Structured Review

Corning Life Sciences a549 cells
Effect of Saps 4–6 on cell viability and lysosome permeability. A ) Viability of <t>A549</t> cells after incubation with isolated Sap 6 at 37°C for a time period as indicated. Cell viability was analyzed by counting trypan blue-stained cells. B–E ) Sap 6 induced lysosome permeability and changed distribution of acridine orange (AO). B ) In control A549 cells, AO accumulated in lysosomes and produced red fluorescence. C ) Cells pretreated with Sap 6 had marked decrease of red fluorescence of lysosomes and increased green fluorescence from the cytosol as compared with the control cells in B. D ) Sap 2 did not significantly change lysosomal AO. E ) Ratios of green to red fluorescence from B–D. F–J ) Sap 4-induced changes in cellular distribution of AO were reversed by GRL-110C, a specific inhibitor of Sap 4. F ) Confocal microscopic image of AO in untreated A549 cells. G ) AO in cells pretreated with Sap 4 showed increased cytosolic green fluorescence, as compared with cells in F. H ) AO fluorescence in cells pretreated with Sap 4 in the presence of Sap 4 inhibitor GRL-110C was significantly redder than those cells in G and approached that in F. I ) Image of AO fluorescence in H 2 O 2 -treated cells (positive control). J ) Ratios of green to red fluorescence in experiments F–I. K–Q ) Saps 4–6, but not Saps 4 mut , 5 mut , and 6 mut , induced changes in cellular distribution of AO. K ) Image of AO in untreated A549 cells. L , N , P ) AO in cells treated separately with Saps 4–6 showed increased cytosolic green fluorescence as compared with untreated cells in K. M , O , Q ) Image of AO in cells treated separately with Saps 4 mut , 5 mut , and 6 mut showed lysosomal red fluorescence similar to the untreated cells in K. R–W ) Confocal microscopic images of Sap-treated A549 cells immunochemically stained for cathepsin D. R ) An untreated cell contains cathepsin D-positive lysosomes (arrow). S ) An H 2 O 2 -treated cell showed no cathepsin D-positive vesicles. T , U , W ) Cells separately treated with Saps 4–6 showed no cathepsin D-positive lysosomes as in S. V ) Cell treated with Sap 5 mut clearly retained cathepsin D-positive lysosomes (arrow). Quantitation on the ratios of green to red fluorescence in experiments K–Q between Saps and Sap mut s are statistically significant in the range of P
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1) Product Images from "Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism"

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism

Journal: The FASEB Journal

doi: 10.1096/fj.12-214353

Effect of Saps 4–6 on cell viability and lysosome permeability. A ) Viability of A549 cells after incubation with isolated Sap 6 at 37°C for a time period as indicated. Cell viability was analyzed by counting trypan blue-stained cells. B–E ) Sap 6 induced lysosome permeability and changed distribution of acridine orange (AO). B ) In control A549 cells, AO accumulated in lysosomes and produced red fluorescence. C ) Cells pretreated with Sap 6 had marked decrease of red fluorescence of lysosomes and increased green fluorescence from the cytosol as compared with the control cells in B. D ) Sap 2 did not significantly change lysosomal AO. E ) Ratios of green to red fluorescence from B–D. F–J ) Sap 4-induced changes in cellular distribution of AO were reversed by GRL-110C, a specific inhibitor of Sap 4. F ) Confocal microscopic image of AO in untreated A549 cells. G ) AO in cells pretreated with Sap 4 showed increased cytosolic green fluorescence, as compared with cells in F. H ) AO fluorescence in cells pretreated with Sap 4 in the presence of Sap 4 inhibitor GRL-110C was significantly redder than those cells in G and approached that in F. I ) Image of AO fluorescence in H 2 O 2 -treated cells (positive control). J ) Ratios of green to red fluorescence in experiments F–I. K–Q ) Saps 4–6, but not Saps 4 mut , 5 mut , and 6 mut , induced changes in cellular distribution of AO. K ) Image of AO in untreated A549 cells. L , N , P ) AO in cells treated separately with Saps 4–6 showed increased cytosolic green fluorescence as compared with untreated cells in K. M , O , Q ) Image of AO in cells treated separately with Saps 4 mut , 5 mut , and 6 mut showed lysosomal red fluorescence similar to the untreated cells in K. R–W ) Confocal microscopic images of Sap-treated A549 cells immunochemically stained for cathepsin D. R ) An untreated cell contains cathepsin D-positive lysosomes (arrow). S ) An H 2 O 2 -treated cell showed no cathepsin D-positive vesicles. T , U , W ) Cells separately treated with Saps 4–6 showed no cathepsin D-positive lysosomes as in S. V ) Cell treated with Sap 5 mut clearly retained cathepsin D-positive lysosomes (arrow). Quantitation on the ratios of green to red fluorescence in experiments K–Q between Saps and Sap mut s are statistically significant in the range of P
Figure Legend Snippet: Effect of Saps 4–6 on cell viability and lysosome permeability. A ) Viability of A549 cells after incubation with isolated Sap 6 at 37°C for a time period as indicated. Cell viability was analyzed by counting trypan blue-stained cells. B–E ) Sap 6 induced lysosome permeability and changed distribution of acridine orange (AO). B ) In control A549 cells, AO accumulated in lysosomes and produced red fluorescence. C ) Cells pretreated with Sap 6 had marked decrease of red fluorescence of lysosomes and increased green fluorescence from the cytosol as compared with the control cells in B. D ) Sap 2 did not significantly change lysosomal AO. E ) Ratios of green to red fluorescence from B–D. F–J ) Sap 4-induced changes in cellular distribution of AO were reversed by GRL-110C, a specific inhibitor of Sap 4. F ) Confocal microscopic image of AO in untreated A549 cells. G ) AO in cells pretreated with Sap 4 showed increased cytosolic green fluorescence, as compared with cells in F. H ) AO fluorescence in cells pretreated with Sap 4 in the presence of Sap 4 inhibitor GRL-110C was significantly redder than those cells in G and approached that in F. I ) Image of AO fluorescence in H 2 O 2 -treated cells (positive control). J ) Ratios of green to red fluorescence in experiments F–I. K–Q ) Saps 4–6, but not Saps 4 mut , 5 mut , and 6 mut , induced changes in cellular distribution of AO. K ) Image of AO in untreated A549 cells. L , N , P ) AO in cells treated separately with Saps 4–6 showed increased cytosolic green fluorescence as compared with untreated cells in K. M , O , Q ) Image of AO in cells treated separately with Saps 4 mut , 5 mut , and 6 mut showed lysosomal red fluorescence similar to the untreated cells in K. R–W ) Confocal microscopic images of Sap-treated A549 cells immunochemically stained for cathepsin D. R ) An untreated cell contains cathepsin D-positive lysosomes (arrow). S ) An H 2 O 2 -treated cell showed no cathepsin D-positive vesicles. T , U , W ) Cells separately treated with Saps 4–6 showed no cathepsin D-positive lysosomes as in S. V ) Cell treated with Sap 5 mut clearly retained cathepsin D-positive lysosomes (arrow). Quantitation on the ratios of green to red fluorescence in experiments K–Q between Saps and Sap mut s are statistically significant in the range of P

Techniques Used: Permeability, Incubation, Isolation, Staining, Produced, Fluorescence, Positive Control, Quantitation Assay

Binding of Saps 4–6 but not Sap 2 or mutant Saps 4–6 to A549 cells. Isolated Saps 2 and 4–6 and mutant Saps (Sap 4 mut , Sap 5 mut , and Sap 6 mut were mutated to change the integrin-binding motifs) were individually conjugated to Alexa Fluor-488 and incubated with A549 cells at 10°C for 1 h. After washing 3 times with PBS, the cells were observed in a Zeiss LSM510 confocal fluorescence microscope to detect cell-bound Alexa-Fluor Saps. Green fluorescence from Alexa-Fluor Saps 4–6 was found to associate with the cells; however, Alexa-Fluor-conjugated Sap 2, Sap 4 mut , Sap 5 mut , and Sap 6 mut displayed only minimal association with the cells. Scale bar = 20 μm.
Figure Legend Snippet: Binding of Saps 4–6 but not Sap 2 or mutant Saps 4–6 to A549 cells. Isolated Saps 2 and 4–6 and mutant Saps (Sap 4 mut , Sap 5 mut , and Sap 6 mut were mutated to change the integrin-binding motifs) were individually conjugated to Alexa Fluor-488 and incubated with A549 cells at 10°C for 1 h. After washing 3 times with PBS, the cells were observed in a Zeiss LSM510 confocal fluorescence microscope to detect cell-bound Alexa-Fluor Saps. Green fluorescence from Alexa-Fluor Saps 4–6 was found to associate with the cells; however, Alexa-Fluor-conjugated Sap 2, Sap 4 mut , Sap 5 mut , and Sap 6 mut displayed only minimal association with the cells. Scale bar = 20 μm.

Techniques Used: Binding Assay, Mutagenesis, Isolation, Incubation, Fluorescence, Microscopy

Binding of C. albicans Saps 4–6 to cell surface integrin. A ) Left panel: confocal microscopic image of an isolated human platelet is virtually without fluorescence. Center and right panels: a platelet that was incubated with Alexa-Fluor 488-labeled Sap 6 (center panel) and a deconvolved fluorescence image from confocal microscopy of an Alexa-Fluor Sap 6-treated platelet (right panel) show fluorescence concentrated at the cell surface. Scale bars = 2 μm. B ) Fluorescence intensity of platelets after incubation with Alexa-Fluor Sap 6 and different concentration of ADP. C ) Fluorescence images from confocal microscopy of an A549 cell after incubation with Saps at 10°C. Integrin β1 chain on the cell surface was revealed by Cy3-conjugated antibody (left panels). Fluorescence images of Alexa-Fluor modified Saps 4–6 are seen on the surface of A549 cells (center panels); thus, Alexa-Fluor-conjugated Sap 4 mut , Sap 5 mut , and Sap 6 mut did not bind the cells and appear as dark fields. Merged images (right panels) show colocalization of integrin β1 chain and Alexa-Fluor Saps 4–6 on the cell surface (arrows). Scale bar = 20 μm. D ) Platelet- and A549-associated fluorescence from bound Alexa-Fluor Sap 6 was inhibited by RGD-containing peptides RGDS and integrilin. E ) Highly purified Saps 4–6 inhibited the binding of integrin β1 antibody to A549 cells. * P
Figure Legend Snippet: Binding of C. albicans Saps 4–6 to cell surface integrin. A ) Left panel: confocal microscopic image of an isolated human platelet is virtually without fluorescence. Center and right panels: a platelet that was incubated with Alexa-Fluor 488-labeled Sap 6 (center panel) and a deconvolved fluorescence image from confocal microscopy of an Alexa-Fluor Sap 6-treated platelet (right panel) show fluorescence concentrated at the cell surface. Scale bars = 2 μm. B ) Fluorescence intensity of platelets after incubation with Alexa-Fluor Sap 6 and different concentration of ADP. C ) Fluorescence images from confocal microscopy of an A549 cell after incubation with Saps at 10°C. Integrin β1 chain on the cell surface was revealed by Cy3-conjugated antibody (left panels). Fluorescence images of Alexa-Fluor modified Saps 4–6 are seen on the surface of A549 cells (center panels); thus, Alexa-Fluor-conjugated Sap 4 mut , Sap 5 mut , and Sap 6 mut did not bind the cells and appear as dark fields. Merged images (right panels) show colocalization of integrin β1 chain and Alexa-Fluor Saps 4–6 on the cell surface (arrows). Scale bar = 20 μm. D ) Platelet- and A549-associated fluorescence from bound Alexa-Fluor Sap 6 was inhibited by RGD-containing peptides RGDS and integrilin. E ) Highly purified Saps 4–6 inhibited the binding of integrin β1 antibody to A549 cells. * P

Techniques Used: Binding Assay, Isolation, Fluorescence, Incubation, Labeling, Confocal Microscopy, Concentration Assay, Modification, Purification

Saps 4–6 induce apoptosis of A549 cells. Cells were incubated with individual Saps at 37°C for 23 h and studied for apoptosis. A ) Typical flow cytometric 2-dimensional dot plots of A549 cells after incubation with individual Saps, Sap mutants, or controls. Early apoptosis of the cells incubated with Saps, Sap mutants, or the controls were analyzed by annexin V-PE staining and flow cytometry. Right panels: Saps 4–6 significantly increased the number of annexin-positive cells (bottom right quadrants) over the untreated control cells. Center panels: Sap 4 mut -, 5 mut -, and 6 mut -treated cells did not show significant increase in the population of the annexin-positive cells. Data are representative of 4 independent experiments. B ) Top panel: results from two experiments in A ; quantitated percentage of cells in early apoptosis as determined by annexin staining and flow cytometry analysis. Camptothecin treatment was an apoptotic-positive control. Bottom panel: relative number of cells in late apoptosis after incubation with Saps. Quantitation was done with Hoechst 33342 staining and confocal microscopy. Apoptosis reagent camptothecin was used as a positive control. Statistical significance (in the range of P
Figure Legend Snippet: Saps 4–6 induce apoptosis of A549 cells. Cells were incubated with individual Saps at 37°C for 23 h and studied for apoptosis. A ) Typical flow cytometric 2-dimensional dot plots of A549 cells after incubation with individual Saps, Sap mutants, or controls. Early apoptosis of the cells incubated with Saps, Sap mutants, or the controls were analyzed by annexin V-PE staining and flow cytometry. Right panels: Saps 4–6 significantly increased the number of annexin-positive cells (bottom right quadrants) over the untreated control cells. Center panels: Sap 4 mut -, 5 mut -, and 6 mut -treated cells did not show significant increase in the population of the annexin-positive cells. Data are representative of 4 independent experiments. B ) Top panel: results from two experiments in A ; quantitated percentage of cells in early apoptosis as determined by annexin staining and flow cytometry analysis. Camptothecin treatment was an apoptotic-positive control. Bottom panel: relative number of cells in late apoptosis after incubation with Saps. Quantitation was done with Hoechst 33342 staining and confocal microscopy. Apoptosis reagent camptothecin was used as a positive control. Statistical significance (in the range of P

Techniques Used: Incubation, Flow Cytometry, Staining, Cytometry, Positive Control, Quantitation Assay, Confocal Microscopy

2) Product Images from "Tailoring Cell Morphomechanical Perturbations Through Metal Oxide Nanoparticles"

Article Title: Tailoring Cell Morphomechanical Perturbations Through Metal Oxide Nanoparticles

Journal: Nanoscale Research Letters

doi: 10.1186/s11671-019-2941-y

Young’s modulus values expressed in kPa, calculated from the nuclear region and the cytoskeletal area of Caco-2 ( a ) and A549 ( b ) cell lines after a treatment to 45 μg/ml of SiO 2 NPs and TiO 2 NPs for 72 h
Figure Legend Snippet: Young’s modulus values expressed in kPa, calculated from the nuclear region and the cytoskeletal area of Caco-2 ( a ) and A549 ( b ) cell lines after a treatment to 45 μg/ml of SiO 2 NPs and TiO 2 NPs for 72 h

Techniques Used:

Integrated density ( a , b ) and coherency ( c , d ) for Caco-2 and A549 cells treated with 45 μg/ml of SiO 2 NPs and TiO 2 NPs after 72 h. The integrated density and coherency values were expressed as a mean value and relative SD, calculated from confocal acquisitions by ImageJ (calculation on 15 cells). The mean values and their standard deviations were reported in the histograms. Results were statistically significant for p
Figure Legend Snippet: Integrated density ( a , b ) and coherency ( c , d ) for Caco-2 and A549 cells treated with 45 μg/ml of SiO 2 NPs and TiO 2 NPs after 72 h. The integrated density and coherency values were expressed as a mean value and relative SD, calculated from confocal acquisitions by ImageJ (calculation on 15 cells). The mean values and their standard deviations were reported in the histograms. Results were statistically significant for p

Techniques Used:

a – d SOD and MDA assays on Caco-2 and A549 cells. Cells were incubated with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, 96 h. The SOD assay used a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The standard curve was used as a positive control (data not shown). The MDA levels was detected by the quantification of MDA-TBA adduct (OD = 532 nm). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value
Figure Legend Snippet: a – d SOD and MDA assays on Caco-2 and A549 cells. Cells were incubated with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, 96 h. The SOD assay used a tetrazolium salt for detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. The standard curve was used as a positive control (data not shown). The MDA levels was detected by the quantification of MDA-TBA adduct (OD = 532 nm). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value

Techniques Used: Multiple Displacement Amplification, Incubation, Generated, Positive Control

Local enlargement of confocal acquisitions acquired in Figs. 6 and 7 showed (more in details) the effect of SiO 2 NPS and TiO 2 NPs on actin network of Caco-2 and A549 cells after the exposure of 45 μg/ml of NPs for 72 h and 96 h
Figure Legend Snippet: Local enlargement of confocal acquisitions acquired in Figs. 6 and 7 showed (more in details) the effect of SiO 2 NPS and TiO 2 NPs on actin network of Caco-2 and A549 cells after the exposure of 45 μg/ml of NPs for 72 h and 96 h

Techniques Used:

Effect of SiO 2 NPS and TiO 2 NPs on actin network on A549 cells. A549 were treated with 15 μg/ml and 45 μg/ml of NPs for 24 h, 48 h, 72 h, and 96 h; successively they were fixed and stained with Phalloidin–ATTO 488 and DAPI. The 2D images of cortical actin were acquired by a Zeiss LSM700 (Zeiss) confocal microscope equipped with an Axio Observer Z1 (Zeiss) inverted microscope using a × 100, 1.46 numerical aperture oil immersion lens. All data were processed by ZEN software (Zeiss)
Figure Legend Snippet: Effect of SiO 2 NPS and TiO 2 NPs on actin network on A549 cells. A549 were treated with 15 μg/ml and 45 μg/ml of NPs for 24 h, 48 h, 72 h, and 96 h; successively they were fixed and stained with Phalloidin–ATTO 488 and DAPI. The 2D images of cortical actin were acquired by a Zeiss LSM700 (Zeiss) confocal microscope equipped with an Axio Observer Z1 (Zeiss) inverted microscope using a × 100, 1.46 numerical aperture oil immersion lens. All data were processed by ZEN software (Zeiss)

Techniques Used: Staining, Microscopy, Inverted Microscopy, Software

TiO 2 NPs and SiO 2 NPs accumulation in Caco-2 and A549 cell lines exposed to 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h. Cells were then harvested, live cells were counted, and Ti and Si content was measured in 360,000 cells (μg Ti and μg Si). Data reported as mean ± SD from three independent experiments; statistical significance of exposed cells vs. control cells for p value
Figure Legend Snippet: TiO 2 NPs and SiO 2 NPs accumulation in Caco-2 and A549 cell lines exposed to 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h. Cells were then harvested, live cells were counted, and Ti and Si content was measured in 360,000 cells (μg Ti and μg Si). Data reported as mean ± SD from three independent experiments; statistical significance of exposed cells vs. control cells for p value

Techniques Used:

LDH ( a – b ) and ROS ( c – d ) assays on Caco-2 and A549 cells. Cells were incubated with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h. Percent of LDH leakage of nanoparticle-treated cells are expressed relative to non-treated control cells. Positive controls (P) consisted in the treatment of cells with 0.9% Triton X-100 showing ca. 500% LDH increase (data not shown). ROS levels were recorded exposing Caco-2 and A549 cells with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h incubated with 100 μM DCFH-DA. Cell fluorescence was measured. As a positive control (P), cells were incubated with 500 μM H 2 O 2 showing a ca. 300% DCFH-DA increase (not show). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value
Figure Legend Snippet: LDH ( a – b ) and ROS ( c – d ) assays on Caco-2 and A549 cells. Cells were incubated with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h. Percent of LDH leakage of nanoparticle-treated cells are expressed relative to non-treated control cells. Positive controls (P) consisted in the treatment of cells with 0.9% Triton X-100 showing ca. 500% LDH increase (data not shown). ROS levels were recorded exposing Caco-2 and A549 cells with 15 μg/ml and 45 μg/ml of TiO 2 NPs and SiO 2 NPs for 24 h, 48 h, 72 h, and 96 h incubated with 100 μM DCFH-DA. Cell fluorescence was measured. As a positive control (P), cells were incubated with 500 μM H 2 O 2 showing a ca. 300% DCFH-DA increase (not show). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value

Techniques Used: Incubation, Fluorescence, Positive Control

Viability assay (WST-8) of Caco-2 ( a ) and A549 ( b ) cells after 24 h, 48 h, 72 h, and 96 h of exposure to two doses (15 μg/ml and 45 μg/ml) of TiO 2 NPs and SiO 2 NPs. Viability of NP-treated cells was normalized to non-treated control cells. As positive control (P), cells were incubated with 5% DMSO (data not shown). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value
Figure Legend Snippet: Viability assay (WST-8) of Caco-2 ( a ) and A549 ( b ) cells after 24 h, 48 h, 72 h, and 96 h of exposure to two doses (15 μg/ml and 45 μg/ml) of TiO 2 NPs and SiO 2 NPs. Viability of NP-treated cells was normalized to non-treated control cells. As positive control (P), cells were incubated with 5% DMSO (data not shown). Data reported as mean ± SD from three independent experiments are considered statistically significant compared with control ( n = 8) for p value

Techniques Used: Viability Assay, Positive Control, Incubation

3) Product Images from "Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes"

Article Title: Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes

Journal: Journal of cellular physiology

doi: 10.1002/jcp.21279

HNP induces MAPK activation A549 and U937 cells incubated with either control solution (0.01% acetic acid, 0 μg/mL) or HNP at indicated concentrations for 15 and 30 min in the presence and absence of either U0126(MEK1/2 inhibitor), SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor) given 30 min prior to HNP. A–C. Representative Western blots of the HNP-induced MAPKs activation in A549 cells. D. The specific MAPK inhibitors decreased the HNP-mediated MAPK activation. E–G. Representative Western blots of the HNP-induced MAPKs activation in U937 cells, and the specific MAPK inhibitors decreased the HNP-mediated MAPK activation. V: DMSO vehicle control; * p
Figure Legend Snippet: HNP induces MAPK activation A549 and U937 cells incubated with either control solution (0.01% acetic acid, 0 μg/mL) or HNP at indicated concentrations for 15 and 30 min in the presence and absence of either U0126(MEK1/2 inhibitor), SB203580 (p38 inhibitor) or SP600125 (JNK inhibitor) given 30 min prior to HNP. A–C. Representative Western blots of the HNP-induced MAPKs activation in A549 cells. D. The specific MAPK inhibitors decreased the HNP-mediated MAPK activation. E–G. Representative Western blots of the HNP-induced MAPKs activation in U937 cells, and the specific MAPK inhibitors decreased the HNP-mediated MAPK activation. V: DMSO vehicle control; * p

Techniques Used: Activation Assay, Incubation, Western Blot

Effect of Src kinase on HNP-induced MAPK activation A549 and U937 cells were treated with PP2 30 min prior to HNP stimulation for additional 30 min. The cell lysates were analyzed to detect phosphorylated and total levels of ERK1/2 and p38. Representative Western blots along with densitometric analysis are illustrated. N = 8 from four independent experiments. V: DMSO vehicle control of PP2; * p
Figure Legend Snippet: Effect of Src kinase on HNP-induced MAPK activation A549 and U937 cells were treated with PP2 30 min prior to HNP stimulation for additional 30 min. The cell lysates were analyzed to detect phosphorylated and total levels of ERK1/2 and p38. Representative Western blots along with densitometric analysis are illustrated. N = 8 from four independent experiments. V: DMSO vehicle control of PP2; * p

Techniques Used: Activation Assay, Western Blot

Src tyrosine kinase is required for HNP-induced IL-8 production A549 cells and U937 cells were incubated with either control solution (0.01% acetic acid, 0 μg/mL) or HNP at indicated concentrations and time points in the presence and absence of PP2, given 30 min prior to HNP stimulation. The cell lysates were analyzed by Western blot to determine phosphorylated and total levels of Src. A. A representative blot showing HNP-induced Src activation and the mean band density analysis from 3 experiments in A549 cells. The use of the Src inhibitor PP2 decreased HNP-induced Src activation ( B ) and attenuated IL-8 production ( C ). D. A representative blot showing HNP-induced Src activation and the mean band density analysis from 3 experiments in U937 cells. The use of PP2 blunted the HNP-induced Src activation and attenuated IL-8 production ( E ). V: 0.01% DMSO vehicle control of PP2; * p
Figure Legend Snippet: Src tyrosine kinase is required for HNP-induced IL-8 production A549 cells and U937 cells were incubated with either control solution (0.01% acetic acid, 0 μg/mL) or HNP at indicated concentrations and time points in the presence and absence of PP2, given 30 min prior to HNP stimulation. The cell lysates were analyzed by Western blot to determine phosphorylated and total levels of Src. A. A representative blot showing HNP-induced Src activation and the mean band density analysis from 3 experiments in A549 cells. The use of the Src inhibitor PP2 decreased HNP-induced Src activation ( B ) and attenuated IL-8 production ( C ). D. A representative blot showing HNP-induced Src activation and the mean band density analysis from 3 experiments in U937 cells. The use of PP2 blunted the HNP-induced Src activation and attenuated IL-8 production ( E ). V: 0.01% DMSO vehicle control of PP2; * p

