legend Snippet: Internalization of peptides into tumor cells and changes in tumor cell morphology. (A) Internalization of peptides. A549 lung carcinoma cells cultured on glass slides, were incubated with peptide-oleate mixtures for 1 hour, fixed and stained with AlexaFluor568-streptavidin, counterstained with WGA and examined by confocal microscopy. Alpha1 and alpha2 peptides, mixed with oleate, were internalized as shown by the red fluorescence. The beta peptide was not internalized. Scale bar 20 µm. (B) Morphological changes in A549 lung carcinoma cells treated with HAMLET, alpha1 peptide+oleate, alpha2 peptide+oleate and beta peptide+oleate recorded by holography imaging. Cells treated with HAMLET started to round up after 30 minutes and after 60 minutes, many cells had detached. Alpha1 peptide+oleate mixture triggers similar morphological changes as that by HAMLET. Alpha2 peptide+oleate mixture triggers similar morphological changes. Beta peptide+oleate mixture did not change cell morphology.

Techniques Used: Cell Culture, Incubation, Staining, Whole Genome Amplification, Confocal Microscopy, Fluorescence, Imaging

Ion fluxes and tumor cell death. (A) Peptides trigger ion fluxes in tumor cells. The free intracellular concentration of Na + , K + and Ca 2+ were measured by fluorescence spectrometry using CoroNa Green, FluxOR and Fluo-4, respectively. HAMLET, alpha1 peptide+oleate and alpha2 peptide+oleate mixtures trigger rapid fluxes of all three ions. Intracellular potassium ion concentrations were reduced due to ion efflux, while those of sodium and calcium were increased. Mean of at least two experiments. P values are explained in the text. (B) Peptide-oleate mixtures kill tumor cells. A549 lung carcinoma cells and Jurkat leukemia cells were incubated with HAMLET, oleate or peptide-oleate mixtures for 3 hours. Cell death was quantified as ATP levels and PrestoBlue, in % of control.
Figure Legend Snippet: Ion fluxes and tumor cell death. (A) Peptides trigger ion fluxes in tumor cells. The free intracellular concentration of Na + , K + and Ca 2+ were measured by fluorescence spectrometry using CoroNa Green, FluxOR and Fluo-4, respectively. HAMLET, alpha1 peptide+oleate and alpha2 peptide+oleate mixtures trigger rapid fluxes of all three ions. Intracellular potassium ion concentrations were reduced due to ion efflux, while those of sodium and calcium were increased. Mean of at least two experiments. P values are explained in the text. (B) Peptide-oleate mixtures kill tumor cells. A549 lung carcinoma cells and Jurkat leukemia cells were incubated with HAMLET, oleate or peptide-oleate mixtures for 3 hours. Cell death was quantified as ATP levels and PrestoBlue, in % of control.

Techniques Used: Concentration Assay, Fluorescence, Incubation

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Article Snippet: .. 40,000 A549 cells were cultured on μ-Slide I coated with ibiTreat (ibidi, Martinsried, Germany) overnight. ..

Incubation:

Article Title: Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway
Article Snippet: .. Briefly, A549 cells were seeded into 35 mm μ-Dishes (Ibidi) at a density of 1.5 × 105 cells ml−1 (3 × 105 cells/dish) and incubated for 24 h. Confluent cell layers were washed with fresh medium and infected for 1.5 h with L . pneumophila strains (MOI 10) and/or treated with 10 μM LAI-1. .. After the infection and/or compound treatment, the cell layer was scratched with a sterile pipette tip and washed with fresh medium to remove detached cells.

Infection:

Article Title: Cell senescence is an antiviral defense mechanism
Article Snippet: .. For time-lapse microscopy, A549 cells grown on culture-insert microwells (80406, Ibidi) were infected with VSV-GFP at a MOI of 10 PFU/cell. .. Pictures were taken every 10 min starting at 5 hours post infection (hpi) and until 18 hpi on a Nikon BioStation microscope using BioStation IM software (v2.12, build 136, Nikon).

