Journal: Oncology Letters

Article Title: Antitumor activity of nimotuzumab in combination with cisplatin in lung cancer cell line A549 in vitro

doi: 10.3892/ol.2018.7923

Figure Lengend Snippet: Cyclin D1 expression in A549 cells in groups A-E following treatment for 48 h. Lanes: A, nimotuzumab; B, cisplatin; C, nimotuzumab followed by cisplatin; D, nimotuzumab and cisplatin simultaneously; and E, untreated control.

Article Snippet: Cell culture The A549 human lung adenocarcinoma epithelial cell line (supplied by the Central Laboratory of Qinhuangdao No. 1 People's Hospital) was cultured in Dulbecco's modified Eagle's medium (Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Zhejiang Tianhang Biotechnology Co., Ltd., Huzhou, China), 1 U/ml penicillin and 1 mg/ml streptomycin at 37°C, with 5% CO 2 and 95% humidity.

Techniques: Expressing, Control

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) Compound A chemical structure (B) A549 cells, transiently transfected with p(IL6κB) 3 50hu.IL6P-luc+, were treated with solvent (Solv), DEX (1µM) or CpdA (1µM or 10µM) for 2h. Subsequently, cells were induced with TNF (2000 IU/ml) for 6h where indicated. β-gal control-corrected results were presented as relative reporter gene activity with the condition Solv/TNF set at 100. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the TNF condition for selected conditions (*** p<0.001). (C) A549 cells, transiently transfected with p(GRE) 2 50-luc+, were treated with Solv, DEX (1µM) or CpdA (1µM or 10µM) for 8h. β-gal control-corrected results were presented as relative reporter gene activity with the condition Solv set at 1. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the Solv condition (ns not significant; *** p<0.001). Results are shown +/−SD. Figures in (B) and (C) are representative of at least 3 independent experiments. (D) Schematic model of gene modulatory GC and CpdA effects. While classic GCs can transactivate GRE-regulated genes and impede gene expression of specific target genes via a tethering transrepression mechanism (left panel), CpdA drives GR into a monomer formation that does not allow transcactivation of GRE-regulated gene expression. CpdA liganded GR can, however, still repress gene expression via transrepression (right panel).

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Transfection, Solvent, Control, Activity Assay, Comparison, Gene Expression

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells, starved for 48h, were pretreated for 1.5h with solvent (Solv), DEX (1µM), CpdA (10µM) or subjected to heat shock treatment (1h at 43°C and 30′ recovery at 37°C), ensued with TNF (2000IU/ml) for 5.5h. Isolated total RNA was subjected to RT-qPCR assaying IL8 mRNA levels, normalized to cyclophilin household gene mRNA levels. The TNF condition was set at 100 and results were recalculated accordingly. These results are representative of 2 independent experiments. Statistical analysis (ANOVA and Tukey multiple comparison post test) were performed for selected pair-wise comparisons. (B) A549 cells, starved for 48h, were pretreated for 2h with solvent (Solv), DEX (1µM) or CpdA (10µM), after which TNF(2000IU/ml) was added as indicated. Western blot analysis of total cells lysates detects IκBα protein, with NF-κB p65 as loading control. This figure is representative for 2 independent experiments. (C) A549 cells, starved for 48h, were untreated or heat-shocked at 43°C for 2h. Ensuing, cells were treated by TNF (2000IU/ml) for the indicated times. Total cell lysates were analyzed as in (B), with tubulin as loading control. Results were obtained on 2 separate blots in one experiment. (D) A549 cells, starved for 48h in Optimem, were pretreated for 2h with Solv, DEX (1µM), CpdA (10µM) or 2h heat shock (HS) at 43°C. Subsequently, TNF (2000IU/ml) was added for 30′, where indicated. After washing, fixation, and permeabilization, indirect immunofluorescence detects endogenous NF-κB p65. DAPI staining indicates the nuclei. Additionally, we present overlays. This figure is representative for 2 independent experiments. (E) ImageJ integrated density analysis of the TNF-treated conditions in (D) allows statistical analysis (Mann-Whitney U test) and we show the P-value of comparisons to the Solv/TNF condition. These results are representative of 2 independent experiments.