Techniques Used: Incubation, Western Blot, Activation Assay

Effect of Src kinase blockade on HNP-induced Akt activation A549 and U937 cells were treated with PP2 for 30 min and stimulated with HNP for additional 30 min. The cell lysates were analyzed to detect phosphorylated and total levels of Akt. Representative blots from three independent experiments, along with densitometric analysis. V: DMSO vehicle control of PP2; * p
Figure Legend Snippet: Effect of Src kinase blockade on HNP-induced Akt activation A549 and U937 cells were treated with PP2 for 30 min and stimulated with HNP for additional 30 min. The cell lysates were analyzed to detect phosphorylated and total levels of Akt. Representative blots from three independent experiments, along with densitometric analysis. V: DMSO vehicle control of PP2; * p

Techniques Used: Activation Assay

Involvement of MAPKs in the HNP-mediated IL-8 production A549 and U937 cells were treated with the specific inhibitors of MAPKs 30 min prior to HNP stimulation for 6 h in A549 cells ( A ) and 4 h in U937 cells ( B ). IL-8 levels were measured in cell supernatants by ELISA. N = 8 from four independent experiments. V: DMSO vehicle control; * p
Figure Legend Snippet: Involvement of MAPKs in the HNP-mediated IL-8 production A549 and U937 cells were treated with the specific inhibitors of MAPKs 30 min prior to HNP stimulation for 6 h in A549 cells ( A ) and 4 h in U937 cells ( B ). IL-8 levels were measured in cell supernatants by ELISA. N = 8 from four independent experiments. V: DMSO vehicle control; * p

Techniques Used: Enzyme-linked Immunosorbent Assay

HNP induce IL-8 release Cells were incubated overnight with serum-free medium, and exposed to either 0.01% acetic acid as control or HNP (10 μg/mL) for 8 h in A549 cells ( A ) or for 4 h in U937 cells ( B ). Cell supernatants were then collected for multiple cytokine assays. A549 cells or U937 cells were incubated in the presence of HNP at indicated concentrations for 8 h ( C ) and 4 h ( D ), respectively. In separate experiments, U937 cells were transfected with P2Y 6 sense and antisense, respectively prior to HNP stimulation for 8 h ( E ). IL-8 levels were measured in cell supernatants by an ELISA specific for human IL-8. N = 6 from three experiments. * p
Figure Legend Snippet: HNP induce IL-8 release Cells were incubated overnight with serum-free medium, and exposed to either 0.01% acetic acid as control or HNP (10 μg/mL) for 8 h in A549 cells ( A ) or for 4 h in U937 cells ( B ). Cell supernatants were then collected for multiple cytokine assays. A549 cells or U937 cells were incubated in the presence of HNP at indicated concentrations for 8 h ( C ) and 4 h ( D ), respectively. In separate experiments, U937 cells were transfected with P2Y 6 sense and antisense, respectively prior to HNP stimulation for 8 h ( E ). IL-8 levels were measured in cell supernatants by an ELISA specific for human IL-8. N = 6 from three experiments. * p

Techniques Used: Incubation, Transfection, Enzyme-linked Immunosorbent Assay

4) Product Images from "Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert"

Article Title: Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert

Journal: BMC Genomics

doi: 10.1186/s12864-018-4895-3

GO term enrichment analysis of cellular component. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells
Figure Legend Snippet: GO term enrichment analysis of cellular component. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells

Techniques Used: Incubation

GO term enrichment analysis of biological process. A549 up-regulated (green) and down-regulated (red) genes after A. fumigatus co-cultivation
Figure Legend Snippet: GO term enrichment analysis of biological process. A549 up-regulated (green) and down-regulated (red) genes after A. fumigatus co-cultivation

Techniques Used:

GO term enrichment analysis of cellular component. A549 down-regulated genes after A. fumigatus co-cultivation
Figure Legend Snippet: GO term enrichment analysis of cellular component. A549 down-regulated genes after A. fumigatus co-cultivation

Techniques Used:

Venn diagram and heatmap of differentially expressed orthologous genes between A. fumigatus and A. niger expressed during 12 h co-incubation with A549 cells
Figure Legend Snippet: Venn diagram and heatmap of differentially expressed orthologous genes between A. fumigatus and A. niger expressed during 12 h co-incubation with A549 cells

Techniques Used: Incubation

GO term enrichment analysis of molecular function. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells
Figure Legend Snippet: GO term enrichment analysis of molecular function. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells

Techniques Used: Incubation

GO term enrichment analysis of biological process. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells
Figure Legend Snippet: GO term enrichment analysis of biological process. A. fumigatus up-regulated (green) and A. niger up- regulated (blue) genes after 12 h of incubation in the presence of A549 epithelial cells

Techniques Used: Incubation

5) Product Images from "Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs"

Article Title: Multilayered Cultures of NSCLC cells grown at the Air-Liquid Interface allow the efficacy testing of inhaled anti-cancer drugs

Journal: Scientific Reports

doi: 10.1038/s41598-018-31332-6

Cell culture architecture influences the response to anti-cancer drugs administered by direct inoculation: ( A ) Details of the four anti-cancer drugs tested in this study. Their half-maximal inhibitory concentration (IC 50 ) is listed as reported in the GDSC database for the A549 cell model (in the text referred to as “nominal IC 50 ”). ( B ) Changes in cell viability of sub-confluent mono-cultures of A549 cells grown on plastic substrates and exposed to the four anti-cancer drugs at their nominal IC 50 concentration for 72 h. GDSC database experimental conditions were reproduced in our assay. Data, shown as average ± standard error of the mean (n replicates = 3; n tests = 3), are normalized to the cell viability of the untreated control (NT). The symbol (***) indicates statistically significant differences from NT (p
Figure Legend Snippet: Cell culture architecture influences the response to anti-cancer drugs administered by direct inoculation: ( A ) Details of the four anti-cancer drugs tested in this study. Their half-maximal inhibitory concentration (IC 50 ) is listed as reported in the GDSC database for the A549 cell model (in the text referred to as “nominal IC 50 ”). ( B ) Changes in cell viability of sub-confluent mono-cultures of A549 cells grown on plastic substrates and exposed to the four anti-cancer drugs at their nominal IC 50 concentration for 72 h. GDSC database experimental conditions were reproduced in our assay. Data, shown as average ± standard error of the mean (n replicates = 3; n tests = 3), are normalized to the cell viability of the untreated control (NT). The symbol (***) indicates statistically significant differences from NT (p

Techniques Used: Cell Culture, Concentration Assay

Comparison of the chemoresistance detected in ALI multilayered mono-cultures and 3D tumour spheroids: ( A,B ) Percentage (%) of live A549 cells ( A ) and ATP levels ( B ) detected in (from left to right) ALI multilayered mono-cultures and 3D tumour spheroids. Both in vitro models were grown for 14 d and then exposed to four anti-cancer drugs (docetaxel, cytarabine, vinblastine and methotrexate) at their nominal IC 50 concentration for 72 h. Cell cultures were exposed to drugs by direct inoculation. Data are reported as average ± standard error of the mean (n replicates = 2; n tests = 3). The symbols (*), (**) and (***) indicate significant differences (p values
Figure Legend Snippet: Comparison of the chemoresistance detected in ALI multilayered mono-cultures and 3D tumour spheroids: ( A,B ) Percentage (%) of live A549 cells ( A ) and ATP levels ( B ) detected in (from left to right) ALI multilayered mono-cultures and 3D tumour spheroids. Both in vitro models were grown for 14 d and then exposed to four anti-cancer drugs (docetaxel, cytarabine, vinblastine and methotrexate) at their nominal IC 50 concentration for 72 h. Cell cultures were exposed to drugs by direct inoculation. Data are reported as average ± standard error of the mean (n replicates = 2; n tests = 3). The symbols (*), (**) and (***) indicate significant differences (p values

Techniques Used: In Vitro, Concentration Assay

Time-dependent phenotype modifications in ALI MCCs: ( A ) Time-dependent changes in: ATP levels, percentage (%) of live A549 cells, % of LY passage and P app values in ALI multilayered mono-cultures grown up to 14 d. Data are shown as average ± standard error of the mean (n replicates = 2; n tests = 3). The symbols (**) and (***) indicate statistically significant changes as compared to the values measured at 24 h (p
Figure Legend Snippet: Time-dependent phenotype modifications in ALI MCCs: ( A ) Time-dependent changes in: ATP levels, percentage (%) of live A549 cells, % of LY passage and P app values in ALI multilayered mono-cultures grown up to 14 d. Data are shown as average ± standard error of the mean (n replicates = 2; n tests = 3). The symbols (**) and (***) indicate statistically significant changes as compared to the values measured at 24 h (p

Techniques Used:

Efficacy of anti-cancer drugs delivered as a liquid aerosol by nebulization in ALI MCCs. Percentage (%) of live A549 cells (top histogram) and cytotoxicity (bottom histogram) detected in ALI multilayered mono-cultures. In vitro models were exposed to four anti-cancer drugs (docetaxel, cytarabine, vinblastine and methotrexate) at their nominal IC 50 concentration for 72 h, by direct inoculation (on the left) or nebulization (on the right). Data are reported as average ± standard error of the mean (n replicates = 2; n tests = 3). p values indicate significant differences (two-way ANOVA and Bonferroni post-test).
Figure Legend Snippet: Efficacy of anti-cancer drugs delivered as a liquid aerosol by nebulization in ALI MCCs. Percentage (%) of live A549 cells (top histogram) and cytotoxicity (bottom histogram) detected in ALI multilayered mono-cultures. In vitro models were exposed to four anti-cancer drugs (docetaxel, cytarabine, vinblastine and methotrexate) at their nominal IC 50 concentration for 72 h, by direct inoculation (on the left) or nebulization (on the right). Data are reported as average ± standard error of the mean (n replicates = 2; n tests = 3). p values indicate significant differences (two-way ANOVA and Bonferroni post-test).

Techniques Used: In Vitro, Concentration Assay

6) Product Images from "Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways"

Article Title: Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways

Journal: Oncology Reports

doi: 10.3892/or.2015.4325

Antitumor effect of Ph in vivo in nude mice. The nude mice were injected with A549 cells and treated with Ph, as described in the Materials and methods. (A) Representative images of Ph-treated nude mice bearing A549 cell xenograft tumors. (B) Weight of the tumors dissected from the nude mice treated with Ph. Ph significantly reduced the tumor weight at the doses of 10 and 20 mg/kg. Data are shown with mean ± SD, n=5, ** P
Figure Legend Snippet: Antitumor effect of Ph in vivo in nude mice. The nude mice were injected with A549 cells and treated with Ph, as described in the Materials and methods. (A) Representative images of Ph-treated nude mice bearing A549 cell xenograft tumors. (B) Weight of the tumors dissected from the nude mice treated with Ph. Ph significantly reduced the tumor weight at the doses of 10 and 20 mg/kg. Data are shown with mean ± SD, n=5, ** P

Techniques Used: In Vivo, Mouse Assay, Injection

Apoptosis induction by Ph is dependent on the regulation of JNK1/2 and P38 MAPK signaling pathways in A549 cells. Effect of ERK1/2, JNK and p38 MAPK inhibitors on Ph-induced caspase activation. (A) A549 cells were pretreated with 25 µ M U0126, SP600125 or SB202190 for 45 min and then treated with 200 µ M Ph for 24 h, respectively. The expression of cleaved caspase-3 and -9 were detected by western blotting. (B and C) Quantitative results of cleaved caspase-3 and -9 protein levels, which were adjusted to the GAPDH protein level. Data are shown with mean ± SD, n=3, ** P
Figure Legend Snippet: Apoptosis induction by Ph is dependent on the regulation of JNK1/2 and P38 MAPK signaling pathways in A549 cells. Effect of ERK1/2, JNK and p38 MAPK inhibitors on Ph-induced caspase activation. (A) A549 cells were pretreated with 25 µ M U0126, SP600125 or SB202190 for 45 min and then treated with 200 µ M Ph for 24 h, respectively. The expression of cleaved caspase-3 and -9 were detected by western blotting. (B and C) Quantitative results of cleaved caspase-3 and -9 protein levels, which were adjusted to the GAPDH protein level. Data are shown with mean ± SD, n=3, ** P

Techniques Used: Activation Assay, Expressing, Western Blot

Activation of JNK1/2, ERK1/2 and P38 MAPK was increased in Ph-treated A549 cells. Stimulation of the phosphorylation of ERK, P38 and JNK in A549 cells by Ph. (A) A549 cells were treated with different concentration of Ph and western blotting were applied for analysis of phosphorylation as described in the Materials and methods. Phosphorylation of ERK, JNK, P38 were significantly elevated with Ph in a concentration-dependent manner. (B) Quantitative results of p-JNK1/2, p-ERK1/2 and p-p38 MAPK protein levels, which were adjusted to GAPDH protein level and expressed as multiples of induction beyond each respective control. Data are expressed as mean ± SD (n=3). * P
Figure Legend Snippet: Activation of JNK1/2, ERK1/2 and P38 MAPK was increased in Ph-treated A549 cells. Stimulation of the phosphorylation of ERK, P38 and JNK in A549 cells by Ph. (A) A549 cells were treated with different concentration of Ph and western blotting were applied for analysis of phosphorylation as described in the Materials and methods. Phosphorylation of ERK, JNK, P38 were significantly elevated with Ph in a concentration-dependent manner. (B) Quantitative results of p-JNK1/2, p-ERK1/2 and p-p38 MAPK protein levels, which were adjusted to GAPDH protein level and expressed as multiples of induction beyond each respective control. Data are expressed as mean ± SD (n=3). * P

Techniques Used: Activation Assay, Concentration Assay, Western Blot

Activation of caspase-3 and -9, PARP, BAX and P53 was increased in Ph-treated A549 cells. (A) A549 cells were treated with 50, 100 and 200 µ M Ph for 24 h and subjected to western blotting with an antibody against cleaved caspase-3 and -9, PARP, BAX, P53, Bcl-2, NF-κB and MMP-9 antibody. (B) Quantitative results of cleaved caspase-3 and -9, PARP, BAX, P53, Bcl-2, NF-κB and MMP-9 protein levels after 200 µ M Ph-treated for 24 h, which were adjusted to GAPDH protein level and expressed as multiples of induction beyond each respective control. Data are expressed as mean ± SD (n=3).
Figure Legend Snippet: Activation of caspase-3 and -9, PARP, BAX and P53 was increased in Ph-treated A549 cells. (A) A549 cells were treated with 50, 100 and 200 µ M Ph for 24 h and subjected to western blotting with an antibody against cleaved caspase-3 and -9, PARP, BAX, P53, Bcl-2, NF-κB and MMP-9 antibody. (B) Quantitative results of cleaved caspase-3 and -9, PARP, BAX, P53, Bcl-2, NF-κB and MMP-9 protein levels after 200 µ M Ph-treated for 24 h, which were adjusted to GAPDH protein level and expressed as multiples of induction beyond each respective control. Data are expressed as mean ± SD (n=3).

Techniques Used: Activation Assay, Western Blot

The role of Ph in Transwell migration of A549 cells. (A) Medium containing 10% FBS was placed in the lower chamber, and that without FBS was placed in the upper chamber; Ph-treated A549 cells were loaded into the upper migration chamber. (B) Migratory cells in the bottom surface of the membrane were counted. Data are expressed as mean ± SD (n=3). ** P
Figure Legend Snippet: The role of Ph in Transwell migration of A549 cells. (A) Medium containing 10% FBS was placed in the lower chamber, and that without FBS was placed in the upper chamber; Ph-treated A549 cells were loaded into the upper migration chamber. (B) Migratory cells in the bottom surface of the membrane were counted. Data are expressed as mean ± SD (n=3). ** P

Techniques Used: Migration

Ph-elicited concentration-dependent A549 cell death as visualized with a fluorescence microscope. Representative Hoechst 33342, propidium iodide (PI) staining of A549 cells indicated concentrations of Ph for 24 h. Scale bar equals 100 mm. (B) Dead cell counting against the total A549 cells treated with indicated concentrations of Ph for 24 h. Data are shown with mean ± SD of three separate experiments; * P
Figure Legend Snippet: Ph-elicited concentration-dependent A549 cell death as visualized with a fluorescence microscope. Representative Hoechst 33342, propidium iodide (PI) staining of A549 cells indicated concentrations of Ph for 24 h. Scale bar equals 100 mm. (B) Dead cell counting against the total A549 cells treated with indicated concentrations of Ph for 24 h. Data are shown with mean ± SD of three separate experiments; * P

Techniques Used: Concentration Assay, Fluorescence, Microscopy, Staining, Cell Counting

Ph induces cell apoptosis in A549 cells. (A) A549 cells were incubated with indicated concentrations of Ph for 24 h, stained with PI and analyzed for DNA content by flow cytometry. (B) A549 cells were incubated with Ph for 24 h, harvested, and then subjected to quantitative analysis of cell apoptosis by Annexin V and PI double-stained flow cytometry. (C) The number of apoptotic cells accounts for the total cells of each group after 24 h Ph treatment. Data are expressed as mean ± SD, n=3, * P
Figure Legend Snippet: Ph induces cell apoptosis in A549 cells. (A) A549 cells were incubated with indicated concentrations of Ph for 24 h, stained with PI and analyzed for DNA content by flow cytometry. (B) A549 cells were incubated with Ph for 24 h, harvested, and then subjected to quantitative analysis of cell apoptosis by Annexin V and PI double-stained flow cytometry. (C) The number of apoptotic cells accounts for the total cells of each group after 24 h Ph treatment. Data are expressed as mean ± SD, n=3, * P

Techniques Used: Incubation, Staining, Flow Cytometry, Cytometry

Cytotoxic effect of Ph on Beas-2b, A549 and H1299 cells. (A) Structure of Ph. (B) Viability analysis of Beas-2b, A549 and H1299 cells incubated in the indicated concentrations of Ph for 48 h, as assayed by MTT as described in the Materials and methods. (C) Viability of A549 cells incubated in 50, 100 and 200 µ M of concentrations of Ph for 6, 24, 36 and 48 h. Each bar represents the mean ± SD, n=3. Bars with different letters are significantly different at P
Figure Legend Snippet: Cytotoxic effect of Ph on Beas-2b, A549 and H1299 cells. (A) Structure of Ph. (B) Viability analysis of Beas-2b, A549 and H1299 cells incubated in the indicated concentrations of Ph for 48 h, as assayed by MTT as described in the Materials and methods. (C) Viability of A549 cells incubated in 50, 100 and 200 µ M of concentrations of Ph for 6, 24, 36 and 48 h. Each bar represents the mean ± SD, n=3. Bars with different letters are significantly different at P

Techniques Used: Incubation, MTT Assay

7) Product Images from "Synergistic induction of cancer cell migration regulated by G?? and phosphatidylinositol 3-kinase"

Article Title: Synergistic induction of cancer cell migration regulated by G?? and phosphatidylinositol 3-kinase

Journal: Experimental & Molecular Medicine

doi: 10.3858/emm.2012.44.8.055

The activation of Gβγ and RTK is critical for LPA-induced cancer cell migration. (A) Akt activation by LPA (10 µM) and EGF (50 ng/ml) for 10 min was detected by western blotting with phospho-Akt (Ser473) and total Akt antibodies. (B) Migration of A549 cells was determined in the absence or presence of various inhibitors such as LPA receptor inhibitor (Ki: Ki16425, 2 nM), Gβγ inhibitor (Gal: gallein, 10 µM), RTK inhibitor (AG: AG1478 100 nM), PI3K inhibitor (LY: LY294002, 10 µM) or gallein together with AG1478 or LY294002 upon LPA (10 µM) or EGF (50 ng/ml) stimulation for 10 h. The chemotactic migration of A549 cells induced by either LPA (10 nM) with the indicated dose of EGF (C) or EGF (50 pg/ml) with the indicated dose of LPA for 10 h (D), and phosphorylation at Ser473 of Akt and total Akt levels were assessed by western blotting (E, F). * P
Figure Legend Snippet: The activation of Gβγ and RTK is critical for LPA-induced cancer cell migration. (A) Akt activation by LPA (10 µM) and EGF (50 ng/ml) for 10 min was detected by western blotting with phospho-Akt (Ser473) and total Akt antibodies. (B) Migration of A549 cells was determined in the absence or presence of various inhibitors such as LPA receptor inhibitor (Ki: Ki16425, 2 nM), Gβγ inhibitor (Gal: gallein, 10 µM), RTK inhibitor (AG: AG1478 100 nM), PI3K inhibitor (LY: LY294002, 10 µM) or gallein together with AG1478 or LY294002 upon LPA (10 µM) or EGF (50 ng/ml) stimulation for 10 h. The chemotactic migration of A549 cells induced by either LPA (10 nM) with the indicated dose of EGF (C) or EGF (50 pg/ml) with the indicated dose of LPA for 10 h (D), and phosphorylation at Ser473 of Akt and total Akt levels were assessed by western blotting (E, F). * P

Techniques Used: Activation Assay, Migration, Western Blot

LPA dramatically induces cancer cell migration compared to EGF. A549 cell migration was stimulated with LPA (10 µM) for the indicated time or at the indicated dose for 10 h (A, B). Akt phosphorylation was treated with LPA (10 µM) for the indicated time or at the indicated dose of LPA for 10 min and detected by western blotting with phospho-Akt (Ser473) and total Akt (C, D). Western blotting (E) and migration (F) were determined by individually treatment with LPA (10 µM) or EGF (50 ng/ml). * P
Figure Legend Snippet: LPA dramatically induces cancer cell migration compared to EGF. A549 cell migration was stimulated with LPA (10 µM) for the indicated time or at the indicated dose for 10 h (A, B). Akt phosphorylation was treated with LPA (10 µM) for the indicated time or at the indicated dose of LPA for 10 min and detected by western blotting with phospho-Akt (Ser473) and total Akt (C, D). Western blotting (E) and migration (F) were determined by individually treatment with LPA (10 µM) or EGF (50 ng/ml). * P

Techniques Used: Migration, Western Blot

Synergistic acceleration of cancer cell migration is regulated by both GPCR and RTK. A549 cell migration was stimulated with S1P and IGF-1 at the indicated dose for 10 h (A, B). Akt phosphorylation was stimulated by the indicated dose of S1P (C) and IGF-1 (D) for 30 min and verified by western blotting with phospho-Akt (Ser473) and total Akt. The chemotactic migration of A549 cells induced by either IGF-1 (500 pg/ml) with the indicated dose of S1P (E) or S1P (1 nM) with the indicated dose of IGF-1 for 10 h (F). Phosphorylation at Ser473 of Akt and total Akt were stimulated by either IGF-1 (500 pg/ml) with the indicated dose of S1P (G) or S1P (1 nM) with the indicated dose of IGF-1 for 30 min (H).
Figure Legend Snippet: Synergistic acceleration of cancer cell migration is regulated by both GPCR and RTK. A549 cell migration was stimulated with S1P and IGF-1 at the indicated dose for 10 h (A, B). Akt phosphorylation was stimulated by the indicated dose of S1P (C) and IGF-1 (D) for 30 min and verified by western blotting with phospho-Akt (Ser473) and total Akt. The chemotactic migration of A549 cells induced by either IGF-1 (500 pg/ml) with the indicated dose of S1P (E) or S1P (1 nM) with the indicated dose of IGF-1 for 10 h (F). Phosphorylation at Ser473 of Akt and total Akt were stimulated by either IGF-1 (500 pg/ml) with the indicated dose of S1P (G) or S1P (1 nM) with the indicated dose of IGF-1 for 30 min (H).