Article Title: Inter-kingdom Signaling by the Legionella Quorum Sensing Molecule LAI-1 Modulates Cell Migration through an IQGAP1-Cdc42-ARHGEF9-Dependent Pathway
Article Snippet: .. Briefly, A549 cells were seeded into 35 mm μ-Dishes (Ibidi) at a density of 1.5 × 105 cells ml−1 (3 × 105 cells/dish) and incubated for 24 h. Confluent cell layers were washed with fresh medium and infected for 1.5 h with L . pneumophila strains (MOI 10) and/or treated with 10 μM LAI-1. .. After the infection and/or compound treatment, the cell layer was scratched with a sterile pipette tip and washed with fresh medium to remove detached cells.

Migration:

Article Title: Curcumin Inhibits LIN-28A through the Activation of miRNA-98 in the Lung Cancer Cell Line A549
Article Snippet: .. To determine the migration ability of A549 cells, IBIDI™ Culture Inserts (IBIDI, Martinsried, Germany) were placed into 35-mm culture dishes and 1 × 105 cells/mL were added into the two reservoirs of the same insert. .. After 24 h, the insert was removed with caution creating a gap of 0.5 mm and cell migration was monitored by bright-field microscopy at specific time points.

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  • 90
    ibidi germination experiments a549 cells
    Aspergillus fumigatus hyphae grow parallel to <t>A549</t> cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.
    Germination Experiments A549 Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/germination experiments a549 cells/product/ibidi
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    93
    ibidi intracellular accumulation a549 cells
    Intracellular accumulation after 24 h incubation of 20 – 22 in <t>A549</t> cells (5 µM, A) visualized by fluorescence microscopy, and in fibroblasts (5 µM, B) visualized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0 × 0.40 DRY UV objective) and Leica DMI3000B. Presented images were obtained from three independent experiments.
    Intracellular Accumulation A549 Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/intracellular accumulation a549 cells/product/ibidi
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    intracellular accumulation a549 cells - by Bioz Stars, 2020-09
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    92
    ibidi a549 cells
    Aspergillus fumigatus hyphae grow parallel to <t>A549</t> cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.
    A549 Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 92/100, based on 7 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/a549 cells/product/ibidi
    Average 92 stars, based on 7 article reviews
    Price from $9.99 to $1999.99
    a549 cells - by Bioz Stars, 2020-09
    92/100 stars
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    92
    ibidi immunofluorescence a549 cells
    The EMT gene expression at baseline and in the presence of TGF-β (100 pM, 24 h incubation) was measured in the presence of vehicle (Veh, 0.1% DMSO) or over a range of concentrations of PF670462 (0.1 – 10 μM), Pirfenidone (1 –100 μM)) or nintedanib (10 –1000 nM) in. Data are presented as mean and SEM of n = 4 independent experiments and show the TGF-β induced fold increase in the expression of genes that change during EMT or in response to TGF-β in <t>A549</t> cells, including N -cadherin ( N -Cad), Vimentin (Vim), E -Cadherin ( E -Cad), α-smooth muscle actin (a-SMA) plasminogen-activator inhibitor-1 (PAI-1). Data were analyzed by two-way ANOVA with repeated measures, followed by comparisons at individual concentrations using Bonferroni’s correction for multiple comparisons. ∗ P
    Immunofluorescence A549 Cells, supplied by ibidi, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    immunofluorescence a549 cells - by Bioz Stars, 2020-09
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    Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques: Staining, Standard Deviation

    Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques:

    Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques:

    Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques: Blocking Assay

    Intracellular accumulation after 24 h incubation of 20 – 22 in A549 cells (5 µM, A) visualized by fluorescence microscopy, and in fibroblasts (5 µM, B) visualized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0 × 0.40 DRY UV objective) and Leica DMI3000B. Presented images were obtained from three independent experiments.

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    Article Title: In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

    doi: 10.1080/14756366.2019.1593159

    Figure Lengend Snippet: Intracellular accumulation after 24 h incubation of 20 – 22 in A549 cells (5 µM, A) visualized by fluorescence microscopy, and in fibroblasts (5 µM, B) visualized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0 × 0.40 DRY UV objective) and Leica DMI3000B. Presented images were obtained from three independent experiments.