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Solvent, Isolation, Quantitative RT-PCR, Comparison, Western Blot, Control, Immunofluorescence, Staining, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells were transfected with siControl or siRNA targeting HSPA1A and HSPA1B (siHsp70). Total RNA or total protein extracts were prepared 48h post transfection. Purified mRNA was subjected to RT-qPCR detecting HSPA1A or HSPA1B gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S, as presented in the left panel. For siControl-transfected samples gene expression levels were set at 100%. SiHsp70-transfected results were recalculated accordingly. Statistical analysis (unpaired t-test) was performed to show significant difference between siControl and siHsp70 conditions (*** p<0.001). In the right panel, total cell lysates were subjected to Western blot analysis to detect Hsp70 protein levels. Detection of tubulin served as loading control. (B) In parallel with (A), A549 cells were transfected with siControl or siHsp70. 41h post transfection, cells were pretreated with Solv or CpdA (10µM) for 2h, after which ensued a 6h TNF (2000IU/ml) treatment. Purified mRNA was subjected to RT-qPCR detecting IL8 gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S. The condition Solv (siControl) was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference for selected pair wise comparisons (ns not significant; ** p<0.01; *** p<0.001).

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Transfection, Purification, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Comparison

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells were left untreated or were induced with heat shock (HS) at 43°C for 2h either or not followed by 4h recovery time (Rec) at 37°C. Isolated total RNA was subjected to RT-qPCR for the detection of HSPA1A, normalized to cyclophilin housekeeping control. The non-induced condition was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; *p<0.05; ** p<0.01). These results are representative of three independent experiments. (B) A549 cells, treated with solvent (Solv) or CpdA (10µM), were either incubated at 37°C for 6 hours or subjected to the following temperature protocol : 2 hours at 37°C (pre-induction), followed by 2 hours at 43°C heat shock (HS) and lastly 2 hours at 37°C (recovery; Rec). Isolated total RNA was subjected to RT-PCR for the detection of HSPA1A and control GAPDH gene expression levels. The displayed bands were detected from one single gel. The respective bands were quantified using ImageJ software and normalized to GAPDH control expression levels. Solv was set as 1 and all other conditions were recalculated relative to this condition and expressed as relative mRNA expression level. The figure is representative for 2 independent experiments. (C) Eight week old female BALB/c mice were injected intraperitoneally with either PBS as a control or CpdA dissolved in PBS (5mg/kg or 10mg/kg) and 24h later total skin samples were resected and their respective mRNA samples were subjected to RT-qPCR analysis assaying for HSPA1A gene expression levels and normalized to RPL13a, HMBS and ACTB housekeeping controls. The non-induced condition was set as 1 to allow ratio comparisons. Statistical analysis (Mann Whitney-U-test) was performed for selected pair wise comparisons (ns not significant; ** p<0.01).

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Isolation, Quantitative RT-PCR, Control, Comparison, Solvent, Incubation, Reverse Transcription Polymerase Chain Reaction, Gene Expression, Software, Expressing, Injection, MANN-WHITNEY

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells, starved for 48h, were left untreated or were pretreated for 30′ with cycloheximide (CHX)(20µg/ml), after which solvent (Solv) or CpdA (10µM) was added for 1.5h. Subsequently, cells were incubated with TNF(2000IU/ml) for 5h. Isolated total mRNA was subjected to RT-qPCR detecting IL8, normalized to GAPDH levels. Solv was set as 1 and other conditions were recalculated accordingly. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (**p<0.001; **p<0.001). This figure is representative for 2 independent experiments. (B) A549 cells were heat-shocked (HS) at 43°C for 2h either or not followed by recovery time (Rec) at 37°C. (C) A549 cells were treated with Solv or CpdA (10µM) for 6h or 24h or heat-shocked at 43°C for 2h, after which cells were left to recover at 37°C for 2h (HS+Rec). (B)(C) Total cell protein extracts were subjected to Western blot analysis detecting Hsp70, with NF-κB p65 or tubulin as loading controls. These images are representative for at least 3 independent experiments. (D) shows the averaged band densitometric analysis (ImageJ) of 8 independent Hsp70 Western blot analyses. Specific Hsp70 signal was corrected for sample loading. Solv was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; ***p<0.001). (E) A549 cells were treated with solvent or CpdA (10µM) for 4h,8h or 24h or heat-shocked at 43°C for 2h, after which cells were left to recover at 37°C for 2h or 4h(HS+Rec). Total cell protein lysates were analyzed via Hsp70 ELISA. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair-wise comparisons (ns not significant; ***p<0.001). This figure represents averaged data of 4 independent experiments.