Techniques Used: Migration, Western Blot

P-Rex1 plays an essential role in synergism of Gβγ- and PI3K-dependent migration. LPA- and EGF-induced dose-dependent activation of Rac for 5 min was determined by measuring the GTP form of Rac as described in 'Materials and methods' (A, B). (C) A549 cells were pretreated for 20 min with various inhibitors such as Ki16425 (2 nM), gallein (10 µM), AG1478 (100 nM), LY294002 (10 µM) or gallein together with AG1478 or LY294002, followed by stimulation with LPA (10 nM) and EGF (50 pg/ml) for 5 min. (D) After knock-down of P-Rex1, activation of Rac was determined by measuring the GTP form of Rac and expression of P-Rex1 was determined by RT-PCR. (E) Motility after silencing P-Rex1 was determined by migration assay for 10 h. (F) LPA (10 nM)- and EGF (50 pg/ml)-induced cancer cell invasion for 24 h was measured as described in 'Materials and methods' section. * P
Figure Legend Snippet: P-Rex1 plays an essential role in synergism of Gβγ- and PI3K-dependent migration. LPA- and EGF-induced dose-dependent activation of Rac for 5 min was determined by measuring the GTP form of Rac as described in 'Materials and methods' (A, B). (C) A549 cells were pretreated for 20 min with various inhibitors such as Ki16425 (2 nM), gallein (10 µM), AG1478 (100 nM), LY294002 (10 µM) or gallein together with AG1478 or LY294002, followed by stimulation with LPA (10 nM) and EGF (50 pg/ml) for 5 min. (D) After knock-down of P-Rex1, activation of Rac was determined by measuring the GTP form of Rac and expression of P-Rex1 was determined by RT-PCR. (E) Motility after silencing P-Rex1 was determined by migration assay for 10 h. (F) LPA (10 nM)- and EGF (50 pg/ml)-induced cancer cell invasion for 24 h was measured as described in 'Materials and methods' section. * P

Techniques Used: Migration, Activation Assay, Expressing, Reverse Transcription Polymerase Chain Reaction

8) Product Images from "Epithelial-mesenchymal crosstalk influences cellular behavior in a 3D alveolus-fibroblast model system"

Article Title: Epithelial-mesenchymal crosstalk influences cellular behavior in a 3D alveolus-fibroblast model system

Journal: Biomaterials

doi: 10.1016/j.biomaterials.2017.11.008

Click-iT Plus EdU proliferation assay. Plot depicts percent of A549 cell nuclei positive for EdU on day 4 in monoculture and the A549/CCL-210 co-cultures, separated by gel and media type. The nondegradable gels contained a peptide crosslinker insensitive to MMP cleavage. GM6001 was added at 10 μM, and the DMSO control media contained 0.05% DMSO. The degradable bars refer to the original experiment in MMP-degradable gels with regular growth media. Results are presented as means ± SEM of three biological replicates of each condition. *p
Figure Legend Snippet: Click-iT Plus EdU proliferation assay. Plot depicts percent of A549 cell nuclei positive for EdU on day 4 in monoculture and the A549/CCL-210 co-cultures, separated by gel and media type. The nondegradable gels contained a peptide crosslinker insensitive to MMP cleavage. GM6001 was added at 10 μM, and the DMSO control media contained 0.05% DMSO. The degradable bars refer to the original experiment in MMP-degradable gels with regular growth media. Results are presented as means ± SEM of three biological replicates of each condition. *p

Techniques Used: Proliferation Assay

9) Product Images from "Nanoparticle Delivery of Pooled siRNA for Effective Treatment of Non-small Cell Lung Caner"

Article Title: Nanoparticle Delivery of Pooled siRNA for Effective Treatment of Non-small Cell Lung Caner

Journal: Molecular pharmaceutics

doi: 10.1021/mp300152v

In vitro oncogene silencing. (a) Relative mRNA level after transfection of A549 cells with pooled siRNA in targeted LCP. (b) Comparison of each oncogene silencing efficiency for single siRNA formulated LCP versus pooled siRNA formulated LCP. (c) Western
Figure Legend Snippet: In vitro oncogene silencing. (a) Relative mRNA level after transfection of A549 cells with pooled siRNA in targeted LCP. (b) Comparison of each oncogene silencing efficiency for single siRNA formulated LCP versus pooled siRNA formulated LCP. (c) Western

Techniques Used: In Vitro, Transfection, Western Blot

Cellular uptake of siRNA in vitro . (a) Fluorescence photographs of cultured A549 cells after treatment with 5′-FAM-labeled oligo in non-targeted or targeted LCP for 4 h. (b) Quantitative measurement of mean fluorescence intensity of cell lysate
Figure Legend Snippet: Cellular uptake of siRNA in vitro . (a) Fluorescence photographs of cultured A549 cells after treatment with 5′-FAM-labeled oligo in non-targeted or targeted LCP for 4 h. (b) Quantitative measurement of mean fluorescence intensity of cell lysate

Techniques Used: In Vitro, Fluorescence, Cell Culture, Labeling

In vivo oncogenes silencing inhibits NSCLC tumor growth. (a) Tumor growth curve of H460 subcutaneous model. (b) Tumor growth curve of A549 subcutaneous model. (c) Dose response curve on A549 subcutaneous model. ED50=205 μg/kg. Data = mean ±
Figure Legend Snippet: In vivo oncogenes silencing inhibits NSCLC tumor growth. (a) Tumor growth curve of H460 subcutaneous model. (b) Tumor growth curve of A549 subcutaneous model. (c) Dose response curve on A549 subcutaneous model. ED50=205 μg/kg. Data = mean ±

Techniques Used: In Vivo

10) Product Images from "Miltefosine Reduces the Cytolytic Activity and Virulence of Acinetobacter baumannii"

Article Title: Miltefosine Reduces the Cytolytic Activity and Virulence of Acinetobacter baumannii

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01409-18

A549 cell culture assays. (A) The effect of the miltefosine dosage, which ranged from 0 to 25 μM, on A549 cell viability was tested using the CellTiter-Glo luminescent cell viability assay. (B) A549 cell monolayers were infected with a subset of A. baumannii isolates, and CellTiter-Glo assays were performed to determine the cytolytic activity of these isolates in the absence or the presence of 300 nM miltefosine. A549 cells were maintained for 20 h at 37°C in 5% CO 2 . Error bars represent the standard error (SE) of the mean for three different biological samples measured in duplicate ( n = 6). RLU, relative luminescence units.
Figure Legend Snippet: A549 cell culture assays. (A) The effect of the miltefosine dosage, which ranged from 0 to 25 μM, on A549 cell viability was tested using the CellTiter-Glo luminescent cell viability assay. (B) A549 cell monolayers were infected with a subset of A. baumannii isolates, and CellTiter-Glo assays were performed to determine the cytolytic activity of these isolates in the absence or the presence of 300 nM miltefosine. A549 cells were maintained for 20 h at 37°C in 5% CO 2 . Error bars represent the standard error (SE) of the mean for three different biological samples measured in duplicate ( n = 6). RLU, relative luminescence units.

Techniques Used: Cell Culture, Cell Viability Assay, Infection, Activity Assay

Cytolytic activity of A. baumannii at a miltefosine dosage of 12 µM. A549 cell monolayers were infected with A. baumannii isolates in the presence of 0 μM or 12 μM miltefosine for 20 h at 37°C in 5% CO 2 . CellTiter-Glo luminescent cell viability assays were used to determine the cytolytic activity of the A. baumannii isolates in the presence of miltefosine. Error bars represent the standard error (SE) of the mean for three different biological samples measured in duplicate ( n = 6). RLU, relative luminescence units.
Figure Legend Snippet: Cytolytic activity of A. baumannii at a miltefosine dosage of 12 µM. A549 cell monolayers were infected with A. baumannii isolates in the presence of 0 μM or 12 μM miltefosine for 20 h at 37°C in 5% CO 2 . CellTiter-Glo luminescent cell viability assays were used to determine the cytolytic activity of the A. baumannii isolates in the presence of miltefosine. Error bars represent the standard error (SE) of the mean for three different biological samples measured in duplicate ( n = 6). RLU, relative luminescence units.

Techniques Used: Activity Assay, Infection

Scanning electron microscopy of polarized A549 cells infected with A. baumannii and treated with miltefosine. (A, B) SEM was performed to visualize uninfected polarized A549 cells cultured in the absence (A) or presence (B) of 300 nM miltefosine, both of which served as negative controls. The white arrow in panel A identifies surfactant. (C to F) Polarized A549 cells were infected with ATCC 19606 T and left untreated (C) or treated with 300 nM miltefosine (D) or infected with AB5075 and left untreated (E) or treated with 300 nM miltefosine (F). The white arrows in panels D and E identify an individual A549 cell and the Transwell membrane of the support plate, respectively. Polarized A549 cells treated with miltefosine were supplemented with this drug at 24-h intervals over the course of 72-h infections. Micrographs were captured at a magnification of ×5,000. Bars, 2 µm.
Figure Legend Snippet: Scanning electron microscopy of polarized A549 cells infected with A. baumannii and treated with miltefosine. (A, B) SEM was performed to visualize uninfected polarized A549 cells cultured in the absence (A) or presence (B) of 300 nM miltefosine, both of which served as negative controls. The white arrow in panel A identifies surfactant. (C to F) Polarized A549 cells were infected with ATCC 19606 T and left untreated (C) or treated with 300 nM miltefosine (D) or infected with AB5075 and left untreated (E) or treated with 300 nM miltefosine (F). The white arrows in panels D and E identify an individual A549 cell and the Transwell membrane of the support plate, respectively. Polarized A549 cells treated with miltefosine were supplemented with this drug at 24-h intervals over the course of 72-h infections. Micrographs were captured at a magnification of ×5,000. Bars, 2 µm.

Techniques Used: Electron Microscopy, Infection, Cell Culture

11) Product Images from "Evaluation of azacitidine and entinostat as sensitization agents to cytotoxic chemotherapy in preclinical models of non-small cell lung cancer"

Article Title: Evaluation of azacitidine and entinostat as sensitization agents to cytotoxic chemotherapy in preclinical models of non-small cell lung cancer

Journal: Oncotarget

doi:

Epigenetic priming does not potentiate the effects chemotherapy on colony growth H358 and A549 cells were seeded on a solidified Matrigel layer six days after epigenetic therapy. Beginning the following day, cells were treated with chemotherapy for 72 hours. Drug was then removed and colonies were permitted to grow 2 – 4 additional days. (A) Representative H358 colonies following treatment with 10 nM gemcitabine or 10 nM 17-AAG. (B) H358 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), calculated from one representative experiment with five replicates. (C) Representative A549 colonies following treatment with 600 nM cisplatin, 30 nM 17-AAG, or 1 nM docetaxel. (D) A549 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), averaged from two independent experiments (total nine replicates). Statistical significance by ANOVA with Tukey's multiple comparison test denoted as follows: * p
Figure Legend Snippet: Epigenetic priming does not potentiate the effects chemotherapy on colony growth H358 and A549 cells were seeded on a solidified Matrigel layer six days after epigenetic therapy. Beginning the following day, cells were treated with chemotherapy for 72 hours. Drug was then removed and colonies were permitted to grow 2 – 4 additional days. (A) Representative H358 colonies following treatment with 10 nM gemcitabine or 10 nM 17-AAG. (B) H358 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), calculated from one representative experiment with five replicates. (C) Representative A549 colonies following treatment with 600 nM cisplatin, 30 nM 17-AAG, or 1 nM docetaxel. (D) A549 percent colony formation (+/− standard deviation) relative to untreated control (PBS or DMSO), averaged from two independent experiments (total nine replicates). Statistical significance by ANOVA with Tukey's multiple comparison test denoted as follows: * p

Techniques Used: Standard Deviation

Epigenetic therapy augments response of A549 xenografts, but abrogrates response of H460 xenografts, to irinotecan, and does not sensitize to cisplatin or docetaxel Subcutaneous hind flank tumors were established in NOD/SCID mice from A549 or H460 cells treated in vitro with mock (M) or the combination of Aza and entinostat (C). (A) Mice bearing pretreated A549 tumors were treated with 2 mg/kg cisplatin (M-C and C-C) on day 2, 10 mg/kg irinotecan (M-I and C-I) on days 2 5, or saline vehicle (M-V and C-V), for three one-week cycles. Curves represent mean tumor volume +/− SEM. Statistical significance determined using a mixed effects model and REML. (B) Mice bearing pretreated H460 tumors were treated with 2.5 mg/kg docetaxel q4d × 2 escalated to 5 mg/kg docetaxel q4d × 2 (M-D and C-D), 10 mg/kg irinotecan q4d × 4 (M-I and C-I), or saline vehicle (M-V and C-V). Curves represent mean tumor volume +/− SEM.
Figure Legend Snippet: Epigenetic therapy augments response of A549 xenografts, but abrogrates response of H460 xenografts, to irinotecan, and does not sensitize to cisplatin or docetaxel Subcutaneous hind flank tumors were established in NOD/SCID mice from A549 or H460 cells treated in vitro with mock (M) or the combination of Aza and entinostat (C). (A) Mice bearing pretreated A549 tumors were treated with 2 mg/kg cisplatin (M-C and C-C) on day 2, 10 mg/kg irinotecan (M-I and C-I) on days 2 5, or saline vehicle (M-V and C-V), for three one-week cycles. Curves represent mean tumor volume +/− SEM. Statistical significance determined using a mixed effects model and REML. (B) Mice bearing pretreated H460 tumors were treated with 2.5 mg/kg docetaxel q4d × 2 escalated to 5 mg/kg docetaxel q4d × 2 (M-D and C-D), 10 mg/kg irinotecan q4d × 4 (M-I and C-I), or saline vehicle (M-V and C-V). Curves represent mean tumor volume +/− SEM.

Techniques Used: Mouse Assay, In Vitro

12) Product Images from "Metabolic Profiling of healthy and cancerous tissues in 2D and 3D"

Article Title: Metabolic Profiling of healthy and cancerous tissues in 2D and 3D

Journal: Scientific Reports

doi: 10.1038/s41598-017-15325-5

Effects of 5FU on 3D spheroid metabolic phenotype versus 2D monolayer in HCT116 colorectal cancer cell line. ( A ) Basal OCR as a percent of control (untreated 2D or 3D) upon treatment with 2 or 20 mM 5-Fluorouracil in 2D or 3D cell culture model. ( B ) Basal ECAR as a percent of control (untreated 2D or 3D) upon treatment with 2 or 20 mM 5-Fluorouracil in 2D or 3D cell culture model. ( C ) Group Key for Panel A,B,D,E. ( D ) OCR:PPR ratio of 2D or 3D cell model with 5FU treatment (2 or 20 mM). ( E ) Percentage viability of cells over time in the 2D or 3D model upon exposure to 2 or 20 mM 5FU using Cell Titer Glo assay. ( F ) A549 spheroid incubated with fluorescent dextran after washing. ( G ) A549 spheroid treated with fluorescent dextran and 5 nM paclitaxel (pseudo-color yellow).
Figure Legend Snippet: Effects of 5FU on 3D spheroid metabolic phenotype versus 2D monolayer in HCT116 colorectal cancer cell line. ( A ) Basal OCR as a percent of control (untreated 2D or 3D) upon treatment with 2 or 20 mM 5-Fluorouracil in 2D or 3D cell culture model. ( B ) Basal ECAR as a percent of control (untreated 2D or 3D) upon treatment with 2 or 20 mM 5-Fluorouracil in 2D or 3D cell culture model. ( C ) Group Key for Panel A,B,D,E. ( D ) OCR:PPR ratio of 2D or 3D cell model with 5FU treatment (2 or 20 mM). ( E ) Percentage viability of cells over time in the 2D or 3D model upon exposure to 2 or 20 mM 5FU using Cell Titer Glo assay. ( F ) A549 spheroid incubated with fluorescent dextran after washing. ( G ) A549 spheroid treated with fluorescent dextran and 5 nM paclitaxel (pseudo-color yellow).

Techniques Used: Cell Culture, Glo Assay, Incubation

13) Product Images from "Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks"

Article Title: Optimization of Early Steps in Oncolytic Adenovirus ONCOS-401 Production in T-175 and HYPERFlasks

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms20030621

Summary of optimization studies in T-175 flasks. The effect of harvesting time on viral productivity. ( a ) Virus productivity from ONCOS-401 infected A549 cells (MOI = 10) in T-175. ( b ) Virus productivity from ONCOS-401 infected A549 cells in T-175 flasks harvested at 66 h. Values are presented as means ± SD, * p
Figure Legend Snippet: Summary of optimization studies in T-175 flasks. The effect of harvesting time on viral productivity. ( a ) Virus productivity from ONCOS-401 infected A549 cells (MOI = 10) in T-175. ( b ) Virus productivity from ONCOS-401 infected A549 cells in T-175 flasks harvested at 66 h. Values are presented as means ± SD, * p

Techniques Used: Infection

The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 30) on the yield of infectious viral particles in HYPERFlasks. ONCOS-401 infected A549 cells (MOI = 30) were incubated in HYPERFlasks for 60, 66, 72, 96 h and the adenoviral particles were harvested by the TrypLE method. The infectious titers were determined by ICC. The yields of infectious ONCOS-401 viral particles were low compared to previous experiments that used the Lysis A method.
Figure Legend Snippet: The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 30) on the yield of infectious viral particles in HYPERFlasks. ONCOS-401 infected A549 cells (MOI = 30) were incubated in HYPERFlasks for 60, 66, 72, 96 h and the adenoviral particles were harvested by the TrypLE method. The infectious titers were determined by ICC. The yields of infectious ONCOS-401 viral particles were low compared to previous experiments that used the Lysis A method.

Techniques Used: Incubation, Infection, Immunocytochemistry, Lysis

The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. The infectious titers were tested at harvesting times of 48, 60, 72, 84 and 96 h. The highest viral titers were obtained at 60 h post infection. Values are presented as means.
Figure Legend Snippet: The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. The infectious titers were tested at harvesting times of 48, 60, 72, 84 and 96 h. The highest viral titers were obtained at 60 h post infection. Values are presented as means.

Techniques Used: Incubation, Infection

The effect of the input inoculum (MOI) on the yield of infectious viral particles from ONCOS-401 infected A549 cells was determined in T-175 flasks. A549 cells were infected with ONCOS-401 at the indicated MOIs (1, 5, 20, 50 and 100), and the viral particles were harvested at 66 h post infection. Infectious titers were determined by ICC. The A549 cells infected with 20 and 50 MOI provided the highest viral titers. Moreover, one log drop of titer was observed at 1 MOI. Values are presented as means ± SD, * p
Figure Legend Snippet: The effect of the input inoculum (MOI) on the yield of infectious viral particles from ONCOS-401 infected A549 cells was determined in T-175 flasks. A549 cells were infected with ONCOS-401 at the indicated MOIs (1, 5, 20, 50 and 100), and the viral particles were harvested at 66 h post infection. Infectious titers were determined by ICC. The A549 cells infected with 20 and 50 MOI provided the highest viral titers. Moreover, one log drop of titer was observed at 1 MOI. Values are presented as means ± SD, * p

Techniques Used: Infection, Immunocytochemistry

The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. ( a ) The infectious titers from ONCOS-401 infected A549 cells (MOI = 10) in T-175 flasks were tested at harvesting times of 66, 72, 78, 84, 90 h: the highest viral titers were obtained at 66 h post infection. ( b ) The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. The yield of infectious viral particles from ONCOS-401 infected A549 cells in T-175 flasks were tested at harvesting times of 56, 60, 66, 72 h: similar titers were obtained at harvest times of 56 to 66 h post infection. Values are presented as means ± SD, * p
Figure Legend Snippet: The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. ( a ) The infectious titers from ONCOS-401 infected A549 cells (MOI = 10) in T-175 flasks were tested at harvesting times of 66, 72, 78, 84, 90 h: the highest viral titers were obtained at 66 h post infection. ( b ) The effect of length of incubation period of ONCOS-401 infected A549 cells (MOI = 10) on the yield of infectious viral particles in T-175 flasks. The yield of infectious viral particles from ONCOS-401 infected A549 cells in T-175 flasks were tested at harvesting times of 56, 60, 66, 72 h: similar titers were obtained at harvest times of 56 to 66 h post infection. Values are presented as means ± SD, * p

Techniques Used: Incubation, Infection

14) Product Images from "Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing"

Article Title: Co-regulatory activity of hnRNP K and NS1-BP in influenza and human mRNA splicing

Journal: Nature Communications

doi: 10.1038/s41467-018-04779-4

Mutation of hnRNP K but not NS1-BP-binding sites alters M mRNA localization. a A549 cells were infected with wild type (WT) and mutant A/WNS/33 viruses at MOI 5. After 8.45 h infection, cells were subjected to RNA-FISH combined with immunofluorescence to detect M mRNA and SON protein. SON protein is a marker for nuclear speckles. Scale bar, 20 µm. The marked rectangular region is enlarged and shown in the right-most panel. Scale bar, 5 µm. Images are representative of at least 12 images from biological triplicates. b Quantification of M mRNA intensity in the cytoplasm (C) and in the nucleus (N) expressed as C/N ratios. Values are mean ± s.d. measured in at least 25 cells. c Quantification of M mRNA intensity at nuclear speckles with respect to total cell intensity. Values are mean ± s.d. measured in at least 25 cells. Statistical significance was determined via two-tailed student’s t test, where ** p
Figure Legend Snippet: Mutation of hnRNP K but not NS1-BP-binding sites alters M mRNA localization. a A549 cells were infected with wild type (WT) and mutant A/WNS/33 viruses at MOI 5. After 8.45 h infection, cells were subjected to RNA-FISH combined with immunofluorescence to detect M mRNA and SON protein. SON protein is a marker for nuclear speckles. Scale bar, 20 µm. The marked rectangular region is enlarged and shown in the right-most panel. Scale bar, 5 µm. Images are representative of at least 12 images from biological triplicates. b Quantification of M mRNA intensity in the cytoplasm (C) and in the nucleus (N) expressed as C/N ratios. Values are mean ± s.d. measured in at least 25 cells. c Quantification of M mRNA intensity at nuclear speckles with respect to total cell intensity. Values are mean ± s.d. measured in at least 25 cells. Statistical significance was determined via two-tailed student’s t test, where ** p

Techniques Used: Mutagenesis, Binding Assay, Infection, Fluorescence In Situ Hybridization, Immunofluorescence, Marker, Two Tailed Test

hnRNP K and NS1-BP co-operatively regulate host alternative splicing events. A549 cells were mock transfected or treated with 50 nM siRNA targeted for hnRNP K or NS1-BP in triplicate. Total cell RNA was then extracted and subjected to RASL-seq. a Overlap of hnRNP K and NS1-BP responsive alternative splicing events with ∆PSI ≥ |10| and p
Figure Legend Snippet: hnRNP K and NS1-BP co-operatively regulate host alternative splicing events. A549 cells were mock transfected or treated with 50 nM siRNA targeted for hnRNP K or NS1-BP in triplicate. Total cell RNA was then extracted and subjected to RASL-seq. a Overlap of hnRNP K and NS1-BP responsive alternative splicing events with ∆PSI ≥ |10| and p

Techniques Used: Transfection

Host proteins hnRNP K and NS1-BP regulate IAV PR8 M segment splicing. a . b Immunoblots of A549 whole cell extract 12 h post PR8 infection in the context of mock, hnRNP K or NS1-BP siRNA knockdown. Molecular weights are indicated to left of each blot. c Diagram of IAV PR8 M segment mRNA with possible splice isoforms and extension primer positions labeled. Boxes denote exons and the line denotes an intron. M3 and M2 indicate potential 5′ss and numbers indicate nucleotide position. d . e Quantification of mRNA primer extension products represented as percentage signal of each isoform relative to signal of total isoforms (M1 + M2 + M3). Values are means ± s.d. from three independent experiments. For all experiments in manuscript, a minimum of 3 experiments were performed to insure appropriateness of statistical tests. Statistical significance was determined via two-tailed student’s t test, where * p
Figure Legend Snippet: Host proteins hnRNP K and NS1-BP regulate IAV PR8 M segment splicing. a . b Immunoblots of A549 whole cell extract 12 h post PR8 infection in the context of mock, hnRNP K or NS1-BP siRNA knockdown. Molecular weights are indicated to left of each blot. c Diagram of IAV PR8 M segment mRNA with possible splice isoforms and extension primer positions labeled. Boxes denote exons and the line denotes an intron. M3 and M2 indicate potential 5′ss and numbers indicate nucleotide position. d . e Quantification of mRNA primer extension products represented as percentage signal of each isoform relative to signal of total isoforms (M1 + M2 + M3). Values are means ± s.d. from three independent experiments. For all experiments in manuscript, a minimum of 3 experiments were performed to insure appropriateness of statistical tests. Statistical significance was determined via two-tailed student’s t test, where * p

Techniques Used: Western Blot, Infection, Labeling, Two Tailed Test

15) Product Images from "NEK4 kinase regulates EMT to promote lung cancer metastasis, et al. NEK4 kinase regulates EMT to promote lung cancer metastasis"

Article Title: NEK4 kinase regulates EMT to promote lung cancer metastasis, et al. NEK4 kinase regulates EMT to promote lung cancer metastasis

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/jcmm.13857

NEK4 regulation of EMT associated transcription factors and signal transduction. (A) WB to detect the EMT transcription factors Zeb1. A549 cells were transfected with siNEK4 mix or siCon supplemented with TGF‐β (2 μg/mL) or not for 48 hours. (B) WB to detect the Smads proteins in A549 cells transfected with siNEK4 mix or siCon and supplement with TGF‐β (2 μg/mL) or not for 48 hours. (C) WB to detect the proteins in A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. (D) WB to detect the proteins in MDA‐MB‐231 cells transfected with siNEK4 mix or siCon for 48 hours. (E) WB to detect the proteins in MCF7 cells transfected with siNEK4 mix or siCon for 48 hours
Figure Legend Snippet: NEK4 regulation of EMT associated transcription factors and signal transduction. (A) WB to detect the EMT transcription factors Zeb1. A549 cells were transfected with siNEK4 mix or siCon supplemented with TGF‐β (2 μg/mL) or not for 48 hours. (B) WB to detect the Smads proteins in A549 cells transfected with siNEK4 mix or siCon and supplement with TGF‐β (2 μg/mL) or not for 48 hours. (C) WB to detect the proteins in A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. (D) WB to detect the proteins in MDA‐MB‐231 cells transfected with siNEK4 mix or siCon for 48 hours. (E) WB to detect the proteins in MCF7 cells transfected with siNEK4 mix or siCon for 48 hours

Techniques Used: Transduction, Western Blot, Transfection, Stable Transfection, Plasmid Preparation, Multiple Displacement Amplification