    Article Snippet: Intracellular accumulation A549 cells (6 × 103 per well) were seeded onto microscopy glass slides with mounted 12-well silicone chamber (Ibidi GmbH, Planegg, Germany) and left to settle for 24 h. They were then treated with the studied 7-MEOTA-THA thio-/ureas 12 – 22 (5 µM) or with the medium alone (control group) for 24 h and washed with pre-warmed PBS in order to remove the unbound fraction.

    Techniques: Incubation, Fluorescence, Microscopy

    Intracellular accumulation after 24 h incubation of 14 – 17 in A549 cells (5 µM, A) visualized by fluorescence microscopy, and in fibroblasts (25 µM, B) visualized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0 × 0.40 DRY UV objective) and Leica DMI3000B. Presented images were obtained from three independent experiments.

    Journal: Journal of Enzyme Inhibition and Medicinal Chemistry

    Article Title: In vitro investigating of anticancer activity of new 7-MEOTA-tacrine heterodimers

    doi: 10.1080/14756366.2019.1593159

    Figure Lengend Snippet: Intracellular accumulation after 24 h incubation of 14 – 17 in A549 cells (5 µM, A) visualized by fluorescence microscopy, and in fibroblasts (25 µM, B) visualized by phase contrast microscopy. Images were obtained by the fluorescence microscope Leica DMI6000B (HCX PL APO CS 10.0 × 0.40 DRY UV objective) and Leica DMI3000B. Presented images were obtained from three independent experiments.

    Article Snippet: Intracellular accumulation A549 cells (6 × 103 per well) were seeded onto microscopy glass slides with mounted 12-well silicone chamber (Ibidi GmbH, Planegg, Germany) and left to settle for 24 h. They were then treated with the studied 7-MEOTA-THA thio-/ureas 12 – 22 (5 µM) or with the medium alone (control group) for 24 h and washed with pre-warmed PBS in order to remove the unbound fraction.

    Techniques: Incubation, Fluorescence, Microscopy

    Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Aspergillus fumigatus hyphae grow parallel to A549 cell layer whereas A. niger hyphae grow more perpendicularly. Direction of hyphal growth of A. fumigatus and A. niger in the presence of A549 cells: (A) Z-plane showing thickness of A549 cell layer, nuclei are stained with Hoechst (blue) and cell contour by CellMask TM (green). (B) A549 cell layer X/Y-plane. Z-plane (C,E) and X/Y-plane (D,F) showing A. fumigatus (C,D) and A. niger growth (E,F) on A549 (Hoechst stained) cells. (G) Hyphal growth of A. fumigatus and A. niger in the Z-plane. Bars represent standard deviation. ∗ Indicates significant difference. Approximately 10 fields per slide from three biological replicas were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques: Staining, Standard Deviation

    Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Germination and hyphal length of A. fumigatus are more effectively decreased in the presence of A549 cells than that of A. niger . (A) Germination and (B) hyphal length. Bar represents standard error of the mean. ∗ Indicates significant difference. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques:

    Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Polymorphonuclear neutrophils reduce A. niger germination and hyphal length at the surface of A549 cells. A. fumigatus and A. niger percentage of germination (A,C) and hyphal length (B,D) in the presence of A549 cells. Bars represent standard error of the mean. ∗ Indicate significant differences. Data are obtained from three separate experiments; at least 100 conidia per condition were analyzed.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques:

    Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.

    Journal: Frontiers in Microbiology

    Article Title: Hide, Keep Quiet, and Keep Low: Properties That Make Aspergillus fumigatus a Successful Lung Pathogen

    doi: 10.3389/fmicb.2016.00438

    Figure Lengend Snippet: Cytochalasin-B and nocodazole block internalization of Aspergillus fumigatus more effectively than that of A. niger . Internalization of A. niger (AN) and A. fumigatus (AF) by A549 after treatment with 10 μM cytochalasin-B (CB), and/or 20 μM nocodazole (Noc). For analysis, each conidium was scored as either inside or outside the epithelial cells. A chi-square proportion test was performed using a z -test (α = 0.01) and adjusting P -values for multiple comparisons using the Bonferroni correction method. ∗ Indicates significant difference.