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Solvent, Incubation, Isolation, Quantitative RT-PCR, Comparison, Western Blot, Enzyme-linked Immunosorbent Assay

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells were preinduced with Solv or MG132 (5µM) for 1.5 h. Subsequently, cells were stimulated with Solv or CpdA (10µM) for 6h or subjected to the following temperature protocol : 2 hours at 43°C followed by 2 hours at 37°C (HS+Rec), as indicated. Total cell protein extracts were subjected to Western blot analysis detecting inducible Hsp70. Detection of PARP served as a loading control. The displayed bands were detected from one single membrane. (B) A549 cells were starved for 48h in 0% DMEM, after which these cells were treated with solvent for 48h or Compound A (CpdA) (10µM) for 2h, 6h, 24h or 48h. Total cell protein extracts were subjected to Western blot analysis detecting β-catenin. Tubulin detection served as a loading control.

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Western Blot, Control, Membrane, Solvent

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) L929sA cells, stably transfected with a mHsp70i-luc reporter gene construct, were left untreated (Untr) or were stimulated with heat shock at 43°C for 2h, followed by a recovery period of 2h at 37°C (HS+Rec). Normalized luciferase levels were presented as relative reporter gene activity with the condition ‘Untr’ set as 1. Statistical analysis (unpaired t-test) was performed. This figure is representative for 6 independent experiments. (B) L929sA cells, stably transfected with mHsp70i-luc, were induced for 8h with various concentrations of CpdA, as indicated. All samples were controlled to a similar amount of solvent. This figure is representative for 4 independent experiments. (C) L929sA cells, stably transfected with mHsp70i-luc, were treated with solvent (Solv) or CpdA (10µM) for 6h, 24h or 48h. Data were presented as relative reporter gene activity with the Solv condition set as 1. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to explore if Solv differs from CpdA treatment for each respective induction time. (D) A549 cells, were treated with solvent or CpdA 10µM for the indicated time period. Total cellular mRNA was subjected to RT-qPCR detecting gene expression levels for HSPA1A, normalized using housekeeping 36B4 and β-actin mRNA levels. Four independent experiments with slightly varying time kinetics all show comparable results. (E) PC-3 cells were starved for 48h in 0% DMEM, after which these cells were left untreated or treated with Compound A (10µM), as indicated. Purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin, GAPDH and 36B4. (B)(D)(E) Solv condition was set as 1 and results recalculated accordingly. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to compare with Solv (ns not significant; ** p<0.01; *** p<0.001).

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Stable Transfection, Transfection, Construct, Luciferase, Activity Assay, Solvent, Comparison, Quantitative RT-PCR, Gene Expression, Purification

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells were transfected with siControl or siRNA targeting GR (siGR). Total RNA or total protein extracts were prepared 48h post-transfection. In the left panel, purified mRNA was subjected to RT-qPCR detecting GR gene expression levels, normalized to housekeeping controls cyclophilin and 28S. For the siControl-transfected sample, signal was set at 100%. Data from SiGR-transfected cells were recalculated accordingly. Statistical analysis (unpaired t-test) was performed to show significant difference between siControl and siGR conditions (*** p<0.001). In the right panel, total cell lysates were subjected to Western blot analysis to detect GR protein, with NF-κB p65 as a loading control. (B) In parallel with (A) A549 cells were transfected with siControl or siGR. 41h post transfection, cells were induced with Solv or CpdA (10µM) for 8h. The derived purified mRNA was subjected to RT-qPCR detecting HSPA1A gene expression levels and specific results were normalized to housekeeping controls cyclophilin and 28S. The condition Solv (siControl) was set as 1 to allow ratio comparisons. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed for selected pair wise comparisons (ns not significant; * p<0.05). This experiment is representative for 2 independent experiments. (C) and (D) A549 cells, serum-starved for48h in 0% DMEM, were treated with Solv, Dex (1µM), CpdA (10 µM) for 2h, or exposed to a 43°C heat shock (HS) for 1h. Total cell extracts were subjected to a ChIP assay targeting GR. Ensuing, qPCR signal of immunoprecipitated HSPA1A and GILZ gene promoter fragments is presented relative to input data. Binding to rabbit IgG represents aspecific binding. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the Solv condition (ns not significant; *** p<0.001). This experiment is representative for 2 independent experiments.