NEK4 in the regulation of EMT molecular markers. (A) Expression of E‐cadherin in A549 cells transfected with siNEK4 mix or siCon supplemented with TGF‐β (2 μg/mL) or not for 48 hours. (B) Detection of EMT marker proteins. A549 cells were transfected with siNEK4 mix or siCon for 48 hours. (C and D) Immunofluorescence technique to analyze E‐cadherin and ZO1 proteins. A549 cells were transfected with siNEK4 mix or siCon for 48 hours and the E‐cadherin and ZO1 proteins were detected by IF. Cells were observed by fluorescence microscope
Figure Legend Snippet: NEK4 in the regulation of EMT molecular markers. (A) Expression of E‐cadherin in A549 cells transfected with siNEK4 mix or siCon supplemented with TGF‐β (2 μg/mL) or not for 48 hours. (B) Detection of EMT marker proteins. A549 cells were transfected with siNEK4 mix or siCon for 48 hours. (C and D) Immunofluorescence technique to analyze E‐cadherin and ZO1 proteins. A549 cells were transfected with siNEK4 mix or siCon for 48 hours and the E‐cadherin and ZO1 proteins were detected by IF. Cells were observed by fluorescence microscope

Techniques Used: Expressing, Transfection, Marker, Immunofluorescence, IF-cells, Fluorescence, Microscopy

The biological function of NEK4 associated with EMT. (A) Representative images of scratch assay at different time points. A549 cells were seeded into 6‐well culture plates and transfected with siNEK4 mix or siRNA control. Cell migration was then observed by microscope at different time points. Data are representative of 3 independent experiments. (B) Graphs showed wound areas in A549 cells transfected with siNEK4 mix or siRNA control. The wound area was analyzed using ImageJ software. Bars represent mean ± SD of three measurements. (C) Representative images of the matrigel‐coated transwell assay at different time points. A549 cells were transfected with siNEK4 mix or siRNA control. Data are representative of 3 independent experiments. (D) Representative images of scratch assay at different time points. A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4 were seeded into 6‐well culture plates. Cell migration was then observed by microscope at different time points. Data are representative of 3 independent experiments. (E) Graphs showed remaining wound areas in A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. The wound area was analyzed using ImageJ software. Bars represent mean ± SD of three measurements. (F) Representative images of the matrigel‐coated transwell assay. A549 cells were stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. Data are representative of 3 independent experiments. (G) Graphs showed the cell numbers below the membrane. A549 cells were stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. Bars represent mean ± SD of three measurements
Figure Legend Snippet: The biological function of NEK4 associated with EMT. (A) Representative images of scratch assay at different time points. A549 cells were seeded into 6‐well culture plates and transfected with siNEK4 mix or siRNA control. Cell migration was then observed by microscope at different time points. Data are representative of 3 independent experiments. (B) Graphs showed wound areas in A549 cells transfected with siNEK4 mix or siRNA control. The wound area was analyzed using ImageJ software. Bars represent mean ± SD of three measurements. (C) Representative images of the matrigel‐coated transwell assay at different time points. A549 cells were transfected with siNEK4 mix or siRNA control. Data are representative of 3 independent experiments. (D) Representative images of scratch assay at different time points. A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4 were seeded into 6‐well culture plates. Cell migration was then observed by microscope at different time points. Data are representative of 3 independent experiments. (E) Graphs showed remaining wound areas in A549 cells stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. The wound area was analyzed using ImageJ software. Bars represent mean ± SD of three measurements. (F) Representative images of the matrigel‐coated transwell assay. A549 cells were stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. Data are representative of 3 independent experiments. (G) Graphs showed the cell numbers below the membrane. A549 cells were stably transfected with plasmid pc‐DNA 3.1+ or pc‐NEK4. Bars represent mean ± SD of three measurements

Techniques Used: Wound Healing Assay, Transfection, Migration, Microscopy, Software, Transwell Assay, Stable Transfection, Plasmid Preparation

NEK4 promotion of invasion and migration in vivo. (A) WB to detect the proteins in A549 cells stably transfected with shNEK4 or shCon. (B) Representative images of scratch assay at different time points. A549 cells were stably transfected with shNEK4 or shCon. (C) Graphs showed remaining wound areas in A549 cells stably transfected with shNEK4 or shCon at different time points. Bars represent mean ± SD of three measurements. Data are representative of 3 independent experiments. (D) Lung tumor clone formation in mouse injected with A549‐shcon or A549‐shNEK4 cells for 1 month. The whole lung specimens were fixed with bouins and observed with stereoscopic microscope. (E) Statistical analysis of tumor clone number. Tumor clones were counted in the whole lung and analyzed between A549‐shcon and A549‐shNEK4 group. (F) HE stain and IHC to evaluate the pathological features. Tissue slides were stained with HE and observed using a microscope to detect every tumor clone in the whole lung. The expression of NEK4 and E‐cadherin in lung tissue slides were detected with IHC. (G) Statistical analysis of the relative NEK4 expression in two groups. The score was given as a sum of each stain intensity multiplied by the percentage of stained cells in every tumor clone. (H) Correlation analysis between the expression of NEK4 and E‐cadherin. The score was given as a sum of each stain intensity multiplied by the percentage of stained cells in every tumor clone
Figure Legend Snippet: NEK4 promotion of invasion and migration in vivo. (A) WB to detect the proteins in A549 cells stably transfected with shNEK4 or shCon. (B) Representative images of scratch assay at different time points. A549 cells were stably transfected with shNEK4 or shCon. (C) Graphs showed remaining wound areas in A549 cells stably transfected with shNEK4 or shCon at different time points. Bars represent mean ± SD of three measurements. Data are representative of 3 independent experiments. (D) Lung tumor clone formation in mouse injected with A549‐shcon or A549‐shNEK4 cells for 1 month. The whole lung specimens were fixed with bouins and observed with stereoscopic microscope. (E) Statistical analysis of tumor clone number. Tumor clones were counted in the whole lung and analyzed between A549‐shcon and A549‐shNEK4 group. (F) HE stain and IHC to evaluate the pathological features. Tissue slides were stained with HE and observed using a microscope to detect every tumor clone in the whole lung. The expression of NEK4 and E‐cadherin in lung tissue slides were detected with IHC. (G) Statistical analysis of the relative NEK4 expression in two groups. The score was given as a sum of each stain intensity multiplied by the percentage of stained cells in every tumor clone. (H) Correlation analysis between the expression of NEK4 and E‐cadherin. The score was given as a sum of each stain intensity multiplied by the percentage of stained cells in every tumor clone

Techniques Used: Migration, In Vivo, Western Blot, Stable Transfection, Transfection, Wound Healing Assay, Injection, Microscopy, Clone Assay, H&E Stain, Immunohistochemistry, Staining, Expressing

Identification of NEK4 protein kinase as regulator of E‐cadherin in A549 cells. (A) EMT model induced by TGF‐β. A549 cells were treated with TGF‐β (2 μg/mL) for 48 hours and taken for photographs under optical microscope. (B) (up panel) High‐throughput siRNA screening against human kinases. The human 720 protein kinase siRNAs were screened using A549 cell line. For each siRNA, triplet wells were set up. (lower panel) 26 potential candidates after second‐round selection. Fold change values for each siRNA were plotted to identify hits with a score > 1.6 or
Figure Legend Snippet: Identification of NEK4 protein kinase as regulator of E‐cadherin in A549 cells. (A) EMT model induced by TGF‐β. A549 cells were treated with TGF‐β (2 μg/mL) for 48 hours and taken for photographs under optical microscope. (B) (up panel) High‐throughput siRNA screening against human kinases. The human 720 protein kinase siRNAs were screened using A549 cell line. For each siRNA, triplet wells were set up. (lower panel) 26 potential candidates after second‐round selection. Fold change values for each siRNA were plotted to identify hits with a score > 1.6 or

Techniques Used: Microscopy, High Throughput Screening Assay, Selection

16) Product Images from "Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation"

Article Title: Transcriptome Analysis of Infected and Bystander Type 2 Alveolar Epithelial Cells during Influenza A Virus Infection Reveals In Vivo Wnt Pathway Downregulation

Journal: Journal of Virology

doi: 10.1128/JVI.01325-18

Wnt signaling within infected epithelial cells is reduced compared to that in bystander uninfected epithelial cells. (A and B) Representative histograms of reduced expression of Wnt3a (A) and Fzd10 (B) in PR8-GFP-infected (GFP + ) A549 cells compared to
Figure Legend Snippet: Wnt signaling within infected epithelial cells is reduced compared to that in bystander uninfected epithelial cells. (A and B) Representative histograms of reduced expression of Wnt3a (A) and Fzd10 (B) in PR8-GFP-infected (GFP + ) A549 cells compared to

Techniques Used: Infection, Expressing

17) Product Images from "EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin"

Article Title: EphA2-Dependent Internalization of A. fumigatus Conidia in A549 Lung Cells Is Modulated by DHN-Melanin

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2020.534118

Model of the role of receptors in internalization and association of wild-type conidia (in dark green) or conidia lacking DHN-melanin (gray with black line). Conidia will associate to various indicated receptors present on the type II A549 epithelial lung cells, and the EphA2, Dectin-1 and at least one other receptor will be activated, which results in the internalization of conidia. In conidia lacking DHN-melanin, association of conidia is increased, and internalization of conidia is facilitated in an EphA2 independent and a Dectin-1 dependent manner.
Figure Legend Snippet: Model of the role of receptors in internalization and association of wild-type conidia (in dark green) or conidia lacking DHN-melanin (gray with black line). Conidia will associate to various indicated receptors present on the type II A549 epithelial lung cells, and the EphA2, Dectin-1 and at least one other receptor will be activated, which results in the internalization of conidia. In conidia lacking DHN-melanin, association of conidia is increased, and internalization of conidia is facilitated in an EphA2 independent and a Dectin-1 dependent manner.

Techniques Used:

18) Product Images from "Injectable Thermosensitive Hydrogel Containing Erlotinib‐Loaded Hollow Mesoporous Silica Nanoparticles as a Localized Drug Delivery System for NSCLC Therapy, Injectable Thermosensitive Hydrogel Containing Erlotinib‐Loaded Hollow Mesoporous Silica Nanoparticles as a Localized Drug Delivery System for NSCLC Therapy"

Article Title: Injectable Thermosensitive Hydrogel Containing Erlotinib‐Loaded Hollow Mesoporous Silica Nanoparticles as a Localized Drug Delivery System for NSCLC Therapy, Injectable Thermosensitive Hydrogel Containing Erlotinib‐Loaded Hollow Mesoporous Silica Nanoparticles as a Localized Drug Delivery System for NSCLC Therapy

Journal: Advanced Science

doi: 10.1002/advs.202001442

The H E sections of hearts, livers, spleens, lungs, kidneys, and skins after different administration in A549 model. Each group ( n = 3) was a) NS; b)ERT@HMSNs (i.t. 50 mg kg −1 ); c) ERT@HMSNs/gel (i.t. 25 mg kg −1 ); d) ERT@HMSNs/gel (i.t. 50 mg kg −1 ); e) ERT@HMSNs/hydrogel (i.t. 100 mg kg −1 ); f) Tarceva (p.o. 25 mg kg −1 per day); g) Tarceva (p.o. 50 mg kg −1 per day); h) Tarceva (p.o. 100 mg kg −1 per day). Scale bar: 50 µm.
Figure Legend Snippet: The H E sections of hearts, livers, spleens, lungs, kidneys, and skins after different administration in A549 model. Each group ( n = 3) was a) NS; b)ERT@HMSNs (i.t. 50 mg kg −1 ); c) ERT@HMSNs/gel (i.t. 25 mg kg −1 ); d) ERT@HMSNs/gel (i.t. 50 mg kg −1 ); e) ERT@HMSNs/hydrogel (i.t. 100 mg kg −1 ); f) Tarceva (p.o. 25 mg kg −1 per day); g) Tarceva (p.o. 50 mg kg −1 per day); h) Tarceva (p.o. 100 mg kg −1 per day). Scale bar: 50 µm.

Techniques Used:

Cellular uptake of different ERT formulations in A549 cells. The FITC‐labeled formulations were added into transwell inserts and then incubated with cells at different time. A) HMSNs accumulation was represented by the fluorescence of FITC which was observed by fluorescence microscopy. B,C) Quantitative analysis of fluorescent intensity of FITC was determined by flow cytometry; D) Viability of A549 cells incubated with ERT@HMSNs and ERT@HMSNs/gel in Transwell co‐culture systems for 24, 48, and 72 h. All quantitative data are given as mean ± SD ( n = 3). “*” and “**” mean p
Figure Legend Snippet: Cellular uptake of different ERT formulations in A549 cells. The FITC‐labeled formulations were added into transwell inserts and then incubated with cells at different time. A) HMSNs accumulation was represented by the fluorescence of FITC which was observed by fluorescence microscopy. B,C) Quantitative analysis of fluorescent intensity of FITC was determined by flow cytometry; D) Viability of A549 cells incubated with ERT@HMSNs and ERT@HMSNs/gel in Transwell co‐culture systems for 24, 48, and 72 h. All quantitative data are given as mean ± SD ( n = 3). “*” and “**” mean p

Techniques Used: Labeling, Incubation, Fluorescence, Microscopy, Flow Cytometry, Co-Culture Assay

Apoptosis (A) and p‐EGFR level (B) of A549 tumors in different treatment groups. a) NS; b) ERT@HMSNs (i.t. 50 mg kg −1 ); c) ERT@HMSNs/gel (i.t. 25 mg kg −1 ); d) ERT@HMSNs/gel (i.t. 50 mg kg −1 ); e) ERT@HMSNs/hydrogel (i.t. 100 mg kg −1 ); f) Tarceva (p.o. 25 mg kg −1 per day); g) Tarceva (p.o. 50 mg kg −1 per day); h) Tarceva (p.o. 100 mg kg −1 per day). Scale bar: 100 µm. All quantitative data are given as mean ± SD ( n = 5). “**” means p
Figure Legend Snippet: Apoptosis (A) and p‐EGFR level (B) of A549 tumors in different treatment groups. a) NS; b) ERT@HMSNs (i.t. 50 mg kg −1 ); c) ERT@HMSNs/gel (i.t. 25 mg kg −1 ); d) ERT@HMSNs/gel (i.t. 50 mg kg −1 ); e) ERT@HMSNs/hydrogel (i.t. 100 mg kg −1 ); f) Tarceva (p.o. 25 mg kg −1 per day); g) Tarceva (p.o. 50 mg kg −1 per day); h) Tarceva (p.o. 100 mg kg −1 per day). Scale bar: 100 µm. All quantitative data are given as mean ± SD ( n = 5). “**” means p

Techniques Used:

In vivo biodistribution and retention studies by NIR imaging. A) The NIR real‐time images of A549 xenograft models after i.t. injection of DiR@HMSNs formulation and DiR@HMSNs/hydrogel composite at 1st, 3rd, 7th, and 14th days B) with the NIR images of ex vivo tumors and mean organs on 14th day after the initial injection; C) Quantitative analysis was conducted to determine the fluorescence in ex vivo mean organs and tumors. All quantitative data are given as mean ± SD ( n = 3). “**” means p
Figure Legend Snippet: In vivo biodistribution and retention studies by NIR imaging. A) The NIR real‐time images of A549 xenograft models after i.t. injection of DiR@HMSNs formulation and DiR@HMSNs/hydrogel composite at 1st, 3rd, 7th, and 14th days B) with the NIR images of ex vivo tumors and mean organs on 14th day after the initial injection; C) Quantitative analysis was conducted to determine the fluorescence in ex vivo mean organs and tumors. All quantitative data are given as mean ± SD ( n = 3). “**” means p

Techniques Used: In Vivo, Imaging, Injection, Ex Vivo, Fluorescence

19) Product Images from "Imaging PD-L1 Expression with ImmunoPET"

Article Title: Imaging PD-L1 Expression with ImmunoPET

Journal: Bioconjugate Chemistry

doi: 10.1021/acs.bioconjchem.7b00631

89 Zr-C4 detects PD-L1 expression levels in a PDX derived from a NSCLC patient that experienced a durable clinical response to anti-PD-1 and anti-CTLA4 therapies. (A) Transaxial CT slices showing a soft tissue lesion in the lung prior to the initiation of pembrolizumab and ipilimumab (left), and a smaller mass 3 months after the start of therapy (right). The position of the tumor is indicated with a white arrow. This patient experienced a partial response for 8 months. The PDX was derived 7 months prior to the first CT scan. (B) Small animal PET/CT data showing the biodistribution of 89 Zr-C4 in mice bearing bilateral PDX tumors in the flank. The tumors can be clearly resolved, and radiotracer uptake in abdominal tissues like the liver is observed, as expected for a large biomolecule. Mice treated with 89 Zr-C4 that was heat denatured (HD) for 10 min prior to injection show no evidence of radiotracer uptake in the tumor. (C) Biodistribution data showing the uptake of 89 Zr-C4 in the PDX tissue 48 h after injection. The uptake is higher in the tumor compared to heat denatured 89 Zr-C4 (HD) and standard reference tissues like the blood and muscle. (D) Biodistribution data acquired 48 h after injection in mice bearing subcutaneous H1975, PC3, A549, and the PDX tumors show the different degree of 89 Zr-C4 uptake in the tumors.
Figure Legend Snippet: 89 Zr-C4 detects PD-L1 expression levels in a PDX derived from a NSCLC patient that experienced a durable clinical response to anti-PD-1 and anti-CTLA4 therapies. (A) Transaxial CT slices showing a soft tissue lesion in the lung prior to the initiation of pembrolizumab and ipilimumab (left), and a smaller mass 3 months after the start of therapy (right). The position of the tumor is indicated with a white arrow. This patient experienced a partial response for 8 months. The PDX was derived 7 months prior to the first CT scan. (B) Small animal PET/CT data showing the biodistribution of 89 Zr-C4 in mice bearing bilateral PDX tumors in the flank. The tumors can be clearly resolved, and radiotracer uptake in abdominal tissues like the liver is observed, as expected for a large biomolecule. Mice treated with 89 Zr-C4 that was heat denatured (HD) for 10 min prior to injection show no evidence of radiotracer uptake in the tumor. (C) Biodistribution data showing the uptake of 89 Zr-C4 in the PDX tissue 48 h after injection. The uptake is higher in the tumor compared to heat denatured 89 Zr-C4 (HD) and standard reference tissues like the blood and muscle. (D) Biodistribution data acquired 48 h after injection in mice bearing subcutaneous H1975, PC3, A549, and the PDX tumors show the different degree of 89 Zr-C4 uptake in the tumors.

Techniques Used: Expressing, Derivative Assay, Computed Tomography, Positron Emission Tomography, Mouse Assay, Injection

20) Product Images from "EPAS-1 Mediates SP-1-Dependent FBI-1 Expression and Regulates Tumor Cell Survival and Proliferation"

Article Title: EPAS-1 Mediates SP-1-Dependent FBI-1 Expression and Regulates Tumor Cell Survival and Proliferation

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms150915689

EPAS-1 potentiates human lung adenocarcinoma cell survival and proliferation. ( A ) EPAS-1 over-expression increased A549 cell viability while its knockdown restricted this effect. Data points were determined in triplicate and shown with the mean ± SD (* p
Figure Legend Snippet: EPAS-1 potentiates human lung adenocarcinoma cell survival and proliferation. ( A ) EPAS-1 over-expression increased A549 cell viability while its knockdown restricted this effect. Data points were determined in triplicate and shown with the mean ± SD (* p

Techniques Used: Over Expression

21) Product Images from "SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *"

Article Title: SHP2 Positively Regulates TGFβ1-induced Epithelial-Mesenchymal Transition Modulated by Its Novel Interacting Protein Hook1 *

Journal: The Journal of Biological Chemistry

doi: 10.1074/jbc.M113.546077

Phosphatase activity of SHP2 was required for EMT. A , Western analysis of effects of SHP2 C459S overexpression on ERK activation with or without TGFβ1 treatment (5 ng/ml) for 48 h. B , quantification of EMT-associated markers ( FN1, ZEB1 , α SMA, COL1A1 ) in empty vector and SHP2 C459S -transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p
Figure Legend Snippet: Phosphatase activity of SHP2 was required for EMT. A , Western analysis of effects of SHP2 C459S overexpression on ERK activation with or without TGFβ1 treatment (5 ng/ml) for 48 h. B , quantification of EMT-associated markers ( FN1, ZEB1 , α SMA, COL1A1 ) in empty vector and SHP2 C459S -transfected A549 cells, with or without TGFβ1 treatment (5 ng/ml) for 24 h. ***, p

Techniques Used: Activity Assay, Western Blot, Over Expression, Activation Assay, Plasmid Preparation, Transfection

SHP2 positively regulated EMT. A , morphology of A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h. B , cell wound assay was used to test the migration of A549 cells, with or without TGFβ1 treatment (5 ng/ml). Images were captured at 0, 16, and 40 h after wounding. C , relative mRNA levels of EMT-related transcription factors ( Snail1 and Snail2 ) and markers ( CDH1, FN1, VIM, COL1A1 ) detected by real-time PCR in A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h (***, p
Figure Legend Snippet: SHP2 positively regulated EMT. A , morphology of A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h. B , cell wound assay was used to test the migration of A549 cells, with or without TGFβ1 treatment (5 ng/ml). Images were captured at 0, 16, and 40 h after wounding. C , relative mRNA levels of EMT-related transcription factors ( Snail1 and Snail2 ) and markers ( CDH1, FN1, VIM, COL1A1 ) detected by real-time PCR in A549 cells with or without TGFβ1 treatment (5 ng/ml) for 24 h (***, p

Techniques Used: Migration, Real-time Polymerase Chain Reaction

Hook1 negatively regulated EMT. A , quantification of the mRNA levels of CDH1 , Hook1, Hook2 , and Hook3 in A549 cells with TGFβ1 treatment (5 ng/ml) for 0, 3, 6, and 12 h (**, p
Figure Legend Snippet: Hook1 negatively regulated EMT. A , quantification of the mRNA levels of CDH1 , Hook1, Hook2 , and Hook3 in A549 cells with TGFβ1 treatment (5 ng/ml) for 0, 3, 6, and 12 h (**, p

Techniques Used:

22) Product Images from "IL‐37 inhibits invasion and metastasis in non‐small cell lung cancer by suppressing the IL‐6/STAT3 signaling pathway"

Article Title: IL‐37 inhibits invasion and metastasis in non‐small cell lung cancer by suppressing the IL‐6/STAT3 signaling pathway

Journal: Thoracic Cancer

doi: 10.1111/1759-7714.12628

IL‐6 promotes non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐6 protein (0, 50, 100, 150 ng/mL). * P
Figure Legend Snippet: IL‐6 promotes non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐6 protein (0, 50, 100, 150 ng/mL). * P

Techniques Used: Transwell Invasion Assay

E‐cadherin, vimentin and N‐cadherin protein expression in A549 cells was determined by Western blot. * P
Figure Legend Snippet: E‐cadherin, vimentin and N‐cadherin protein expression in A549 cells was determined by Western blot. * P

Techniques Used: Expressing, Western Blot

Invasion and metastasis of A549 cells under different treatments. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells in the four groups. * P
Figure Legend Snippet: Invasion and metastasis of A549 cells under different treatments. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells in the four groups. * P

Techniques Used: Transwell Invasion Assay

Gene expression in A549 cells under different treatments. ( a ) STAT3, E‐cadherin, vimentin and N‐cadherin messenger RNA (mRNA) expression in A549 cells was determined by RT‐PCR. ( b ) STAT3 and pSTAT3 protein expression in A549 cells was determined by Western blot. * P
Figure Legend Snippet: Gene expression in A549 cells under different treatments. ( a ) STAT3, E‐cadherin, vimentin and N‐cadherin messenger RNA (mRNA) expression in A549 cells was determined by RT‐PCR. ( b ) STAT3 and pSTAT3 protein expression in A549 cells was determined by Western blot. * P

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

IL‐37 suppresses non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐37 protein (0, 10, 100, 500 ng/mL). * P
Figure Legend Snippet: IL‐37 suppresses non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐37 protein (0, 10, 100, 500 ng/mL). * P

Techniques Used: Transwell Invasion Assay

23) Product Images from "?-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells"

Article Title: ?-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021468

Interference of endogenous PLD host cell expression reduces the internalization of A. fumigatus . A549 cells were transfected with non-specific small interfering RNAs (siRNAs) (Control), PLD1-specific siRNAs (A, C), or PLD2-specific siRNAs (B, D). After 48 h, cells were infected with the swollen conidia of A. fumigatus 13073 at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, B) and PLD activities (C, D) were measured. Differences in [ 3 H] PtdEtOH formation and A. fumigatus internalization between the untransfected cells (Control) and PLD-silenced cells were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Interference of endogenous PLD host cell expression reduces the internalization of A. fumigatus . A549 cells were transfected with non-specific small interfering RNAs (siRNAs) (Control), PLD1-specific siRNAs (A, C), or PLD2-specific siRNAs (B, D). After 48 h, cells were infected with the swollen conidia of A. fumigatus 13073 at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, B) and PLD activities (C, D) were measured. Differences in [ 3 H] PtdEtOH formation and A. fumigatus internalization between the untransfected cells (Control) and PLD-silenced cells were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Expressing, Transfection, Infection

Heat-killed swollen conidia stimulate PLD activity and internalize in A549 cells. A549 cells were infected with heat-killed (HK) resting conidia, HK swollen conidia (germinated for 6 h), and HK hyphae (germinated for 12 h) of A. fumigatus 13073 (at an MOI of 10 each). The PLD activities were measured (A) and A. fumigatus internalization was determined by immunofluorescent staining (B). Differences in [ 3 H] PtdEtOH formation and in the internalization indices between groups were compared as indicated in the figure. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Heat-killed swollen conidia stimulate PLD activity and internalize in A549 cells. A549 cells were infected with heat-killed (HK) resting conidia, HK swollen conidia (germinated for 6 h), and HK hyphae (germinated for 12 h) of A. fumigatus 13073 (at an MOI of 10 each). The PLD activities were measured (A) and A. fumigatus internalization was determined by immunofluorescent staining (B). Differences in [ 3 H] PtdEtOH formation and in the internalization indices between groups were compared as indicated in the figure. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Infection, Staining