    Article Snippet: Germination and Directionality of Hyphal Growth upon Interaction with A549 Cells For germination experiments A549 cells were grown on 8-mm glass coverslips (WPI international BV, Europe) and in μ-slide eight well chambers (Ibidi® , Munich, Germany) for observing hyphal growth directionality.

    Techniques: Blocking Assay

    The EMT gene expression at baseline and in the presence of TGF-β (100 pM, 24 h incubation) was measured in the presence of vehicle (Veh, 0.1% DMSO) or over a range of concentrations of PF670462 (0.1 – 10 μM), Pirfenidone (1 –100 μM)) or nintedanib (10 –1000 nM) in. Data are presented as mean and SEM of n = 4 independent experiments and show the TGF-β induced fold increase in the expression of genes that change during EMT or in response to TGF-β in A549 cells, including N -cadherin ( N -Cad), Vimentin (Vim), E -Cadherin ( E -Cad), α-smooth muscle actin (a-SMA) plasminogen-activator inhibitor-1 (PAI-1). Data were analyzed by two-way ANOVA with repeated measures, followed by comparisons at individual concentrations using Bonferroni’s correction for multiple comparisons. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis

    doi: 10.3389/fphar.2018.00738

    Figure Lengend Snippet: The EMT gene expression at baseline and in the presence of TGF-β (100 pM, 24 h incubation) was measured in the presence of vehicle (Veh, 0.1% DMSO) or over a range of concentrations of PF670462 (0.1 – 10 μM), Pirfenidone (1 –100 μM)) or nintedanib (10 –1000 nM) in. Data are presented as mean and SEM of n = 4 independent experiments and show the TGF-β induced fold increase in the expression of genes that change during EMT or in response to TGF-β in A549 cells, including N -cadherin ( N -Cad), Vimentin (Vim), E -Cadherin ( E -Cad), α-smooth muscle actin (a-SMA) plasminogen-activator inhibitor-1 (PAI-1). Data were analyzed by two-way ANOVA with repeated measures, followed by comparisons at individual concentrations using Bonferroni’s correction for multiple comparisons. ∗ P

    Article Snippet: Immunofluorescence A549 cells for immunofluorescence staining were seeded in ibiTreat 8-chamber slides (Ibidi) and left to adhere overnight.

    Techniques: Expressing, Incubation

    (A) Effect of PF670462 on EMT associated gene induction in A549 alveolar epithelial cells 4 and 24 h after TGF-β (100 pM) stimulation ( n = 4). (B) Immunofluorescence of membrane E -cadherin expression in A549 alveolar epithelial cells. Cells were treated with TGF-β (100 pM) for 48 h with PF670462 added 30 min prior to TGF-β. Nuclei were stained with DAPI. Quantification of n = 4 experiments is shown in (C) with > 50 fields analyzed per well. Data are presented as mean ± SEM from 4 independent experiments on A549 epithelial cells. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Casein Kinase 1δ/ε Inhibitor, PF670462 Attenuates the Fibrogenic Effects of Transforming Growth Factor-β in Pulmonary Fibrosis

    doi: 10.3389/fphar.2018.00738

    Figure Lengend Snippet: (A) Effect of PF670462 on EMT associated gene induction in A549 alveolar epithelial cells 4 and 24 h after TGF-β (100 pM) stimulation ( n = 4). (B) Immunofluorescence of membrane E -cadherin expression in A549 alveolar epithelial cells. Cells were treated with TGF-β (100 pM) for 48 h with PF670462 added 30 min prior to TGF-β. Nuclei were stained with DAPI. Quantification of n = 4 experiments is shown in (C) with > 50 fields analyzed per well. Data are presented as mean ± SEM from 4 independent experiments on A549 epithelial cells. ∗ P

    Article Snippet: Immunofluorescence A549 cells for immunofluorescence staining were seeded in ibiTreat 8-chamber slides (Ibidi) and left to adhere overnight.

    Techniques: Immunofluorescence, Expressing, Staining