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Transfection, Purification, Quantitative RT-PCR, Gene Expression, Western Blot, Control, Derivative Assay, Comparison, Immunoprecipitation, Binding Assay

Journal: PLoS ONE

Article Title: Compound A, a Selective Glucocorticoid Receptor Modulator, Enhances Heat Shock Protein Hsp70 Gene Promoter Activation

doi: 10.1371/journal.pone.0069115

Figure Lengend Snippet: (A) A549 cells, starved for 48h in Optimem, were treated with solvent (Solv) or CpdA (10µM) for 4h or heat-shocked (HS) at 43°C for 1h. Via indirect immunofluorescence using an α-HSF1 Ab, endogenous HSF1 was visualized (green) and DAPI staining indicates the nuclei of the cells (blue). We also present an overlay and in the panel below, we digitally zoom in on one cell. White arrow heads indicate nuclear stress granules or foci of HSF1. This experiment is representative for 2 independent experiments. (B) A549 cells, starved for 48h in 0% DMEM, were treated with Solv, CpdA (10µM) or DEX (1µM) for 2h or HS at 43°C for 1h. Total cell extracts were subjected to a ChIP assay targeting HSF1. Ensuing, qPCR signal of immunoprecipitated HSPA1A gene promoter fragments is presented relative to input data. Binding to rabbit IgG represents aspecific binding. Statistical analysis (ANOVA with Tukey’s multiple comparison post test) was performed to show significant difference with the Solv condition (ns not significant; *** p<0.001). This experiment is representative for 2 independent experiments.

Article Snippet: A549 adenocarcinoma human alveolar basal epithelial cells were a kind gift from Dr. Ian Adcock .

Techniques: Solvent, Immunofluorescence, Staining, Immunoprecipitation, Binding Assay, Comparison

Journal: Journal of the Royal Society Interface

Article Title: Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

doi: 10.1098/rsif.2009.0161.focus

Figure Lengend Snippet: EFTEM analysis of yttrium as a constituent of the CNT pellet catalyst in an epithelial cell. (a) Overview of one A549 epithelial cell in the triple cell co-cultures with intracellular CNT pellet. (b) EFTEM detection of yttrium (red points).

Article Snippet: The A549 epithelial cell line ( Lieber et al . 1976 ) from the American Tissue Type Culture Collection (LGC Prochem, Molsheim, France) was used.

Techniques:

Journal: Journal of the Royal Society Interface

Article Title: Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

doi: 10.1098/rsif.2009.0161.focus

Figure Lengend Snippet: TEM pictures of the triple cell co-cultures with intracellular CNT pellet. Overview of the cell (left) and details of the part in white frame (right). (a) A549 epithelial cell, (b) MDM and (c) MDDC.

Article Snippet: The A549 epithelial cell line ( Lieber et al . 1976 ) from the American Tissue Type Culture Collection (LGC Prochem, Molsheim, France) was used.

Techniques:

Journal: Journal of the Royal Society Interface

Article Title: Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

doi: 10.1098/rsif.2009.0161.focus

Figure Lengend Snippet: TEM pictures of the triple cell co-cultures with intracellular DEPs. Overview of the cell (left) and details of the part in white frame (right). (a) A549 epithelial cell, (b) MDM and (c) MDDC.

Article Snippet: The A549 epithelial cell line ( Lieber et al . 1976 ) from the American Tissue Type Culture Collection (LGC Prochem, Molsheim, France) was used.

Techniques:

Journal: Journal of the Royal Society Interface

Article Title: Oxidative stress and inflammation response after nanoparticle exposure: differences between human lung cell monocultures and an advanced three-dimensional model of the human epithelial airways

doi: 10.1098/rsif.2009.0161.focus

Figure Lengend Snippet: TAC equivalent, TNF-α and IL-8 concentrations in monocultures and triple cell co-cultures following exposure to different nanosized particles for 24 h. All values are expressed as a percentage of control. (a) A549 epithelial cell monocultures ...

Article Snippet: The A549 epithelial cell line ( Lieber et al . 1976 ) from the American Tissue Type Culture Collection (LGC Prochem, Molsheim, France) was used.

Techniques: Control

Journal: Journal of Nanobiotechnology

Article Title: PINK1/TAX1BP1-directed mitophagy attenuates vascular endothelial injury induced by copper oxide nanoparticles

doi: 10.1186/s12951-022-01338-4

Figure Lengend Snippet: Toxic effects and mechanisms of CuONPs

Article Snippet: Intrinsiq Materials Ltd , 28.2 ± 13.7 nm , 10 μg/ml , 0, 1, 3, 6, 12, 24, 48 h , Alveolar epithelial cell line (A549) , Oxidative stress; Apoptosis , 10.1186/s12989-016-0160-6.

Techniques: In Vitro, Activation Assay