A. fumigatus stimulates PLD activity during its internalization into A549 cells. A. A549 cells were prelabeled with [ 3 H] oleic acid and infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods. Then, ethanol was added to determine the PLD activity. B. A549 cells were infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods, and the internalization of A. fumigatus was analyzed by the nystatin protection assay. Differences in [ 3 H] PtdEtOH formation between the 0 h time point and the other time points (A) and differences in the internalization of A. fumigatus between the 2 h time point and the other time points (B) were compared. In parallel, the cells were lysed for immunoblotting with the indicated antibody (C) and the densitometric analysis of immunoblots for three independent experiments is shown (D). Data are represented as the mean ± SE (n = 3–4), and the blots are characteristic of 3 independent experiments. *P
Figure Legend Snippet: A. fumigatus stimulates PLD activity during its internalization into A549 cells. A. A549 cells were prelabeled with [ 3 H] oleic acid and infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods. Then, ethanol was added to determine the PLD activity. B. A549 cells were infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods, and the internalization of A. fumigatus was analyzed by the nystatin protection assay. Differences in [ 3 H] PtdEtOH formation between the 0 h time point and the other time points (A) and differences in the internalization of A. fumigatus between the 2 h time point and the other time points (B) were compared. In parallel, the cells were lysed for immunoblotting with the indicated antibody (C) and the densitometric analysis of immunoblots for three independent experiments is shown (D). Data are represented as the mean ± SE (n = 3–4), and the blots are characteristic of 3 independent experiments. *P

Techniques Used: Activity Assay, Infection, Western Blot

Host cell PLD activity is stimulated by swollen conidia, but not resting conidia. A. A549 cells were infected with the live resting conidia, swollen conidia (germinated for 6 h), and hyphae (germinated for 12 h) of A. fumigatus 13073 at an MOI of 10 for 30 min. B. A549 cells were infected with the swollen conidia of A. fumigatus 13073 for 30 min at the indicated MOI. C. A549 cells were infected with live resting conidia, swollen conidia and hyphae of A. fumigatus AF293 at an MOI of 10 for 30 min. Thereafter, the PLD activity in the A549 cells was measured, and the differences in [ 3 H] PtdEtOH formation between uninfected group (Control) and infected group or between groups were compared as indicated in the figure (A, B, C). D. A549 cells were infected with resting conidia (germinating time = 0), or conidia of A. fumigatus 13073 germinated for the indicated time periods at an MOI of 10 for 60 min. A. fumigatus internalization was determined by immunofluorescent staining. Differences in the internalization index between resting conidia and germinated conidia were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Host cell PLD activity is stimulated by swollen conidia, but not resting conidia. A. A549 cells were infected with the live resting conidia, swollen conidia (germinated for 6 h), and hyphae (germinated for 12 h) of A. fumigatus 13073 at an MOI of 10 for 30 min. B. A549 cells were infected with the swollen conidia of A. fumigatus 13073 for 30 min at the indicated MOI. C. A549 cells were infected with live resting conidia, swollen conidia and hyphae of A. fumigatus AF293 at an MOI of 10 for 30 min. Thereafter, the PLD activity in the A549 cells was measured, and the differences in [ 3 H] PtdEtOH formation between uninfected group (Control) and infected group or between groups were compared as indicated in the figure (A, B, C). D. A549 cells were infected with resting conidia (germinating time = 0), or conidia of A. fumigatus 13073 germinated for the indicated time periods at an MOI of 10 for 60 min. A. fumigatus internalization was determined by immunofluorescent staining. Differences in the internalization index between resting conidia and germinated conidia were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Infection, Staining

Inhibition of A. fumigatus internalization by PLD chemical inhibitors. A549 cells were incubated for 30 min with 1% (v/v) 1-butanol or tert-butanol (A, B), 2 nM VU0359595 (PLD1-specific inhibitor), 100 nM VU0285655-1 (PLD2-specific inhibitor), or both (C, D). Subsequently, the cells were infected with A. fumigatus 13073 swollen conidia at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, C) and PLD activities (B, D) were measured. Differences in [ 3 H] PtdEtOH formation or A. fumigatus internalization between the untreated (control) cells and inhibitor-pretreated cells were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Inhibition of A. fumigatus internalization by PLD chemical inhibitors. A549 cells were incubated for 30 min with 1% (v/v) 1-butanol or tert-butanol (A, B), 2 nM VU0359595 (PLD1-specific inhibitor), 100 nM VU0285655-1 (PLD2-specific inhibitor), or both (C, D). Subsequently, the cells were infected with A. fumigatus 13073 swollen conidia at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, C) and PLD activities (B, D) were measured. Differences in [ 3 H] PtdEtOH formation or A. fumigatus internalization between the untreated (control) cells and inhibitor-pretreated cells were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Inhibition, Incubation, Infection

β-1,3-glucan induces PLD activity in A549 cells. A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [ 3 H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). A. fumigatus internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in A. fumigatus internalization and [ 3 H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P
Figure Legend Snippet: β-1,3-glucan induces PLD activity in A549 cells. A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [ 3 H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). A. fumigatus internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in A. fumigatus internalization and [ 3 H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Incubation, Infection, Staining, FACS, Flow Cytometry, Cytometry, Fluorescence, Software, Expressing, Western Blot

Both PLD1 and PLD2 are associated with A. fumigatus conidia during internalization into A549 cells. A549 cells were infected with the swollen conidia of A. fumigatus 13073 stably expressing green fluorescence protein (at an MOI of 10) for 30 min. The internalization of A. fumigatus into A549 cells was monitored by differential interference contrast (DIC) microscopy or immunofluorescence using laser confocal microscopy Olympus FluoView FV1000 (green, A. fumigatus conidia; red, PLD1 or PLD2). The images were processed with Olympus FluoView ver. 1.6., and the merged fluorescence images are shown. The data represent 3 similar experiments, and the arrows (white) indicate the association of internalized conidia with PLD. Scale bar, 10 µm.
Figure Legend Snippet: Both PLD1 and PLD2 are associated with A. fumigatus conidia during internalization into A549 cells. A549 cells were infected with the swollen conidia of A. fumigatus 13073 stably expressing green fluorescence protein (at an MOI of 10) for 30 min. The internalization of A. fumigatus into A549 cells was monitored by differential interference contrast (DIC) microscopy or immunofluorescence using laser confocal microscopy Olympus FluoView FV1000 (green, A. fumigatus conidia; red, PLD1 or PLD2). The images were processed with Olympus FluoView ver. 1.6., and the merged fluorescence images are shown. The data represent 3 similar experiments, and the arrows (white) indicate the association of internalized conidia with PLD. Scale bar, 10 µm.

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

24) Product Images from "?-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells"

Article Title: ?-1,3-Glucan-Induced Host Phospholipase D Activation Is Involved in Aspergillus fumigatus Internalization into Type II Human Pneumocyte A549 Cells

Journal: PLoS ONE

doi: 10.1371/journal.pone.0021468

Interference of endogenous PLD host cell expression reduces the internalization of A. fumigatus . A549 cells were transfected with non-specific small interfering RNAs (siRNAs) (Control), PLD1-specific siRNAs (A, C), or PLD2-specific siRNAs (B, D). After 48 h, cells were infected with the swollen conidia of A. fumigatus 13073 at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, B) and PLD activities (C, D) were measured. Differences in [ 3 H] PtdEtOH formation and A. fumigatus internalization between the untransfected cells (Control) and PLD-silenced cells were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Interference of endogenous PLD host cell expression reduces the internalization of A. fumigatus . A549 cells were transfected with non-specific small interfering RNAs (siRNAs) (Control), PLD1-specific siRNAs (A, C), or PLD2-specific siRNAs (B, D). After 48 h, cells were infected with the swollen conidia of A. fumigatus 13073 at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, B) and PLD activities (C, D) were measured. Differences in [ 3 H] PtdEtOH formation and A. fumigatus internalization between the untransfected cells (Control) and PLD-silenced cells were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Expressing, Transfection, Infection

Heat-killed swollen conidia stimulate PLD activity and internalize in A549 cells. A549 cells were infected with heat-killed (HK) resting conidia, HK swollen conidia (germinated for 6 h), and HK hyphae (germinated for 12 h) of A. fumigatus 13073 (at an MOI of 10 each). The PLD activities were measured (A) and A. fumigatus internalization was determined by immunofluorescent staining (B). Differences in [ 3 H] PtdEtOH formation and in the internalization indices between groups were compared as indicated in the figure. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Heat-killed swollen conidia stimulate PLD activity and internalize in A549 cells. A549 cells were infected with heat-killed (HK) resting conidia, HK swollen conidia (germinated for 6 h), and HK hyphae (germinated for 12 h) of A. fumigatus 13073 (at an MOI of 10 each). The PLD activities were measured (A) and A. fumigatus internalization was determined by immunofluorescent staining (B). Differences in [ 3 H] PtdEtOH formation and in the internalization indices between groups were compared as indicated in the figure. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Infection, Staining

A. fumigatus stimulates PLD activity during its internalization into A549 cells. A. A549 cells were prelabeled with [ 3 H] oleic acid and infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods. Then, ethanol was added to determine the PLD activity. B. A549 cells were infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods, and the internalization of A. fumigatus was analyzed by the nystatin protection assay. Differences in [ 3 H] PtdEtOH formation between the 0 h time point and the other time points (A) and differences in the internalization of A. fumigatus between the 2 h time point and the other time points (B) were compared. In parallel, the cells were lysed for immunoblotting with the indicated antibody (C) and the densitometric analysis of immunoblots for three independent experiments is shown (D). Data are represented as the mean ± SE (n = 3–4), and the blots are characteristic of 3 independent experiments. *P
Figure Legend Snippet: A. fumigatus stimulates PLD activity during its internalization into A549 cells. A. A549 cells were prelabeled with [ 3 H] oleic acid and infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods. Then, ethanol was added to determine the PLD activity. B. A549 cells were infected with the resting conidia of A. fumigatus 13073 at an MOI of 10 for the indicated time periods, and the internalization of A. fumigatus was analyzed by the nystatin protection assay. Differences in [ 3 H] PtdEtOH formation between the 0 h time point and the other time points (A) and differences in the internalization of A. fumigatus between the 2 h time point and the other time points (B) were compared. In parallel, the cells were lysed for immunoblotting with the indicated antibody (C) and the densitometric analysis of immunoblots for three independent experiments is shown (D). Data are represented as the mean ± SE (n = 3–4), and the blots are characteristic of 3 independent experiments. *P

Techniques Used: Activity Assay, Infection, Western Blot

Host cell PLD activity is stimulated by swollen conidia, but not resting conidia. A. A549 cells were infected with the live resting conidia, swollen conidia (germinated for 6 h), and hyphae (germinated for 12 h) of A. fumigatus 13073 at an MOI of 10 for 30 min. B. A549 cells were infected with the swollen conidia of A. fumigatus 13073 for 30 min at the indicated MOI. C. A549 cells were infected with live resting conidia, swollen conidia and hyphae of A. fumigatus AF293 at an MOI of 10 for 30 min. Thereafter, the PLD activity in the A549 cells was measured, and the differences in [ 3 H] PtdEtOH formation between uninfected group (Control) and infected group or between groups were compared as indicated in the figure (A, B, C). D. A549 cells were infected with resting conidia (germinating time = 0), or conidia of A. fumigatus 13073 germinated for the indicated time periods at an MOI of 10 for 60 min. A. fumigatus internalization was determined by immunofluorescent staining. Differences in the internalization index between resting conidia and germinated conidia were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Host cell PLD activity is stimulated by swollen conidia, but not resting conidia. A. A549 cells were infected with the live resting conidia, swollen conidia (germinated for 6 h), and hyphae (germinated for 12 h) of A. fumigatus 13073 at an MOI of 10 for 30 min. B. A549 cells were infected with the swollen conidia of A. fumigatus 13073 for 30 min at the indicated MOI. C. A549 cells were infected with live resting conidia, swollen conidia and hyphae of A. fumigatus AF293 at an MOI of 10 for 30 min. Thereafter, the PLD activity in the A549 cells was measured, and the differences in [ 3 H] PtdEtOH formation between uninfected group (Control) and infected group or between groups were compared as indicated in the figure (A, B, C). D. A549 cells were infected with resting conidia (germinating time = 0), or conidia of A. fumigatus 13073 germinated for the indicated time periods at an MOI of 10 for 60 min. A. fumigatus internalization was determined by immunofluorescent staining. Differences in the internalization index between resting conidia and germinated conidia were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Infection, Staining

Inhibition of A. fumigatus internalization by PLD chemical inhibitors. A549 cells were incubated for 30 min with 1% (v/v) 1-butanol or tert-butanol (A, B), 2 nM VU0359595 (PLD1-specific inhibitor), 100 nM VU0285655-1 (PLD2-specific inhibitor), or both (C, D). Subsequently, the cells were infected with A. fumigatus 13073 swollen conidia at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, C) and PLD activities (B, D) were measured. Differences in [ 3 H] PtdEtOH formation or A. fumigatus internalization between the untreated (control) cells and inhibitor-pretreated cells were compared. Data are represented as the mean ± SE (n = 3–4). *P
Figure Legend Snippet: Inhibition of A. fumigatus internalization by PLD chemical inhibitors. A549 cells were incubated for 30 min with 1% (v/v) 1-butanol or tert-butanol (A, B), 2 nM VU0359595 (PLD1-specific inhibitor), 100 nM VU0285655-1 (PLD2-specific inhibitor), or both (C, D). Subsequently, the cells were infected with A. fumigatus 13073 swollen conidia at an MOI of 10. A. fumigatus internalization was analyzed by the nystatin protection assay (A, C) and PLD activities (B, D) were measured. Differences in [ 3 H] PtdEtOH formation or A. fumigatus internalization between the untreated (control) cells and inhibitor-pretreated cells were compared. Data are represented as the mean ± SE (n = 3–4). *P

Techniques Used: Inhibition, Incubation, Infection

β-1,3-glucan induces PLD activity in A549 cells. A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [ 3 H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). A. fumigatus internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in A. fumigatus internalization and [ 3 H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P
Figure Legend Snippet: β-1,3-glucan induces PLD activity in A549 cells. A549 cells were stimulated with the indicated concentrations of β-1,3-glucan for 30 min (A) or for the indicated duration of time with 50 µg/mL of β-1,3-glucan (B). Thereafter, the PLD activity was determined and differences in [ 3 H] PtdEtOH formation between the control (indicated by “0”) and other groups were compared. In C and D, A549 cells were first incubated with HBSS (Control), HBSS containing 5 µg/mL of isotype control antibody and HBSS containing 5 µg/mL of anti-dectin-1 mAb GE2 (ab82888) for 30 min, respectively. Then, the cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 (C) or stimulated by HBSS and HBSS containing 50 µg/mL of β-1,3-glucan for 30 min, respectively (D). A. fumigatus internalization was analyzed by the nystatin protection assay (C) and the PLD activity (D) was measured. Differences in A. fumigatus internalization and [ 3 H] PtdEtOH formation between the untreated cells (Control) and antibody-treated cells were compared. In E and F, A549 cells were infected with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Subsequently, the cells were stained with isotype (IC) antibody or the primary anti-dectin-1 mAb GE2 (ab82888) and analyzed by FACS Calibur flow cytometer. The geometric mean fluorescence intensity was determined by Cell Quest Pro software. Differences between uninfected cells (Control) and conidia-infected cells were compared. G. A549 cells were incubated with swollen conidia of A. fumigatus 13073 at an MOI of 10 or incubated with PBS (Control) for 30 min. Cells were analyzed for dectin-1 expression by immunoblotting using an anti-dectin-1 antibody (sc-26094). FACS profiles and immunoblots shown here are characteristic of 3 independent experiments. Data are represented as mean ± SE (n = 3–4). *P

Techniques Used: Activity Assay, Incubation, Infection, Staining, FACS, Flow Cytometry, Cytometry, Fluorescence, Software, Expressing, Western Blot

Both PLD1 and PLD2 are associated with A. fumigatus conidia during internalization into A549 cells. A549 cells were infected with the swollen conidia of A. fumigatus 13073 stably expressing green fluorescence protein (at an MOI of 10) for 30 min. The internalization of A. fumigatus into A549 cells was monitored by differential interference contrast (DIC) microscopy or immunofluorescence using laser confocal microscopy Olympus FluoView FV1000 (green, A. fumigatus conidia; red, PLD1 or PLD2). The images were processed with Olympus FluoView ver. 1.6., and the merged fluorescence images are shown. The data represent 3 similar experiments, and the arrows (white) indicate the association of internalized conidia with PLD. Scale bar, 10 µm.
Figure Legend Snippet: Both PLD1 and PLD2 are associated with A. fumigatus conidia during internalization into A549 cells. A549 cells were infected with the swollen conidia of A. fumigatus 13073 stably expressing green fluorescence protein (at an MOI of 10) for 30 min. The internalization of A. fumigatus into A549 cells was monitored by differential interference contrast (DIC) microscopy or immunofluorescence using laser confocal microscopy Olympus FluoView FV1000 (green, A. fumigatus conidia; red, PLD1 or PLD2). The images were processed with Olympus FluoView ver. 1.6., and the merged fluorescence images are shown. The data represent 3 similar experiments, and the arrows (white) indicate the association of internalized conidia with PLD. Scale bar, 10 µm.

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

25) Product Images from "Profiling ribonucleotide and deoxyribonucleotide pools perturbed by gemcitabine in human non-small cell lung cancer cells"

Article Title: Profiling ribonucleotide and deoxyribonucleotide pools perturbed by gemcitabine in human non-small cell lung cancer cells

Journal: Scientific Reports

doi: 10.1038/srep37250

Effects of dFdC on cell cycle arrest in A549 cells at different times. ( A ) 4 h; ( B ) 10 h; ( C ) 16 h; ( D ) 24 h. P -value of less than 0.05 (* P
Figure Legend Snippet: Effects of dFdC on cell cycle arrest in A549 cells at different times. ( A ) 4 h; ( B ) 10 h; ( C ) 16 h; ( D ) 24 h. P -value of less than 0.05 (* P

Techniques Used:

Effects of dFdC on dNTP pool sizes after treatment of A549 cells with 0.5 and 2.0 μM of dFdC. Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation values.
Figure Legend Snippet: Effects of dFdC on dNTP pool sizes after treatment of A549 cells with 0.5 and 2.0 μM of dFdC. Each data point is an average of two independent experiments (done in triplicate) and is reported as mean ± standard deviation values.

Techniques Used: Standard Deviation

26) Product Images from "Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza"

Article Title: Development of CRISPR as a prophylactic strategy to combat novel coronavirus and influenza

Journal: bioRxiv

doi: 10.1101/2020.03.13.991307

Inhibition of IAV infection using CRISPR PAC-MAN. (A) Workflow used to challenge Cas13d A549 lung epithelial cells with PR8 mNeon IAV. (B) Screen of pools of 6 crRNAs targeting each of the eight IAV genome segments. % of mNeon+ cells in each of the eight crRNA conditions are compared to a pool of non-targeting (NT) crRNAs at an MOI of 2.5 (left) or 5 (right). (C) Microscopy quantification of % of mNeon+ cells per field of view (FOV) at an MOI of 2.5 (p = 1×10 −5 , left) and 5 (p = 5×10 −9 , right). Each dot represents the % for a single microscopy FOV. N = 48 FOV. (D) Flow cytometry evaluation of % of mNeon+ cells at an MOI of 2.5 (p = 0.04, left) and 5 (p = 0.006, right). N = 6 biological samples.
Figure Legend Snippet: Inhibition of IAV infection using CRISPR PAC-MAN. (A) Workflow used to challenge Cas13d A549 lung epithelial cells with PR8 mNeon IAV. (B) Screen of pools of 6 crRNAs targeting each of the eight IAV genome segments. % of mNeon+ cells in each of the eight crRNA conditions are compared to a pool of non-targeting (NT) crRNAs at an MOI of 2.5 (left) or 5 (right). (C) Microscopy quantification of % of mNeon+ cells per field of view (FOV) at an MOI of 2.5 (p = 1×10 −5 , left) and 5 (p = 5×10 −9 , right). Each dot represents the % for a single microscopy FOV. N = 48 FOV. (D) Flow cytometry evaluation of % of mNeon+ cells at an MOI of 2.5 (p = 0.04, left) and 5 (p = 0.006, right). N = 6 biological samples.

Techniques Used: Inhibition, Infection, CRISPR, Microscopy, Flow Cytometry

Bioinformatic analysis of CRISPR-Cas13d target sites for SARS-CoV-2 and the PAC-MAN system design. (A) Alignment of sequences from SARS-CoV-2 genomes from 47 patients along with SARS- and MERS-causing coronaviruses. Top: predicted abundance of crRNAs that are able to target SARS-CoV-2 genomes and SARS or MERS; Middle: annotation of genes in the SARS-CoV-2 genomes, along with conserved regions chosen to be synthesized into the SARS-CoV-2 reporters (in magenta and purple). Color regions indicate the synthesized SARS-CoV-2 fragments; Bottom: the percent of conservation between aligned viral genomes. See Supplemental Table 2 for the designed crRNA sequences and Supplemental Table 3 for synthesized SARS-CoV-2 fragments. (B) Schematic of the two reporters (SARS-CoV-2-F1/F2) created with synthesized viral sequences. SARS-CoV-2-F1 contains GFP fused to a portion of RdRP (RdRP-F1) and SARS-CoV-2-F2 contains GFP fused to portions of both RdRP (RDRP-F2) and N. (C) Schematics for the constructs used to express Cas13d or its crRNAs. (D) Workflow used to challenge Cas13d A549 lung epithelial cells with SARS-CoV-2 reporters.
Figure Legend Snippet: Bioinformatic analysis of CRISPR-Cas13d target sites for SARS-CoV-2 and the PAC-MAN system design. (A) Alignment of sequences from SARS-CoV-2 genomes from 47 patients along with SARS- and MERS-causing coronaviruses. Top: predicted abundance of crRNAs that are able to target SARS-CoV-2 genomes and SARS or MERS; Middle: annotation of genes in the SARS-CoV-2 genomes, along with conserved regions chosen to be synthesized into the SARS-CoV-2 reporters (in magenta and purple). Color regions indicate the synthesized SARS-CoV-2 fragments; Bottom: the percent of conservation between aligned viral genomes. See Supplemental Table 2 for the designed crRNA sequences and Supplemental Table 3 for synthesized SARS-CoV-2 fragments. (B) Schematic of the two reporters (SARS-CoV-2-F1/F2) created with synthesized viral sequences. SARS-CoV-2-F1 contains GFP fused to a portion of RdRP (RdRP-F1) and SARS-CoV-2-F2 contains GFP fused to portions of both RdRP (RDRP-F2) and N. (C) Schematics for the constructs used to express Cas13d or its crRNAs. (D) Workflow used to challenge Cas13d A549 lung epithelial cells with SARS-CoV-2 reporters.

Techniques Used: CRISPR, Synthesized, Construct

27) Product Images from "Rickettsia Sca4 reduces vinculin-mediated intercellular tension to promote spread"

Article Title: Rickettsia Sca4 reduces vinculin-mediated intercellular tension to promote spread

Journal: Cell

doi: 10.1016/j.cell.2016.09.023

Sca4 acts in trans to target vinculin in the donor cell (A) Mixed cell assay schematic, as described in methods. (B) Images of infectious foci detected in the mixed cell assay showing A549-TRT (soluble TagRFP-T, red), R. parkeri (green) and β-catenin (blue). Scale bar, 10 μm. (C) Mixed cell assay results showing percentage of bacteria that spread to recipient cells in a focus. Donor or recipient cells were reverse transfected with control siRNA (NT) or siVCL #1 and infected with WT (Rp-GFP) or sca4::tn . Mean ± SEM, one-way ANOVA: ** p
Figure Legend Snippet: Sca4 acts in trans to target vinculin in the donor cell (A) Mixed cell assay schematic, as described in methods. (B) Images of infectious foci detected in the mixed cell assay showing A549-TRT (soluble TagRFP-T, red), R. parkeri (green) and β-catenin (blue). Scale bar, 10 μm. (C) Mixed cell assay results showing percentage of bacteria that spread to recipient cells in a focus. Donor or recipient cells were reverse transfected with control siRNA (NT) or siVCL #1 and infected with WT (Rp-GFP) or sca4::tn . Mean ± SEM, one-way ANOVA: ** p

Techniques Used: Transfection, Infection

28) Product Images from "Biocompatible and Label-Free Separation of Cancer Cells of Cell Culture Lines from White Blood Cells in Ferrofluids"

Article Title: Biocompatible and Label-Free Separation of Cancer Cells of Cell Culture Lines from White Blood Cells in Ferrofluids

Journal: Lab on a chip

doi: 10.1039/c7lc00327g

(a) Short-term cell viability comparison between before vs. after separation groups. No significant difference was found between the two. (b) Representative images of Live/Dead staining of the before (top) and after separation (bottom) groups. Calcein-AM (green) and PI (red) channels were merged in these images. Scale bars: 100 µ m. (c) Bright field images of cultured A549 cells collected after separation from day 1 to day 4. A Live/Dead staining of the cultured cells on day 4 showed excellent viability. A control group of cell culture was used for comparison. No significant difference was found in cell proliferation between cells in the control group and cells collected after device separation. Scale bars: 50 µ m.
Figure Legend Snippet: (a) Short-term cell viability comparison between before vs. after separation groups. No significant difference was found between the two. (b) Representative images of Live/Dead staining of the before (top) and after separation (bottom) groups. Calcein-AM (green) and PI (red) channels were merged in these images. Scale bars: 100 µ m. (c) Bright field images of cultured A549 cells collected after separation from day 1 to day 4. A Live/Dead staining of the cultured cells on day 4 showed excellent viability. A control group of cell culture was used for comparison. No significant difference was found in cell proliferation between cells in the control group and cells collected after device separation. Scale bars: 50 µ m.

Techniques Used: Staining, Cell Culture

29) Product Images from "The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment"

Article Title: The Interferon Type I/III Response to Respiratory Syncytial Virus Infection in Airway Epithelial Cells Can Be Attenuated or Amplified by Antiviral Treatment

Journal: Journal of Virology

doi: 10.1128/JVI.02417-15

Helicase sensing of RSV-2 infection and polymerase inhibition in A549 cells. A549 cells infected for 24 h before treatment and harvested at 48 h postinfection were subjected to Western blot analysis of proteins in the innate viral RNA sensing pathway.
Figure Legend Snippet: Helicase sensing of RSV-2 infection and polymerase inhibition in A549 cells. A549 cells infected for 24 h before treatment and harvested at 48 h postinfection were subjected to Western blot analysis of proteins in the innate viral RNA sensing pathway.

Techniques Used: Infection, Inhibition, Western Blot

Antiviral activities of RSV inhibitors in A549 cells. (A) The EC 50 concentration of each compound was determined in a colorimetric RSV F protein cell-based ELISA. The x axis data show the concentrations of compound, and the y axis data represent the measured
Figure Legend Snippet: Antiviral activities of RSV inhibitors in A549 cells. (A) The EC 50 concentration of each compound was determined in a colorimetric RSV F protein cell-based ELISA. The x axis data show the concentrations of compound, and the y axis data represent the measured

Techniques Used: Concentration Assay, In-Cell ELISA

Compound time-of-addition (ToA) profiles for RSV inhibitors. (A) Single-cycle RSV-A2 infection of A549 (dashed lines) or HEp-2 (solid lines) cells. TMC-353121, RSV-604, YM-53403, or BI-D and ribavirin (80 μM) were added at −4, 0, 2, 4,
Figure Legend Snippet: Compound time-of-addition (ToA) profiles for RSV inhibitors. (A) Single-cycle RSV-A2 infection of A549 (dashed lines) or HEp-2 (solid lines) cells. TMC-353121, RSV-604, YM-53403, or BI-D and ribavirin (80 μM) were added at −4, 0, 2, 4,

Techniques Used: Infection

30) Product Images from "Pulmonary impact of titanium dioxide nanorods: examination of nanorod-exposed rat lungs and human alveolar cells"

Article Title: Pulmonary impact of titanium dioxide nanorods: examination of nanorod-exposed rat lungs and human alveolar cells

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S179159

Internalization of TiO 2 nanorods by A549 cells. Notes: TEM of a control cell ( A , magnification 1,500×) with intact organelles ( B , 10,000×) and a nano-TiO 2 -treated cell ( C , 4,000×), showing internalized NPs in multivesicle bodies ( D , 12,000×). Images of another treated cell ( E , 2,000×) reveal a caveola being formed on the cell surface near a cluster of NPs ( F , 20,000×). The white frames in A , C , and E show the cutouts seen magnified in B , D , and F . Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.
Figure Legend Snippet: Internalization of TiO 2 nanorods by A549 cells. Notes: TEM of a control cell ( A , magnification 1,500×) with intact organelles ( B , 10,000×) and a nano-TiO 2 -treated cell ( C , 4,000×), showing internalized NPs in multivesicle bodies ( D , 12,000×). Images of another treated cell ( E , 2,000×) reveal a caveola being formed on the cell surface near a cluster of NPs ( F , 20,000×). The white frames in A , C , and E show the cutouts seen magnified in B , D , and F . Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.

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

SEM images of control ( A ) and TiO 2 nanorod-treated A549 cell ( B ). The cluster of white particles at the lower left region of the cell appeared to be nano-TiO 2 , which was verified by the density of the red color, corresponding to titanium ( C ), based on the EDS result ( D ) from the same region. Nanorods in the region with lower red density ( C ) were probably attached to the plasma membrane, while where red density was maximal, they might have already been internalized. Abbreviations: EDS, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.
Figure Legend Snippet: SEM images of control ( A ) and TiO 2 nanorod-treated A549 cell ( B ). The cluster of white particles at the lower left region of the cell appeared to be nano-TiO 2 , which was verified by the density of the red color, corresponding to titanium ( C ), based on the EDS result ( D ) from the same region. Nanorods in the region with lower red density ( C ) were probably attached to the plasma membrane, while where red density was maximal, they might have already been internalized. Abbreviations: EDS, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.

Techniques Used: Spectroscopy, Electron Microscopy

31) Product Images from "Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens"

Article Title: Allergen Delivery Inhibitors: Characterisation of Potent and Selective Inhibitors of Der p 1 and Their Attenuation of Airway Responses to House Dust Mite Allergens

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19103166

Release of IL-8 or IL-6 from cultured A549 cells and the effect of ADZ 50,000. ( A , B ) Release of IL-8 or IL-6 following stimulation by Der p 1 (20 nM), Der p 2 (14 nM), TPCK-treated trypsin (9 nM), or thrombin (0.5 U/mL) all applied apically. Cells were exposed for 8 h to serum-free EMEM containing the treatments, supplemented with 5 mM cysteine in the case of Der p 1. Cells were then washed and incubated with serum-free EMEM alone for a further 16 h prior to collection of samples. Data are shown as results from individual experiments with cells of different passage number. * p
Figure Legend Snippet: Release of IL-8 or IL-6 from cultured A549 cells and the effect of ADZ 50,000. ( A , B ) Release of IL-8 or IL-6 following stimulation by Der p 1 (20 nM), Der p 2 (14 nM), TPCK-treated trypsin (9 nM), or thrombin (0.5 U/mL) all applied apically. Cells were exposed for 8 h to serum-free EMEM containing the treatments, supplemented with 5 mM cysteine in the case of Der p 1. Cells were then washed and incubated with serum-free EMEM alone for a further 16 h prior to collection of samples. Data are shown as results from individual experiments with cells of different passage number. * p

Techniques Used: Cell Culture, Incubation

32) Product Images from "Pulmonary impact of titanium dioxide nanorods: examination of nanorod-exposed rat lungs and human alveolar cells"

Article Title: Pulmonary impact of titanium dioxide nanorods: examination of nanorod-exposed rat lungs and human alveolar cells

Journal: International Journal of Nanomedicine

doi: 10.2147/IJN.S179159

SEM images of control ( A ) and TiO 2 nanorod-treated A549 cell ( B ). The cluster of white particles at the lower left region of the cell appeared to be nano-TiO 2 , which was verified by the density of the red color, corresponding to titanium ( C ), based on the EDS result ( D ) from the same region. Nanorods in the region with lower red density ( C ) were probably attached to the plasma membrane, while where red density was maximal, they might have already been internalized. Abbreviations: EDS, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.
Figure Legend Snippet: SEM images of control ( A ) and TiO 2 nanorod-treated A549 cell ( B ). The cluster of white particles at the lower left region of the cell appeared to be nano-TiO 2 , which was verified by the density of the red color, corresponding to titanium ( C ), based on the EDS result ( D ) from the same region. Nanorods in the region with lower red density ( C ) were probably attached to the plasma membrane, while where red density was maximal, they might have already been internalized. Abbreviations: EDS, energy-dispersive X-ray spectroscopy; SEM, scanning electron microscopy.

Techniques Used: Spectroscopy, Electron Microscopy

Internalization of TiO 2 nanorods by A549 cells. Notes: TEM of a control cell ( A , magnification 1,500×) with intact organelles ( B , 10,000×) and a nano-TiO 2 -treated cell ( C , 4,000×), showing internalized NPs in multivesicle bodies ( D , 12,000×). Images of another treated cell ( E , 2,000×) reveal a caveola being formed on the cell surface near a cluster of NPs ( F , 20,000×). The white frames in A , C , and E show the cutouts seen magnified in B , D , and F . Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.
Figure Legend Snippet: Internalization of TiO 2 nanorods by A549 cells. Notes: TEM of a control cell ( A , magnification 1,500×) with intact organelles ( B , 10,000×) and a nano-TiO 2 -treated cell ( C , 4,000×), showing internalized NPs in multivesicle bodies ( D , 12,000×). Images of another treated cell ( E , 2,000×) reveal a caveola being formed on the cell surface near a cluster of NPs ( F , 20,000×). The white frames in A , C , and E show the cutouts seen magnified in B , D , and F . Abbreviations: NPs, nanoparticles; TEM, transmission electron microscopy.

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

33) Product Images from "Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus "

Article Title: Transcription Factor PrtT Controls Expression of Multiple Secreted Proteases in the Human Pathogenic Mold Aspergillus fumigatus

Journal: Infection and Immunity

doi: 10.1128/IAI.00426-09

CF collected from the prtTΔ mutant shows decreased killing of A549 alveolar cells in culture and reduced hemolytic activity. (A) Confluent A549 cells were incubated in the presence of WT (left) and prtTΔ (center) CF for 12 h at 37°C
Figure Legend Snippet: CF collected from the prtTΔ mutant shows decreased killing of A549 alveolar cells in culture and reduced hemolytic activity. (A) Confluent A549 cells were incubated in the presence of WT (left) and prtTΔ (center) CF for 12 h at 37°C

Techniques Used: Mutagenesis, Activity Assay, Incubation

34) Product Images from "Overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibits migration and invasion via targeting EYA2"

Article Title: Overexpression of miR-30a in lung adenocarcinoma A549 cell line inhibits migration and invasion via targeting EYA2

Journal: Acta Biochimica et Biophysica Sinica

doi: 10.1093/abbs/gmv139

miR-30a suppresses EYA2 protein expression (A) The results of the western blot of the proteins extracted from the A549 and BEAS-2B cell lines. GAPDH was used as an internal reference protein for loading control. (B) Immunohistochemical analysis of EYA2 expression in 14 pairs of lung adenocarcinoma or the corresponding normal lung tissues (×200). (C) The A549 and BEAS-2B cells were transfected with 50 nM mimics NC, inhibitors NC, miR-30a mimics or inhibitors and cultured for additional 48 h. The cells were digested by trypsin for western blot analysis. The protein levels were normalized by GAPDH. (D) The fold of change of miR-30a expression in A549 or BEAS-2B cells under the same conditions was evaluated by qRT-PCR. The experiments were repeated at least three times with similar trends. * P
Figure Legend Snippet: miR-30a suppresses EYA2 protein expression (A) The results of the western blot of the proteins extracted from the A549 and BEAS-2B cell lines. GAPDH was used as an internal reference protein for loading control. (B) Immunohistochemical analysis of EYA2 expression in 14 pairs of lung adenocarcinoma or the corresponding normal lung tissues (×200). (C) The A549 and BEAS-2B cells were transfected with 50 nM mimics NC, inhibitors NC, miR-30a mimics or inhibitors and cultured for additional 48 h. The cells were digested by trypsin for western blot analysis. The protein levels were normalized by GAPDH. (D) The fold of change of miR-30a expression in A549 or BEAS-2B cells under the same conditions was evaluated by qRT-PCR. The experiments were repeated at least three times with similar trends. * P

Techniques Used: Expressing, Western Blot, Immunohistochemistry, Transfection, Cell Culture, Quantitative RT-PCR

EYA2 gene is a direct downstream target of miR-30a in A549 cells (A) The EYA2-wild-type with putative binding site of miR-30a and the mutant generated by replacing the seed region of miR-30a with complementary bases were cloned into the pmirGLO vector, respectively. The restriction enzyme cutting sites of the Xho I and Xba I were chosen as the cloning sites. (B) For luciferase assay, A549 cells were transiently cotransfected with 200 ng luciferase vectors and 50 nM miR-30a mimics. Luciferase activities were measured 24 h after transfection. Firefly luciferase activities were normalized by the activity of renilla luciferase. The experiments were performed in triplicate with similar trend. ** P
Figure Legend Snippet: EYA2 gene is a direct downstream target of miR-30a in A549 cells (A) The EYA2-wild-type with putative binding site of miR-30a and the mutant generated by replacing the seed region of miR-30a with complementary bases were cloned into the pmirGLO vector, respectively. The restriction enzyme cutting sites of the Xho I and Xba I were chosen as the cloning sites. (B) For luciferase assay, A549 cells were transiently cotransfected with 200 ng luciferase vectors and 50 nM miR-30a mimics. Luciferase activities were measured 24 h after transfection. Firefly luciferase activities were normalized by the activity of renilla luciferase. The experiments were performed in triplicate with similar trend. ** P

Techniques Used: Binding Assay, Mutagenesis, Generated, Clone Assay, Plasmid Preparation, Luciferase, Transfection, Activity Assay

Overexpression of miR-30a in A549 cells inhibited migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively, were seeded into the up chambers at 8000 cells per well. After culturing for 24 h, the invasive cells were stained by Giemsa. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments±SE was shown. ** P
Figure Legend Snippet: Overexpression of miR-30a in A549 cells inhibited migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) A549 cells transfected with 20 nM mimics NC or miR-30a mimics, respectively, were seeded into the up chambers at 8000 cells per well. After culturing for 24 h, the invasive cells were stained by Giemsa. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments±SE was shown. ** P

Techniques Used: Over Expression, Migration, Wound Healing Assay, Transfection, Staining

Overexpression of miR-30a in A549 cells has no effect on the cell proliferation and cell cycle distribution. (A) For cell proliferation analysis, A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively. Twenty-four hours after transfection, cells were collected and seeded into a 96-well plate (1000 cells per well), and cultured for 120 h. The growth of the cells was analyzed by MTS assay, and all values were shown as the mean ± SE of triplicate measurements. (B) A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively, as indicated. Forty-eight hours later, 10 6 cells were collected for cell cycle analysis by PI staining and flow cytometer assay. (C) Western blot analysis of p27 kip1, cyclin D1, cyclin E1, and cyclin A1 in A549 cells after transfection with 20 nM mimics NC or miR-30a mimics. GAPDH was used as a loading control. All experiments were repeated at least three times with similar trends.
Figure Legend Snippet: Overexpression of miR-30a in A549 cells has no effect on the cell proliferation and cell cycle distribution. (A) For cell proliferation analysis, A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively. Twenty-four hours after transfection, cells were collected and seeded into a 96-well plate (1000 cells per well), and cultured for 120 h. The growth of the cells was analyzed by MTS assay, and all values were shown as the mean ± SE of triplicate measurements. (B) A549 cells were transfected with 20 nM mimics NC or miR-30a mimics, respectively, as indicated. Forty-eight hours later, 10 6 cells were collected for cell cycle analysis by PI staining and flow cytometer assay. (C) Western blot analysis of p27 kip1, cyclin D1, cyclin E1, and cyclin A1 in A549 cells after transfection with 20 nM mimics NC or miR-30a mimics. GAPDH was used as a loading control. All experiments were repeated at least three times with similar trends.

Techniques Used: Over Expression, Transfection, Cell Culture, MTS Assay, Cell Cycle Assay, Staining, Flow Cytometry, Cytometry, Western Blot

Downregulation of EYA2 in A549 cells suppressed migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM siRNAs NC or EYA2 siRNAs, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) The transwell cell invasion assay of A549 cells transfected with 20 nM siRNAs NC or EYA2 siRNAs, respectively. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments ±SE was shown. ** P
Figure Legend Snippet: Downregulation of EYA2 in A549 cells suppressed migration and invasion of the cells (A) The wound-healing assay of A549 cells transfected with 20 nM siRNAs NC or EYA2 siRNAs, respectively. (B) The rate of wound closure ±SE was shown. The experiment was performed in triplicate with similar trend. (C) The transwell cell invasion assay of A549 cells transfected with 20 nM siRNAs NC or EYA2 siRNAs, respectively. Representative fields of invasive cells on membrane are captured. (D) The average number of invasive cells per field from three independent experiments ±SE was shown. ** P

Techniques Used: Migration, Wound Healing Assay, Transfection, Invasion Assay

Overexpression of EYA2 reverses the inhibition of cell invasion and migration induced by upregulated miR-30a in A549 cells (A) The expression of EGFP cotransfected with p-EYA2 + mimics NC, p-EYA2 + miR-30a mimics, p-empty + mimics NC, or p-empty + miR-30a mimics for 48 h was observed using a fluorescent inverted microscope (×200). Hoechst was used for cell staining. (B,C) Wound-healing assay and (D,E) matrigel invasion assay demonstrated functional effects in A549 cells after cotransfection. Experimental values presented as the mean ± SE. The experiments were repeated at least three times with similar trends. ** P
Figure Legend Snippet: Overexpression of EYA2 reverses the inhibition of cell invasion and migration induced by upregulated miR-30a in A549 cells (A) The expression of EGFP cotransfected with p-EYA2 + mimics NC, p-EYA2 + miR-30a mimics, p-empty + mimics NC, or p-empty + miR-30a mimics for 48 h was observed using a fluorescent inverted microscope (×200). Hoechst was used for cell staining. (B,C) Wound-healing assay and (D,E) matrigel invasion assay demonstrated functional effects in A549 cells after cotransfection. Experimental values presented as the mean ± SE. The experiments were repeated at least three times with similar trends. ** P

Techniques Used: Over Expression, Inhibition, Migration, Expressing, Inverted Microscopy, Staining, Wound Healing Assay, Invasion Assay, Functional Assay, Cotransfection

35) Product Images from "miR-154 inhibits migration and invasion of human non-small cell lung cancer by targeting ZEB2"

Article Title: miR-154 inhibits migration and invasion of human non-small cell lung cancer by targeting ZEB2

Journal: Oncology Letters

doi: 10.3892/ol.2016.4577

Overexpression of miR-154 inhibited A549 cell migration and invasion. Transwell assay was used to evaluate the (A) migration and (B) invasion abilities of A549 cells transfected with miR-154 mimic or miR-NC (magnification, ×200). **P
Figure Legend Snippet: Overexpression of miR-154 inhibited A549 cell migration and invasion. Transwell assay was used to evaluate the (A) migration and (B) invasion abilities of A549 cells transfected with miR-154 mimic or miR-NC (magnification, ×200). **P

Techniques Used: Over Expression, Migration, Transwell Assay, Transfection

Overexpression of ZEB2 attenuated the suppressive effect of miR-154 on the migration and invasion abilities of A549 cells. (A) The messenger RNA levels of ZEB2 were quantified by reverse transcription-quantitative polymerase chain reaction in A549 cells
Figure Legend Snippet: Overexpression of ZEB2 attenuated the suppressive effect of miR-154 on the migration and invasion abilities of A549 cells. (A) The messenger RNA levels of ZEB2 were quantified by reverse transcription-quantitative polymerase chain reaction in A549 cells

Techniques Used: Over Expression, Migration, Real-time Polymerase Chain Reaction

Inhibition of ZEB2 expression inhibited migration and invasion in A549 cells. (A) The messenger RNA levels of ZEB2 were evaluated by reverse transcription-quantitative polymerase chain reaction in A549 cells transfected with si-ZEB2 or si-Scramble. (B)
Figure Legend Snippet: Inhibition of ZEB2 expression inhibited migration and invasion in A549 cells. (A) The messenger RNA levels of ZEB2 were evaluated by reverse transcription-quantitative polymerase chain reaction in A549 cells transfected with si-ZEB2 or si-Scramble. (B)

Techniques Used: Inhibition, Expressing, Migration, Real-time Polymerase Chain Reaction, Transfection

Overexpression of miR-154 inhibited ZEB2 expression and epithelial-mesenchymal transition. (A) Messenger RNA levels of ZEB2 were determined by reverse transcription-quantitative polymerase chain reaction in A549 cells transfected with miR-154 or miR-NC.
Figure Legend Snippet: Overexpression of miR-154 inhibited ZEB2 expression and epithelial-mesenchymal transition. (A) Messenger RNA levels of ZEB2 were determined by reverse transcription-quantitative polymerase chain reaction in A549 cells transfected with miR-154 or miR-NC.

Techniques Used: Over Expression, Expressing, Real-time Polymerase Chain Reaction, Transfection

36) Product Images from "IL‐37 inhibits invasion and metastasis in non‐small cell lung cancer by suppressing the IL‐6/STAT3 signaling pathway"

Article Title: IL‐37 inhibits invasion and metastasis in non‐small cell lung cancer by suppressing the IL‐6/STAT3 signaling pathway

Journal: Thoracic Cancer

doi: 10.1111/1759-7714.12628

IL‐6 promotes non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐6 protein (0, 50, 100, 150 ng/mL). * P
Figure Legend Snippet: IL‐6 promotes non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐6 protein (0, 50, 100, 150 ng/mL). * P

Techniques Used: Transwell Invasion Assay

E‐cadherin, vimentin and N‐cadherin protein expression in A549 cells was determined by Western blot. * P
Figure Legend Snippet: E‐cadherin, vimentin and N‐cadherin protein expression in A549 cells was determined by Western blot. * P

Techniques Used: Expressing, Western Blot

Invasion and metastasis of A549 cells under different treatments. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells in the four groups. * P
Figure Legend Snippet: Invasion and metastasis of A549 cells under different treatments. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells in the four groups. * P

Techniques Used: Transwell Invasion Assay

Gene expression in A549 cells under different treatments. ( a ) STAT3, E‐cadherin, vimentin and N‐cadherin messenger RNA (mRNA) expression in A549 cells was determined by RT‐PCR. ( b ) STAT3 and pSTAT3 protein expression in A549 cells was determined by Western blot. * P
Figure Legend Snippet: Gene expression in A549 cells under different treatments. ( a ) STAT3, E‐cadherin, vimentin and N‐cadherin messenger RNA (mRNA) expression in A549 cells was determined by RT‐PCR. ( b ) STAT3 and pSTAT3 protein expression in A549 cells was determined by Western blot. * P

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

IL‐37 suppresses non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐37 protein (0, 10, 100, 500 ng/mL). * P
Figure Legend Snippet: IL‐37 suppresses non‐small cell lung cancer (NSCLC) in a dose‐dependent manner. ( a ) Scratch wound healing and ( b ) Transwell invasion assay of A549 cells with different concentrations of rhIL‐37 protein (0, 10, 100, 500 ng/mL). * P

Techniques Used: Transwell Invasion Assay

37) Product Images from "Circ‐ IGF1R inhibits cell invasion and migration in non‐small cell lung cancer"

Article Title: Circ‐ IGF1R inhibits cell invasion and migration in non‐small cell lung cancer

Journal: Thoracic Cancer

doi: 10.1111/1759-7714.13329

Circ‐IGF1R inhibited migration of lung cancer A549 and PC9 cell lines. The mRNA expression of circ‐IGF1R was detected using qRT‐PCR after overexpression or interference with circ‐IGF1R in lung cancer A549 ( a ) and PC9 ( b ). A wound‐healing assay was performed in A549 ( c ) and PC9 cells ( d ) after overexpressing circ‐IGF1R. ( P = 0.0017 vs. NC; and P = 0.0037 vs. NC). Cell migration was detected using a wound‐healing assay after interference with circ‐IGF1R in A549 ( e ) and PC9 ( f ) cell lines ( P = 0.0006 vs. siNC; and P = 0.0001 vs. siNC).
Figure Legend Snippet: Circ‐IGF1R inhibited migration of lung cancer A549 and PC9 cell lines. The mRNA expression of circ‐IGF1R was detected using qRT‐PCR after overexpression or interference with circ‐IGF1R in lung cancer A549 ( a ) and PC9 ( b ). A wound‐healing assay was performed in A549 ( c ) and PC9 cells ( d ) after overexpressing circ‐IGF1R. ( P = 0.0017 vs. NC; and P = 0.0037 vs. NC). Cell migration was detected using a wound‐healing assay after interference with circ‐IGF1R in A549 ( e ) and PC9 ( f ) cell lines ( P = 0.0006 vs. siNC; and P = 0.0001 vs. siNC).

Techniques Used: Migration, Expressing, Quantitative RT-PCR, Over Expression, Wound Healing Assay

mRNAs regulated by circ‐IGF1R. ( a ) The cluster heat map showing the differentially expressed mRNAs. ( b ) A scatter plot was used to evaluate the variation in mRNA expression between ov‐NC and ov‐circ‐IGF1R of PC9 ( ) upregulated tags, ( ) downregulated tags, and ( ) Not DETs and A549 cells ( ) upregulated tags, ( ) downregulated tags, and ( ) Not DETs. ( c ) Venn diagrams show common differentially expressed mRNAs between ov‐NC and ov‐circ‐IGF1R in PC9 and A549 cells.
Figure Legend Snippet: mRNAs regulated by circ‐IGF1R. ( a ) The cluster heat map showing the differentially expressed mRNAs. ( b ) A scatter plot was used to evaluate the variation in mRNA expression between ov‐NC and ov‐circ‐IGF1R of PC9 ( ) upregulated tags, ( ) downregulated tags, and ( ) Not DETs and A549 cells ( ) upregulated tags, ( ) downregulated tags, and ( ) Not DETs. ( c ) Venn diagrams show common differentially expressed mRNAs between ov‐NC and ov‐circ‐IGF1R in PC9 and A549 cells.

Techniques Used: Expressing

The expression level of VANGL2, miR‐127, CTNNB1, and vimentin in A549 and PC9 lung cancer cell lines was verified using qRT‐PCR and Western Blotting. The expression level of VANGL2 ( a ) is consistent with the overexpression and interference with circ‐IGF1R in A549 and PC9 lung cancer cell lines using qRT‐PCR ( a ) and western blotting ( b ). The expression level of miR‐1270 ( c ) is opposite to the overexpression and interference of circ‐IGF1R in A549 and PC9 lung cancer cell lines. The expression of CTNNB1 and vimentin was downregulated after overexpressing circ‐IGF1R in cells, while the expression of related proteins was upregulated after interference with circ‐IGF1R expression ( d ) ( ) NC, and ( ) OV‐circRNA; ( ) NC, and ( ) si‐circRNA; ( ) NC, and ( ) OV‐circRNA; ( ) NC, and ( ) si‐circRNA.
Figure Legend Snippet: The expression level of VANGL2, miR‐127, CTNNB1, and vimentin in A549 and PC9 lung cancer cell lines was verified using qRT‐PCR and Western Blotting. The expression level of VANGL2 ( a ) is consistent with the overexpression and interference with circ‐IGF1R in A549 and PC9 lung cancer cell lines using qRT‐PCR ( a ) and western blotting ( b ). The expression level of miR‐1270 ( c ) is opposite to the overexpression and interference of circ‐IGF1R in A549 and PC9 lung cancer cell lines. The expression of CTNNB1 and vimentin was downregulated after overexpressing circ‐IGF1R in cells, while the expression of related proteins was upregulated after interference with circ‐IGF1R expression ( d ) ( ) NC, and ( ) OV‐circRNA; ( ) NC, and ( ) si‐circRNA; ( ) NC, and ( ) OV‐circRNA; ( ) NC, and ( ) si‐circRNA.

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

Detection of circ‐IGF1R in lung cancer cells. ( a ) The relative expression levels of circ‐IGF1R in six cell lines. ( b ) Schematic demonstrating that circ‐IGF1R is produced at the IGF1R gene locus exon2. ( c ) qRT‐PCR assay with divergent or convergent primers affirming the existence of circ‐IGF1R in A549 cells. GAPDH was used as the negative control. ( d ) Sanger sequencing of circ‐IGF1R verified the back‐splice junction.
Figure Legend Snippet: Detection of circ‐IGF1R in lung cancer cells. ( a ) The relative expression levels of circ‐IGF1R in six cell lines. ( b ) Schematic demonstrating that circ‐IGF1R is produced at the IGF1R gene locus exon2. ( c ) qRT‐PCR assay with divergent or convergent primers affirming the existence of circ‐IGF1R in A549 cells. GAPDH was used as the negative control. ( d ) Sanger sequencing of circ‐IGF1R verified the back‐splice junction.

Techniques Used: Expressing, Produced, Quantitative RT-PCR, Negative Control, Sequencing

Circ‐IGF1R inhibited invasion of lung cancer A549 and PC9 cell lines. Invasion of cells after overexpression of circ‐IGF1R by Transwell assay in lung cancer A549 ( a ) and PC9 ( c ) cell lines was significantly inhibited ( P
Figure Legend Snippet: Circ‐IGF1R inhibited invasion of lung cancer A549 and PC9 cell lines. Invasion of cells after overexpression of circ‐IGF1R by Transwell assay in lung cancer A549 ( a ) and PC9 ( c ) cell lines was significantly inhibited ( P

Techniques Used: Over Expression, Transwell Assay

38) Product Images from "Apurinic endonuclease 1 promotes the cisplatin resistance of lung cancer cells by inducing Parkin-mediated mitophagy"

Article Title: Apurinic endonuclease 1 promotes the cisplatin resistance of lung cancer cells by inducing Parkin-mediated mitophagy

Journal: Oncology Reports

doi: 10.3892/or.2019.7345

Parkin-mediated mitophagy plays an important role in the APE1-induced cisplatin resistance of A549 cells. A549 cells were first transfected with an APE1 overexpression plasmid. Next, the APE1-overexpressing A549 cells were transfected with small interfering RNA (si-Parkin or si-NC) or treated with 3-MA. (A) A549 cells after the relevant transfections (APE1, si-Parkin or si-NC) and their indicated treatment were subjected to fractionation to obtain the cytosolic fraction. Western blot analysis was performed to analyze the levels of LC3, total cytochrome c , and Parkin in the cytosolic fraction. GAPDH was used as loading control. (B) Following the indicated transfection and treatment, the A549 cells were co-cultured with 3 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (C) Colony formation assays were performed to analyze the colony formation efficiency of the transfected A549 cells after they had received their indicated treatment. (D) The cell apoptosis rates are shown in a histogram. (E) The apoptosis rates of the transfected A549 cells treated or not with 3-MA were analyzed by flow cytometry. (F) The distribution of JC-1 in the transfected A549 cells was analyzed by flow cytometry. *P
Figure Legend Snippet: Parkin-mediated mitophagy plays an important role in the APE1-induced cisplatin resistance of A549 cells. A549 cells were first transfected with an APE1 overexpression plasmid. Next, the APE1-overexpressing A549 cells were transfected with small interfering RNA (si-Parkin or si-NC) or treated with 3-MA. (A) A549 cells after the relevant transfections (APE1, si-Parkin or si-NC) and their indicated treatment were subjected to fractionation to obtain the cytosolic fraction. Western blot analysis was performed to analyze the levels of LC3, total cytochrome c , and Parkin in the cytosolic fraction. GAPDH was used as loading control. (B) Following the indicated transfection and treatment, the A549 cells were co-cultured with 3 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (C) Colony formation assays were performed to analyze the colony formation efficiency of the transfected A549 cells after they had received their indicated treatment. (D) The cell apoptosis rates are shown in a histogram. (E) The apoptosis rates of the transfected A549 cells treated or not with 3-MA were analyzed by flow cytometry. (F) The distribution of JC-1 in the transfected A549 cells was analyzed by flow cytometry. *P

Techniques Used: Transfection, Over Expression, Plasmid Preparation, Small Interfering RNA, Fractionation, Western Blot, Cell Culture, CCK-8 Assay, Flow Cytometry, Cytometry

APE1 knockdown increases the cisplatin sensitivity of A549/DDP cells. A549/DDP cells were transfected with small interfering RNA (si-APE1 or si-NC). (A) Transfection efficiency was assessed by qPCR. (B) The levels of mitochondrial APE1 (m-APE1) in the transfected A549/DDP cells were analyzed by western blotting. COX4 was used as loading control for mitochondrial APE1. (C) The total APE1 (t-APE1) protein levels in transfected A549/DDP cells were analyzed by western blotting. GAPDH was used as loading control. (D) Immunofluorescence assays were performed to assess the total APE1 levels in A549/DDP cells after transfection. (E) Transfected A549/DDP cells were treated with 15 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (F) Colony formation assays were performed to analyze the colony formation efficiency of the transfected A549/DDP cells. (G) The transfected A549/DDP cells were treated with cisplatin, and their rate of apoptosis was analyzed by flow cytometry. (H) Flow cytometry was used to analyze the distribution of JC-1 after transfection. (I) The transfected A549/DDP cells were subjected to fractionation to obtain the cytosolic fraction. Western blot analysis was performed to analyze the levels of LC3, total cytochrome c , and Parkin. (J) The cell apoptosis rate is shown in a histogram. GAPDH was used as loading control. Data represent the results obtained from three independent experiments (mean ± SEM of triplicate samples). *P
Figure Legend Snippet: APE1 knockdown increases the cisplatin sensitivity of A549/DDP cells. A549/DDP cells were transfected with small interfering RNA (si-APE1 or si-NC). (A) Transfection efficiency was assessed by qPCR. (B) The levels of mitochondrial APE1 (m-APE1) in the transfected A549/DDP cells were analyzed by western blotting. COX4 was used as loading control for mitochondrial APE1. (C) The total APE1 (t-APE1) protein levels in transfected A549/DDP cells were analyzed by western blotting. GAPDH was used as loading control. (D) Immunofluorescence assays were performed to assess the total APE1 levels in A549/DDP cells after transfection. (E) Transfected A549/DDP cells were treated with 15 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (F) Colony formation assays were performed to analyze the colony formation efficiency of the transfected A549/DDP cells. (G) The transfected A549/DDP cells were treated with cisplatin, and their rate of apoptosis was analyzed by flow cytometry. (H) Flow cytometry was used to analyze the distribution of JC-1 after transfection. (I) The transfected A549/DDP cells were subjected to fractionation to obtain the cytosolic fraction. Western blot analysis was performed to analyze the levels of LC3, total cytochrome c , and Parkin. (J) The cell apoptosis rate is shown in a histogram. GAPDH was used as loading control. Data represent the results obtained from three independent experiments (mean ± SEM of triplicate samples). *P

Techniques Used: Transfection, Small Interfering RNA, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, CCK-8 Assay, Flow Cytometry, Cytometry, Fractionation

APE1 and Mia40 overexpression increases the cisplatin resistance of A549 cells. A549 cells were transfected with vectors containing an APE1 overexpression plasmid and Mia40 overexpression plasmid as indicated. (A) Transfection efficiency was assessed by qPCR. (B) Western blot analysis of the mitochondrial APE1 (m-APE1) protein levels in A549 cells after transfection. COX4 was used as loading control for mitochondrial APE1. (C) Western blot analysis was used to analyze the total APE1 (t-APE1) and Mia40 protein levels in A549 cells after transfection. GAPDH was used as a loading control. (D) Immunofluorescence assays were performed to assess the total APE1 levels in A549 cells after transfection. (E) The transfected A549 cells were treated with 0, 1, 2, 3, 4, 5, and 6 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (F) Colony-formation assays were performed to analyze the colony formation efficiency of the transfected A549 cells. *P
Figure Legend Snippet: APE1 and Mia40 overexpression increases the cisplatin resistance of A549 cells. A549 cells were transfected with vectors containing an APE1 overexpression plasmid and Mia40 overexpression plasmid as indicated. (A) Transfection efficiency was assessed by qPCR. (B) Western blot analysis of the mitochondrial APE1 (m-APE1) protein levels in A549 cells after transfection. COX4 was used as loading control for mitochondrial APE1. (C) Western blot analysis was used to analyze the total APE1 (t-APE1) and Mia40 protein levels in A549 cells after transfection. GAPDH was used as a loading control. (D) Immunofluorescence assays were performed to assess the total APE1 levels in A549 cells after transfection. (E) The transfected A549 cells were treated with 0, 1, 2, 3, 4, 5, and 6 µmol/l cisplatin for 24 h, and their viability was assessed by the CCK-8 assay. (F) Colony-formation assays were performed to analyze the colony formation efficiency of the transfected A549 cells. *P

Techniques Used: Over Expression, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Western Blot, Immunofluorescence, CCK-8 Assay

Cisplatin-resistant A549 cells exhibit high levels of APE1 and autophagy. (A) A549 cells were treated with 0, 1, 2, 3, 4, 5 and 6 µmol/l cisplatin for 24 h, and the A549/DDP cells were treated with 0, 10, 20, 30, 40, 50 and 60 µmol/l cisplatin for 24 h. Cell viability was assessed by the CCK-8 assay. (B) Western blot analysis was performed to analyze the levels of mitochondrial APE1 (m-APE1) protein in A549/DDP and A549 cells. COX4 was used as loading control for the mitochondrial APE1 protein. (C) Western blot analysis of the total APE1 (t-APE1) and Mia40 protein levels in A549/DDP and A549 cells. GAPDH was used as a loading control. (D) Western blot analysis of the LC3 protein levels in A549/DDP and A549 cells. (E and F) Immunofluorescence assays were performed to assess the total APE1 and total LC3 levels in A549 and A549/DDP cells. Data represent results obtained from three independent experiments (mean ± SEM of triplicate samples). ***P
Figure Legend Snippet: Cisplatin-resistant A549 cells exhibit high levels of APE1 and autophagy. (A) A549 cells were treated with 0, 1, 2, 3, 4, 5 and 6 µmol/l cisplatin for 24 h, and the A549/DDP cells were treated with 0, 10, 20, 30, 40, 50 and 60 µmol/l cisplatin for 24 h. Cell viability was assessed by the CCK-8 assay. (B) Western blot analysis was performed to analyze the levels of mitochondrial APE1 (m-APE1) protein in A549/DDP and A549 cells. COX4 was used as loading control for the mitochondrial APE1 protein. (C) Western blot analysis of the total APE1 (t-APE1) and Mia40 protein levels in A549/DDP and A549 cells. GAPDH was used as a loading control. (D) Western blot analysis of the LC3 protein levels in A549/DDP and A549 cells. (E and F) Immunofluorescence assays were performed to assess the total APE1 and total LC3 levels in A549 and A549/DDP cells. Data represent results obtained from three independent experiments (mean ± SEM of triplicate samples). ***P

Techniques Used: CCK-8 Assay, Western Blot, Immunofluorescence

39) Product Images from "mRNA display with library of even-distribution reveals cellular interactors of influenza virus NS1"

Article Title: mRNA display with library of even-distribution reveals cellular interactors of influenza virus NS1

Journal: Nature Communications

doi: 10.1038/s41467-020-16140-9

FASN is required for viral replication and regulated by NS1. a Interactions between NS1 protein and FASN were examined by endogenous immunoprecipitation (IP)-western. Three biological replicates were performed, and a representative experiment is shown. b The gene expression level and protein expression level of FASN was examined post-NS1 overexpression in 293T cells ( N = 4 biologically independent samples). c The effect of FASN on viral replication was examined using shRNA knock-down (KD) in A549 cells. The expression level of FASN upon KD was examined by western blot (upper panel). Control cells or KD cells were infected with WT WSN at MOI 0.1. The viral titer in supernatant were determined using TCID50 assays ( N = 3 biologically independent samples). d The effect of FASN on viral replication in A549 cells was examined using FASN inhibitors, including C75, Fasnall and GSK2195069. Indicated concentrations of inhibitors were used at the time of infection. The effect of inhibitor treatment on viral replication at 24-h post infection was examined by TCID50 assay ( N = 3 biologically independent samples). e The levels of newly synthesized fatty acids or cholesterol upon expression of indicated viral proteins were examined by GC/MS. Ctl is transfection with GFP-expressing vector ( N = 4 biologically independent samples). f Interactions between WT and mutant (R38A/K41A) NS1 protein with FASN were examined by Co-immunoprecipitation assay, using Strep-tagged NS1 and FLAG-tagged FASN in 293T cells. g Interactions between NS1 protein and endogenous FASN were examined in A549 cells, which were transduced to be over-expressing indicated proteins. NP and GFP were used as control. Three biological replicates were performed, and a representative experiment is shown. h The levels of newly synthesized fatty acids upon WT or mutant (R38A/K41A) NS1 protein expression were examined by GC/MS ( N = 4 for NS1 WT and mutant, N = 3 for NP, biologically independent samples). Data are presented as mean values +/− SD for all panels. * P
Figure Legend Snippet: FASN is required for viral replication and regulated by NS1. a Interactions between NS1 protein and FASN were examined by endogenous immunoprecipitation (IP)-western. Three biological replicates were performed, and a representative experiment is shown. b The gene expression level and protein expression level of FASN was examined post-NS1 overexpression in 293T cells ( N = 4 biologically independent samples). c The effect of FASN on viral replication was examined using shRNA knock-down (KD) in A549 cells. The expression level of FASN upon KD was examined by western blot (upper panel). Control cells or KD cells were infected with WT WSN at MOI 0.1. The viral titer in supernatant were determined using TCID50 assays ( N = 3 biologically independent samples). d The effect of FASN on viral replication in A549 cells was examined using FASN inhibitors, including C75, Fasnall and GSK2195069. Indicated concentrations of inhibitors were used at the time of infection. The effect of inhibitor treatment on viral replication at 24-h post infection was examined by TCID50 assay ( N = 3 biologically independent samples). e The levels of newly synthesized fatty acids or cholesterol upon expression of indicated viral proteins were examined by GC/MS. Ctl is transfection with GFP-expressing vector ( N = 4 biologically independent samples). f Interactions between WT and mutant (R38A/K41A) NS1 protein with FASN were examined by Co-immunoprecipitation assay, using Strep-tagged NS1 and FLAG-tagged FASN in 293T cells. g Interactions between NS1 protein and endogenous FASN were examined in A549 cells, which were transduced to be over-expressing indicated proteins. NP and GFP were used as control. Three biological replicates were performed, and a representative experiment is shown. h The levels of newly synthesized fatty acids upon WT or mutant (R38A/K41A) NS1 protein expression were examined by GC/MS ( N = 4 for NS1 WT and mutant, N = 3 for NP, biologically independent samples). Data are presented as mean values +/− SD for all panels. * P

Techniques Used: Immunoprecipitation, Western Blot, Expressing, Over Expression, shRNA, Infection, TCID50 Assay, Synthesized, Gas Chromatography-Mass Spectrometry, Transfection, Plasmid Preparation, Mutagenesis, Co-Immunoprecipitation Assay

40) Product Images from "Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection"

Article Title: Bacterial RecA Protein Promotes Adenoviral Recombination during In Vitro Infection

Journal: mSphere

doi: 10.1128/mSphere.00105-18

Relative viral replication and recombination in the presence of bacterial protein. Using primers specific to hexon sequence in HAdV-D19 (A and C) and HAdV-D29 (B and D), quantitative PCR was used to quantify total viral DNA from 1 to 8 days postinfection in PBS-treated versus E . coli K-12 lysate-treated C2BBe1 cells (A and B) and A549 cells (C and D). DNA quantity is graphed relative to the levels at 1 day postinfection. (E and F) C2BBe1 (E) and A549 (F) cells pretreated with PBS, K-12 lysate, DH5α lysate, or K-12 lysate depleted of RecA were coinfected with HAdV-D19 and HAdV-D29 and subjected to quantitative PCR at 5 to 8 days postinfection, with primers chosen to amplify only the HVL2 recombinant. Values that are significantly different ( P
Figure Legend Snippet: Relative viral replication and recombination in the presence of bacterial protein. Using primers specific to hexon sequence in HAdV-D19 (A and C) and HAdV-D29 (B and D), quantitative PCR was used to quantify total viral DNA from 1 to 8 days postinfection in PBS-treated versus E . coli K-12 lysate-treated C2BBe1 cells (A and B) and A549 cells (C and D). DNA quantity is graphed relative to the levels at 1 day postinfection. (E and F) C2BBe1 (E) and A549 (F) cells pretreated with PBS, K-12 lysate, DH5α lysate, or K-12 lysate depleted of RecA were coinfected with HAdV-D19 and HAdV-D29 and subjected to quantitative PCR at 5 to 8 days postinfection, with primers chosen to amplify only the HVL2 recombinant. Values that are significantly different ( P

Techniques Used: Sequencing, Real-time Polymerase Chain Reaction, Recombinant

Related Articles

Cell Culture:

Article Title: Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert
Article Snippet: .. A549 cells were seeded at a concentration of 2 × 105 cells mL− 1 in 6-well plates (Corning® Costar ®, New York, USA) and cultured in 2 mL Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FCS at 37 °C and 5% CO2. ..

Article Title: Candida albicans secreted aspartic proteases 4–6 induce apoptosis of epithelial cells by a novel Trojan horse mechanism
Article Snippet: .. A549 cells were cultured at 37°C in 24-well cell culture plates (Corning, Corning, NY, USA) either with or without 25 μM Sap 6. .. At time intervals, cells were trypsinized and mixed with an equal volume of 0.4% trypan blue and counted for staining using a hemacytometer (Hausser Scientific, Horsham, PA, USA).

Produced:

Article Title: Synergistic induction of cancer cell migration regulated by G?? and phosphatidylinositol 3-kinase
Article Snippet: .. For invasion assays, A549 cells were serum-starved for 12 h (1 × 105 ) and overlaid on top of a 24-well Trans-well plate (Corning Costar Corp., Cambridge, MA) containing artificial basement membrane produced by 1 mg/ml of Matrigel (BD Bioscience, San Jose, CA). ..

Concentration Assay:

Article Title: Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert
Article Snippet: .. A549 cells were seeded at a concentration of 2 × 105 cells mL− 1 in 6-well plates (Corning® Costar ®, New York, USA) and cultured in 2 mL Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FCS at 37 °C and 5% CO2. ..

Article Title: Tailoring Cell Morphomechanical Perturbations Through Metal Oxide Nanoparticles
Article Snippet: .. AFM Analysis Caco-2 and A549 cells were seeded in plastic Petri dishes (Corning) at a concentration of 105 cell/well and grown until a 70–80% of confluence. ..

Migration:

Article Title: Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways
Article Snippet: .. Transwell migration assay The effect of Ph on migration of A549 cells was further analyzed using Transwell chambers with 8-mm porous membrane (Corning, Corning, NY, USA). .. Cells were treated with PBS control and different concentrations (10, 20 and 40 µ M) of Ph for 24 h, and then loaded into the migration chamber at 1×105 .

Transwell Migration Assay:

Article Title: Phloretin induces apoptosis of non-small cell lung carcinoma A549 cells via JNK1/2 and p38 MAPK pathways
Article Snippet: .. Transwell migration assay The effect of Ph on migration of A549 cells was further analyzed using Transwell chambers with 8-mm porous membrane (Corning, Corning, NY, USA). .. Cells were treated with PBS control and different concentrations (10, 20 and 40 µ M) of Ph for 24 h, and then loaded into the migration chamber at 1×105 .

Modification:

Article Title: Expression profile analysis reveals that Aspergillus fumigatus but not Aspergillus niger makes type II epithelial lung cells less immunological alert
Article Snippet: .. A549 cells were seeded at a concentration of 2 × 105 cells mL− 1 in 6-well plates (Corning® Costar ®, New York, USA) and cultured in 2 mL Dulbecco’s Modified Eagle’s Medium (DMEM) with 10% FCS at 37 °C and 5% CO2. ..

Western Blot:

Article Title: Differential signaling mechanisms of HNP-induced IL-8 production in human lung epithelial cells and monocytes
Article Snippet: .. For Western blot analysis, A549 cells in 6-well plates (Corning Costar, Cambridge, MA) after reaching confluence or U937 cells in suspension (1×106 cells/mL) were serum-starved overnight. ..

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    Corning Life Sciences a549
    High miR-200a and low HGF expression promotes apoptosis, DNA double strand breaks, and inhibits the cloning rate of NSCLC cells following irradiation. (A and B) immunofluorescence detection of γ-H2AX foci in miR-200a transfected or siRNA HGF-transfected <t>A549</t> and H1299 cells treated with or without irradiation. Magnification, ×100. *P
    A549, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 92/100, based on 2 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Corning Life Sciences a549 nf κb luc reporter cells
    IKK2 inhibitors enhance GR activity. ( A ) IKK2 inhibitors enhance NF-κB repression by GR. The reporter cells were treated with 5 ng/mL IL-1β ± 0.5 nM Dex, and inhibitors as indicated for 18 h. Luciferase assays were performed, and NF-κB repression was calculated relative to the cells treated with IL1β. The synergy ratio is shown. ( B,C ) TPCA-1 enhances IL-6 release inhibition by GR. <t>A549</t> cells ( B ) or human airway smooth muscle cells ( C ) were treated as in ( A ), and the level of IL-6 in medium was assayed by ELISA. ( D ) IKK2 inhibitor enhances trans-activation by GR. A549 cells were transfected with <t>pGILZ-luc</t> plasmid for 48 h, and then2.5 nM Dex and TPCA-1 were added as indicated. After 18 h treatment, luciferase assays were performed. (E) A549 cells were treated with 0.8 μM TPCA-1 and 2.5 nM Dex for 18 h, and qPCR was used to measure GILZ expression. The relative mRNA levels are shown. *P
    A549 Nf κb Luc Reporter Cells, supplied by Corning Life Sciences, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    High miR-200a and low HGF expression promotes apoptosis, DNA double strand breaks, and inhibits the cloning rate of NSCLC cells following irradiation. (A and B) immunofluorescence detection of γ-H2AX foci in miR-200a transfected or siRNA HGF-transfected A549 and H1299 cells treated with or without irradiation. Magnification, ×100. *P

    Journal: Oncology Reports

    Article Title: MicroRNA-200a suppresses migration and invasion and enhances the radiosensitivity of NSCLC cells by inhibiting the HGF/c-Met signaling pathway

    doi: 10.3892/or.2018.6925

    Figure Lengend Snippet: High miR-200a and low HGF expression promotes apoptosis, DNA double strand breaks, and inhibits the cloning rate of NSCLC cells following irradiation. (A and B) immunofluorescence detection of γ-H2AX foci in miR-200a transfected or siRNA HGF-transfected A549 and H1299 cells treated with or without irradiation. Magnification, ×100. *P

    Article Snippet: A549 and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Corning, Inc., Corning, NY, USA) containing 10% certified fetal bovine serum (FBS)-heat inactivated (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (100 U/ml), and streptomycin (100 U/ml) and maintained in an incubator at 37°C with 5% CO2 in a humidified atmosphere.

    Techniques: Expressing, Clone Assay, Irradiation, Immunofluorescence, Transfection

    Downregulation of HGF expression inhibits NSCLC cell migration and invasion. (A) Western blotting and (B) quantitative analysis revealed that the expression level of HGF and its receptor c-Met were decreased in A549 and H1299 cells following the transfection of siRNA HGF. *P

    Journal: Oncology Reports

    Article Title: MicroRNA-200a suppresses migration and invasion and enhances the radiosensitivity of NSCLC cells by inhibiting the HGF/c-Met signaling pathway

    doi: 10.3892/or.2018.6925

    Figure Lengend Snippet: Downregulation of HGF expression inhibits NSCLC cell migration and invasion. (A) Western blotting and (B) quantitative analysis revealed that the expression level of HGF and its receptor c-Met were decreased in A549 and H1299 cells following the transfection of siRNA HGF. *P

    Article Snippet: A549 and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Corning, Inc., Corning, NY, USA) containing 10% certified fetal bovine serum (FBS)-heat inactivated (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (100 U/ml), and streptomycin (100 U/ml) and maintained in an incubator at 37°C with 5% CO2 in a humidified atmosphere.

    Techniques: Expressing, Migration, Western Blot, Transfection

    Upregulation of miR-200a reduces the migration and invasion of NSCLC cells. (A) Western blotting and (B) quantitative analysis revealed that the expression levels of HGF and its receptor c-Met were significantly decreased in A549 and H1299 cells following transfection of miR-200a. *P

    Journal: Oncology Reports

    Article Title: MicroRNA-200a suppresses migration and invasion and enhances the radiosensitivity of NSCLC cells by inhibiting the HGF/c-Met signaling pathway

    doi: 10.3892/or.2018.6925

    Figure Lengend Snippet: Upregulation of miR-200a reduces the migration and invasion of NSCLC cells. (A) Western blotting and (B) quantitative analysis revealed that the expression levels of HGF and its receptor c-Met were significantly decreased in A549 and H1299 cells following transfection of miR-200a. *P

    Article Snippet: A549 and H1299 cells were grown in Dulbecco's modified Eagle's medium (DMEM; Corning, Inc., Corning, NY, USA) containing 10% certified fetal bovine serum (FBS)-heat inactivated (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (100 U/ml), and streptomycin (100 U/ml) and maintained in an incubator at 37°C with 5% CO2 in a humidified atmosphere.

    Techniques: Migration, Western Blot, Expressing, Transfection

    Effect of CT on A549 lung epithelial cells infected with P. aeruginosa CF strains. Uninfected confluent 24 h-old A549 cells (Column 1) were infected with the above P. aeruginosa strains for 48 h. This resulted in the destruction of the A549 cell architecture and colonization of the remains by the bacteria (Column 3). When, A549 cells were incubated with bacteria and CT together, A549 cells were still adherent and maintained their confluence on well plates, with no evidence of bacterial colonization (Column 4), 48 h incubation with CT alone showed maintenance of almost full A549 confluence, (Column 5) similar to the untreated control (Column 2). Scale bar = 50 μm. Image shown is representative of all results, n = 3 experiments performed in biological replicate.

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Effect of CT on A549 lung epithelial cells infected with P. aeruginosa CF strains. Uninfected confluent 24 h-old A549 cells (Column 1) were infected with the above P. aeruginosa strains for 48 h. This resulted in the destruction of the A549 cell architecture and colonization of the remains by the bacteria (Column 3). When, A549 cells were incubated with bacteria and CT together, A549 cells were still adherent and maintained their confluence on well plates, with no evidence of bacterial colonization (Column 4), 48 h incubation with CT alone showed maintenance of almost full A549 confluence, (Column 5) similar to the untreated control (Column 2). Scale bar = 50 μm. Image shown is representative of all results, n = 3 experiments performed in biological replicate.

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Infection, Incubation

    Effect of pyocyanin and GSH on a marker of oxidative stress within A549 cells. The degree of oxidative stress in 72 h old pre-established A549 cells subjected to pyocyanin, pyocyanin + GSH or GSH over 3 and 5 days was examined using the CellROX oxidative stress assay. Extent of red color indicates level of oxidative stress. (B,G) : 72 h pre-established A549 were considerably stressed when incubated with pyocyanin for 3 and 5 days in comparison to control/untreated A549 cells (A,F) . (C,H) : Pyocyanin + GSH resulted in a considerable reduction in oxidative stress. (D,I) : GSH did not result in oxidative stress even after 5 days. (E,J) : When A549 cells were treated with pyocyanin + GSH followed by treatment with GSH every 24 h for 3 (72 h) and 5 days (120 h), a dramatic reduction in oxidative stress was noted compared to a single treatment with pyocyanin + GSH. Bar = 50 μm. Images shown are representative of results taken from n = 3 independent experiments. (K) Shows mean fluorescence intensity (MFI) of A549 cells after they were subjected to different treatment regimens. Pyocyanin-alone treated cells exhibited significantly higher (MFI = 16115) relative to control (MFI = 7732) and pyocyanin + single-GSH treatment (MFI = 11833) and GSH alone treatment (MFI = 7700). Whereas, pyocyanin treated A549 cells subjected to daily GSH treatment showed MFI = 8619 slightly higher (insignificant) than control (K) . ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Effect of pyocyanin and GSH on a marker of oxidative stress within A549 cells. The degree of oxidative stress in 72 h old pre-established A549 cells subjected to pyocyanin, pyocyanin + GSH or GSH over 3 and 5 days was examined using the CellROX oxidative stress assay. Extent of red color indicates level of oxidative stress. (B,G) : 72 h pre-established A549 were considerably stressed when incubated with pyocyanin for 3 and 5 days in comparison to control/untreated A549 cells (A,F) . (C,H) : Pyocyanin + GSH resulted in a considerable reduction in oxidative stress. (D,I) : GSH did not result in oxidative stress even after 5 days. (E,J) : When A549 cells were treated with pyocyanin + GSH followed by treatment with GSH every 24 h for 3 (72 h) and 5 days (120 h), a dramatic reduction in oxidative stress was noted compared to a single treatment with pyocyanin + GSH. Bar = 50 μm. Images shown are representative of results taken from n = 3 independent experiments. (K) Shows mean fluorescence intensity (MFI) of A549 cells after they were subjected to different treatment regimens. Pyocyanin-alone treated cells exhibited significantly higher (MFI = 16115) relative to control (MFI = 7732) and pyocyanin + single-GSH treatment (MFI = 11833) and GSH alone treatment (MFI = 7700). Whereas, pyocyanin treated A549 cells subjected to daily GSH treatment showed MFI = 8619 slightly higher (insignificant) than control (K) . ∗ P

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Marker, Incubation, Fluorescence

    Schematic diagram illustrating the mechanism of pyocyanin mediated H 2 O 2 production, conversion of GSH to GSSG via GPx and recycling of GSSG to GSH via GRed in A549 cell.

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Schematic diagram illustrating the mechanism of pyocyanin mediated H 2 O 2 production, conversion of GSH to GSSG via GPx and recycling of GSSG to GSH via GRed in A549 cell.

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques:

    Quantification of glutathione peroxidase, glutathione reductase, total glutathione, and glutathione disulphide in pre-established A549 cells. (A) A549 cells treated for 24 h with pyocyanin or pyocyanin + GSSG showed a significantly higher glutathione peroxidase (GPx) activity compared to control, pyocyanin + GSH, GSSG and GSH. (B) Glutathione reductase (GRed) activity of pyocyanin and pyocyanin + GSSG treated A549 cells was significantly lower compared to control, pyocyanin + GSH, GSSG and GSH-treated cells. (C) Total intracellular GSH levels of A549 cells exposed to pyocyanin and pyocyanin + GSSG were significantly lower compared to A549 cell exposure to control, pyocyanin + GSH or GSSG or GSH. (D) . Intracellular GSSG level was elevated in pyocyanin and pyocyanin + GSSG treated A549 cells in comparison to control, pyocyanin + GSH, GSSG or GSH-alone treatment. (A–C) ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Quantification of glutathione peroxidase, glutathione reductase, total glutathione, and glutathione disulphide in pre-established A549 cells. (A) A549 cells treated for 24 h with pyocyanin or pyocyanin + GSSG showed a significantly higher glutathione peroxidase (GPx) activity compared to control, pyocyanin + GSH, GSSG and GSH. (B) Glutathione reductase (GRed) activity of pyocyanin and pyocyanin + GSSG treated A549 cells was significantly lower compared to control, pyocyanin + GSH, GSSG and GSH-treated cells. (C) Total intracellular GSH levels of A549 cells exposed to pyocyanin and pyocyanin + GSSG were significantly lower compared to A549 cell exposure to control, pyocyanin + GSH or GSSG or GSH. (D) . Intracellular GSSG level was elevated in pyocyanin and pyocyanin + GSSG treated A549 cells in comparison to control, pyocyanin + GSH, GSSG or GSH-alone treatment. (A–C) ∗ P

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Activity Assay

    Effect of pyocyanin, GSSG, and GSH on A549 cell growth and confluence over different time periods (A) . A549 adherence and confluence in six-well plates at 24, 72, 120, and 168 h post-treatment, with example of adherence at 0 h. Pyocyanin considerably reduced the confluence of A549 and pyocyanin + GSSG did not increase A549 confluence; however, addition of pyocyanin + GSH resulted in an increase in A549 confluence. GSSG or GSH alone resulted in A549 confluence similar to that of the control. Scale bar = 50 μm. (B) A549 cells exposed to pyocyanin showed a significant decrease in cell number, to a level similar to that of pyocyanin + GSSG, while pyocyanin + GSH showed a significant increase in cell number, but still lower than the untreated control, GSSG and GSH-alone. (C) Resazurin assay showed pyocyanin exposed A549 cells rapidly decreased in viability to a level similar to pyocyanin + GSSG, whereas, A549 cells treated with pyocyanin + GSH showed a significant increase in viability. The percentage of live/respiring cells remained at more than 90% for untreated control, GSSG and GSH alone, from 0 to 168 h. (D) The percentage of A549 confluence over the 240 h period showed a significant increase in confluence for the control, GSH and pyocyanin + GSH-cells compared to pyocyanin alone and pyocyanin + GSSG, at all time-points. (B–D) ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Effect of pyocyanin, GSSG, and GSH on A549 cell growth and confluence over different time periods (A) . A549 adherence and confluence in six-well plates at 24, 72, 120, and 168 h post-treatment, with example of adherence at 0 h. Pyocyanin considerably reduced the confluence of A549 and pyocyanin + GSSG did not increase A549 confluence; however, addition of pyocyanin + GSH resulted in an increase in A549 confluence. GSSG or GSH alone resulted in A549 confluence similar to that of the control. Scale bar = 50 μm. (B) A549 cells exposed to pyocyanin showed a significant decrease in cell number, to a level similar to that of pyocyanin + GSSG, while pyocyanin + GSH showed a significant increase in cell number, but still lower than the untreated control, GSSG and GSH-alone. (C) Resazurin assay showed pyocyanin exposed A549 cells rapidly decreased in viability to a level similar to pyocyanin + GSSG, whereas, A549 cells treated with pyocyanin + GSH showed a significant increase in viability. The percentage of live/respiring cells remained at more than 90% for untreated control, GSSG and GSH alone, from 0 to 168 h. (D) The percentage of A549 confluence over the 240 h period showed a significant increase in confluence for the control, GSH and pyocyanin + GSH-cells compared to pyocyanin alone and pyocyanin + GSSG, at all time-points. (B–D) ∗ P

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Resazurin Assay

    Effect of pyocyanin and GSH on A549 cell eccentricity over different time periods, monitored using IncuCyte Zoom live cell imaging. (A) Quantification of the average eccentricity of A549 confluence over 120 h. Control (untreated), GSH alone and pyocyanin + 2600 μM GSH-treated cells showed stable eccentricity, and the difference is significant in comparison to pyocyanin alone at all time-points. (B) Example showing 72 h images of A549 adherence, confluence and eccentricity in 96-well plates grown under different conditions. In comparison to the control, GSH alone and the pyocyanin + 2600 μM GSH, the pyocyanin only treated A549 cells showed considerably change in cell morphology (eccentricity-red circle). (A) ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Effect of pyocyanin and GSH on A549 cell eccentricity over different time periods, monitored using IncuCyte Zoom live cell imaging. (A) Quantification of the average eccentricity of A549 confluence over 120 h. Control (untreated), GSH alone and pyocyanin + 2600 μM GSH-treated cells showed stable eccentricity, and the difference is significant in comparison to pyocyanin alone at all time-points. (B) Example showing 72 h images of A549 adherence, confluence and eccentricity in 96-well plates grown under different conditions. In comparison to the control, GSH alone and the pyocyanin + 2600 μM GSH, the pyocyanin only treated A549 cells showed considerably change in cell morphology (eccentricity-red circle). (A) ∗ P

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Live Cell Imaging

    Effect of pyocyanin and GSH on 72 h old pre-established A549 cells. (A) Phase contrast microscopy of 72 h pre-established A549 cells subjected to a one-time treatment with pyocyanin resulted in decrease in A549 adherence levels and confluence especially over both the 3 and 5 days periods, whereas treatment with pyocyanin + GSH resulted in decrease in adherence and confluence over 5 days. Red spots in images indicate empty spaces on well plate surfaces due to non-adherence of cells. Treatment of A549 cells with GSH alone resulted in a confluence level similar to that of the untreated control. Interestingly, pyocyanin + GSH at day 1, followed by daily treatment with GSH for 3 and 5 days resulted in increased recovery of A549 adherence and confluence compared to pyocyanin + one-time GSH treatment. Bar = 50 μm. (B) OD 570 nm of A549 cells quantified from microscopy images confirms that pyocyanin resulted in a significant decrease in A549 cell density over 5 days compared to control, GSH and pyocyanin + GSH at all three time points. Daily GSH treatment resulted in a significant increase in OD in comparison to a pyocyanin + single GSH treatment. (B) ∗ P

    Journal: Frontiers in Microbiology

    Article Title: Glutathione Enhances Antibiotic Efficiency and Effectiveness of DNase I in Disrupting Pseudomonas aeruginosa Biofilms While Also Inhibiting Pyocyanin Activity, Thus Facilitating Restoration of Cell Enzymatic Activity, Confluence and Viability

    doi: 10.3389/fmicb.2017.02429

    Figure Lengend Snippet: Effect of pyocyanin and GSH on 72 h old pre-established A549 cells. (A) Phase contrast microscopy of 72 h pre-established A549 cells subjected to a one-time treatment with pyocyanin resulted in decrease in A549 adherence levels and confluence especially over both the 3 and 5 days periods, whereas treatment with pyocyanin + GSH resulted in decrease in adherence and confluence over 5 days. Red spots in images indicate empty spaces on well plate surfaces due to non-adherence of cells. Treatment of A549 cells with GSH alone resulted in a confluence level similar to that of the untreated control. Interestingly, pyocyanin + GSH at day 1, followed by daily treatment with GSH for 3 and 5 days resulted in increased recovery of A549 adherence and confluence compared to pyocyanin + one-time GSH treatment. Bar = 50 μm. (B) OD 570 nm of A549 cells quantified from microscopy images confirms that pyocyanin resulted in a significant decrease in A549 cell density over 5 days compared to control, GSH and pyocyanin + GSH at all three time points. Daily GSH treatment resulted in a significant increase in OD in comparison to a pyocyanin + single GSH treatment. (B) ∗ P

    Article Snippet: Confluent A549 cells were harvested, were plated at final seed density 10 × 105 cells/mL into 24-well plates (Corning, Sydney, Australia).

    Techniques: Microscopy

    Cell harvesting efficiency. ( a ) Average cell harvests, expressed as percentages of cells captured for PANC1 ( n = 12), A375 ( n = 12), PC‐3 ( n = 25), A549 ( n = 12) and T24 ( n = 12). Counts of CFSE‐labeled fluorescent cells successfully removed from the device subsequent to separation and capture. The lines at 70% and 42% are the absolute ranges of percent capture in individual experiments. ( b ) Frequency distribution of percentages of cells harvested. ( c ) Representative images of tumor cells (MDA‐MB‐468 and MDA‐MB‐231) and contaminating leukocytes after harvesting and immune staining. Four panels showing composite and separate fluorescent channels for each type of staining, as noted on the images: DAPI, Keratin and CD45. ( d ) Numbers of nucleated blood cells present in harvest eluents. Graphical summary of numbers of nucleated blood cells present in the harvest eluent from six experiments. The numbers of eluted nucleated cells range from less than 200 to in excess of 5,000. The split scale on the diagram, and four of the six data points are below 1,000 residual cells.

    Journal: International Journal of Cancer

    Article Title: A novel microfluidic platform for size and deformability based separation and the subsequent molecular characterization of viable circulating tumor cells

    doi: 10.1002/ijc.30007

    Figure Lengend Snippet: Cell harvesting efficiency. ( a ) Average cell harvests, expressed as percentages of cells captured for PANC1 ( n = 12), A375 ( n = 12), PC‐3 ( n = 25), A549 ( n = 12) and T24 ( n = 12). Counts of CFSE‐labeled fluorescent cells successfully removed from the device subsequent to separation and capture. The lines at 70% and 42% are the absolute ranges of percent capture in individual experiments. ( b ) Frequency distribution of percentages of cells harvested. ( c ) Representative images of tumor cells (MDA‐MB‐468 and MDA‐MB‐231) and contaminating leukocytes after harvesting and immune staining. Four panels showing composite and separate fluorescent channels for each type of staining, as noted on the images: DAPI, Keratin and CD45. ( d ) Numbers of nucleated blood cells present in harvest eluents. Graphical summary of numbers of nucleated blood cells present in the harvest eluent from six experiments. The numbers of eluted nucleated cells range from less than 200 to in excess of 5,000. The split scale on the diagram, and four of the six data points are below 1,000 residual cells.

    Article Snippet: Cell capture We tested the Parsortix™ system's capability for epitope independent separation by performing spiking experiments with different cell numbers (10, 50 and 100; n = 4 for each spike level) from the cultured cancer cell lines PANC‐1, PC3, A375, A549 and T24.

    Techniques: Cell Harvesting, Labeling, Multiple Displacement Amplification, Staining

    IKK2 inhibitors enhance GR activity. ( A ) IKK2 inhibitors enhance NF-κB repression by GR. The reporter cells were treated with 5 ng/mL IL-1β ± 0.5 nM Dex, and inhibitors as indicated for 18 h. Luciferase assays were performed, and NF-κB repression was calculated relative to the cells treated with IL1β. The synergy ratio is shown. ( B,C ) TPCA-1 enhances IL-6 release inhibition by GR. A549 cells ( B ) or human airway smooth muscle cells ( C ) were treated as in ( A ), and the level of IL-6 in medium was assayed by ELISA. ( D ) IKK2 inhibitor enhances trans-activation by GR. A549 cells were transfected with pGILZ-luc plasmid for 48 h, and then2.5 nM Dex and TPCA-1 were added as indicated. After 18 h treatment, luciferase assays were performed. (E) A549 cells were treated with 0.8 μM TPCA-1 and 2.5 nM Dex for 18 h, and qPCR was used to measure GILZ expression. The relative mRNA levels are shown. *P

    Journal: Scientific Reports

    Article Title: A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor

    doi: 10.1038/s41598-017-07565-2

    Figure Lengend Snippet: IKK2 inhibitors enhance GR activity. ( A ) IKK2 inhibitors enhance NF-κB repression by GR. The reporter cells were treated with 5 ng/mL IL-1β ± 0.5 nM Dex, and inhibitors as indicated for 18 h. Luciferase assays were performed, and NF-κB repression was calculated relative to the cells treated with IL1β. The synergy ratio is shown. ( B,C ) TPCA-1 enhances IL-6 release inhibition by GR. A549 cells ( B ) or human airway smooth muscle cells ( C ) were treated as in ( A ), and the level of IL-6 in medium was assayed by ELISA. ( D ) IKK2 inhibitor enhances trans-activation by GR. A549 cells were transfected with pGILZ-luc plasmid for 48 h, and then2.5 nM Dex and TPCA-1 were added as indicated. After 18 h treatment, luciferase assays were performed. (E) A549 cells were treated with 0.8 μM TPCA-1 and 2.5 nM Dex for 18 h, and qPCR was used to measure GILZ expression. The relative mRNA levels are shown. *P

    Article Snippet: For screening, A549/NF-κB-luc reporter cells were seeded into two 384-well white plates (Corning) at 4000 cells per well in 30 μL of DMEM/F12(1:1) medium without red dye, supplemented with 3% dialyzed FBS.

    Techniques: Activity Assay, Luciferase, Inhibition, Enzyme-linked Immunosorbent Assay, Activation Assay, Transfection, Plasmid Preparation, Real-time Polymerase Chain Reaction, Expressing

    A high-through chemical screen to identify GR modulators. ( A ) Establishment of a stable reporter cell line A549/NF-κB-luc to assay tethered trans-repression of NF-κB activation by GR. A plasmid in which five tandem NF-κB responsive elements (5 × NF-κB-RE) was put upstream of mini promoter (MP) to control Luc2P expression was stably transfected into A549 cells. The individual colonies were selected, expanded and characterized. ( B ) A549/NF-κB-luc reporter cells were sensitive to IL-1β in NFκB activation and sensitive to Dex in NF-κB repression by GR. Cells were treated with 5 ng/mL IL-1β ± Dex as indicated for 18 h and Luciferase assay was performed. ( C ) Schematic representation of the chemical screen design. A549/NF-κB-luc reporter cells were seeded in 384 well plates, and library compounds were added to the cell plates. One hour later, 5 ng/mL IL-1β was added to assay I plates and 5 ng/mL IL-1β + 2.5 nM Dex was added to assay II plates. Luciferase assay was performed after 18 h treatment and MAD based Z scores for each well and ratio II/I of each tested compound were calculated. The inhibitor and enhancer hits were selected based on predefined criteria (see Results ). ( D ) Summary of the GR inhibitors and enhancers identified in the screen.

    Journal: Scientific Reports

    Article Title: A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor

    doi: 10.1038/s41598-017-07565-2

    Figure Lengend Snippet: A high-through chemical screen to identify GR modulators. ( A ) Establishment of a stable reporter cell line A549/NF-κB-luc to assay tethered trans-repression of NF-κB activation by GR. A plasmid in which five tandem NF-κB responsive elements (5 × NF-κB-RE) was put upstream of mini promoter (MP) to control Luc2P expression was stably transfected into A549 cells. The individual colonies were selected, expanded and characterized. ( B ) A549/NF-κB-luc reporter cells were sensitive to IL-1β in NFκB activation and sensitive to Dex in NF-κB repression by GR. Cells were treated with 5 ng/mL IL-1β ± Dex as indicated for 18 h and Luciferase assay was performed. ( C ) Schematic representation of the chemical screen design. A549/NF-κB-luc reporter cells were seeded in 384 well plates, and library compounds were added to the cell plates. One hour later, 5 ng/mL IL-1β was added to assay I plates and 5 ng/mL IL-1β + 2.5 nM Dex was added to assay II plates. Luciferase assay was performed after 18 h treatment and MAD based Z scores for each well and ratio II/I of each tested compound were calculated. The inhibitor and enhancer hits were selected based on predefined criteria (see Results ). ( D ) Summary of the GR inhibitors and enhancers identified in the screen.

    Article Snippet: For screening, A549/NF-κB-luc reporter cells were seeded into two 384-well white plates (Corning) at 4000 cells per well in 30 μL of DMEM/F12(1:1) medium without red dye, supplemented with 3% dialyzed FBS.

    Techniques: Activation Assay, Plasmid Preparation, Expressing, Stable Transfection, Transfection, Luciferase

    Validation of GR inhibitor hits. A549/NF-κB-luc reporter cells were treated with 5 ng/mL IL-1β ± 10 nM Dex and a pan-kinase inhibitor or kinase GSK3β inhibitors ( A ) or other compounds ( B ) as indicated. Luciferase assays were performed after 18 h treatment. The luciferase activity in control cells with 5 ng/mL IL-1β treatment was normalized to 1. The luciferase activity of cells treated with IL-1β + Dex and hits was calculated and compared to that of cells treated with IL-1β + Dex. *P

    Journal: Scientific Reports

    Article Title: A high-throughput chemical screen identifies novel inhibitors and enhancers of anti-inflammatory functions of the glucocorticoid receptor

    doi: 10.1038/s41598-017-07565-2

    Figure Lengend Snippet: Validation of GR inhibitor hits. A549/NF-κB-luc reporter cells were treated with 5 ng/mL IL-1β ± 10 nM Dex and a pan-kinase inhibitor or kinase GSK3β inhibitors ( A ) or other compounds ( B ) as indicated. Luciferase assays were performed after 18 h treatment. The luciferase activity in control cells with 5 ng/mL IL-1β treatment was normalized to 1. The luciferase activity of cells treated with IL-1β + Dex and hits was calculated and compared to that of cells treated with IL-1β + Dex. *P

    Article Snippet: For screening, A549/NF-κB-luc reporter cells were seeded into two 384-well white plates (Corning) at 4000 cells per well in 30 μL of DMEM/F12(1:1) medium without red dye, supplemented with 3% dialyzed FBS.

    Techniques: Luciferase, Activity Assay