Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: A549 cells were either not stimulated (NS) or treated with IL-1β (1 ng/ml) and/or budesonide (300 nM; Bud) for the indicated times. (A) RNA from 4 independent experiments was prepared prior to RNA sequencing and results for unstimulated cells (left panel) or following treatments for the indicated times (right panel) are presented as log 2 transcripts/million (tpm). (B) Following either no stimulation or treatment with IL-1β (1ng/ml) for the indicated times, cells from 4–5 independent experiments ( N ) were harvested for total protein and western blot analysis. All data are plotted mean ± SE or box-and-whisker plots. Using tpm or normalized IRF/GAPDH values, significance was tested by one-way ANOVA with Tukey’s post-hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 indicates significance relative to NS.

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: RNA Sequencing, Western Blot, Whisker Assay

Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: (A) A549 cells stably transfected with an IRF luciferase reporter were treated with increasing concentrations (0.01, 0.1, 1 or 10 nM) of; control siRNA pool (C4), the indicated IRF siRNA pool, or a lipid control (lipid) prior to either no stimulation or treatment with IL-1β (1 ng/ml). Cells were harvested at 0 h (i.e., no stimulation) for IRF2 and IRF3 protein or at 2 h for IRF1 protein, 24 h for IRF9 protein, and 8 h for luciferase determination. Blots representative of N independent experiments are shown. Following normalization to GAPDH, western blot data were plotted as log 2 IRF/GAPDH. IRF reporter data are plotted as log 2 fold of no stimulation. All data are shown as either mean ± SE or box-and-whisker plots. Using normalized IRF/GAPDH values or relative light units, significance was tested by one-way ANOVA with Tukey’s post-hoc test. * P ≤ 0.05, ** P ≤ 0.01 or *** P ≤ 0.001 indicates significance relative to IL-1β treated cells (IRF1, IRF9) or non-stimulated cells (IRF2, IRF3). (B) A549 cells were treated with IL-1β (1 ng/ml) at the indicated times prior to fractionation into ‘Cytoplasmic’ and ‘Nuclear’ lysates, or harvested for total protein (T), prior to western blotting using fluorescently tagged antibodies. Images are representative of 3 or more fluorescent, or equivalent chemiluminescent, blots.

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: Stable Transfection, Transfection, Luciferase, Control, Western Blot, Whisker Assay, Fractionation

Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: (A) A549 cells were either not stimulated (NS) or treated with 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3 and 10 ng/ml of IL-1β or 0.03, 0.1, 0.3, 1, 3, 10, 30 and 100 ng/ml TNFα for 2 h before harvesting for western blot analysis. (B) A549 or (C) BEAS-2B cells were either NS or treated with IL-1β (1 ng/ml), TNFα (10 ng/ml) and/or dexamethasone (1 μM; Dex) for 2 or 6 h as indicated prior to harvesting for western blot analysis. Data from N independent experiments are plotted mean ± SE or box-and-whisker plots. Using normalized IRF1/GAPDH values significance was tested by one-way ANOVA with Tukey’s post-hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 indicates significance relative to NS cells (black), or IL-1β/TNFα stimulated cells as indicated in red and yellow respectively.

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: Western Blot, Whisker Assay

Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: A549 cells were infected with Ad-IκBαΔN or control Ad-GFP at a MOI of 30 for 24 h prior to treatment with IL-1β (1 ng/ml) and/or budesonide (300 nM), as indicated. (A) Total protein was harvested at 2 h for western blot analysis of GFP, IκBαΔN, IRF1 and GAPDH. Representative blots are shown and data from N independent experiments are plotted log 2 fold (IRF1/GAPDH). (B) Cells were harvested at 2 h and RNA was extracted for qPCR analysis of IRF1 and GAPDH mRNA. Following normalization to GAPDH, data were expressed as log 2 fold of untreated. All data are shown as box-and-whisker plots. Using normalized IRF1/GAPDH values, significance was tested by one-way ANOVA with a Tukey’s post-hoc test. * P ≤ 0.05, *** P ≤ 0.001 indicates significance relative to the no-virus (naïve) control within each treatment group (not-stimulated, IL-1β, or IL-1β-Bud treated).

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: Infection, Control, Western Blot, Whisker Assay, Virus

Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: (A) A549 cells were either not treated or treated with lipid or lipid in the presence of 1 nM of control siRNA pool (C4) or p65 siRNA pool. After stimulation with IL-1β (1 ng/ml), total protein was harvested at 2 h for western blot analysis. Representative blots are shown. Following normalization to GAPDH, data were plotted as log 2 fold over not-stimulated (B) A549 cells were treated with 1 nM of each siRNA pool as in A prior to treatment with IL-1β (1 ng/ml) or TNFα (10 ng/ml) in the absence or presence of dexamethasone (1 μM, Dex), as indicated. Total RNA was extracted after 2 h for qPCR analysis of IRF1 and GAPDH. All data are shown as box-and-whisker plots. Using normalized IRF1/GAPDH values, significance was tested by one-way ANOVA with a Tukey’s post-hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 indicates significance relative to IL-1β-stimulated cells within each siRNA group (naïve, C4 or p65).

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: Control, Western Blot, Whisker Assay

Journal: PLOS One

Article Title: Inflammatory cytokines promote interferon regulatory factor (IRF) transcriptional activity in human pulmonary epithelial cells through the induction of IRF1 by nuclear factor-κB

doi: 10.1371/journal.pone.0329244

Figure Lengend Snippet: A549 cells were either incubated with DMSO at a final concentration of 0.3%, or incubated with TPCA-1, ML-120B or PS-1145, at ( A ) the indicated concentrations, or at ( B ) 30 μM, for 1.5 h prior to no stimulation (NS) or treatment with TNFα (10 ng/ml) or IL-1β (1 ng/ml) as indicated. After 2 h, cells were harvested for qPCR and western blot analysis of IRF1 and GAPDH. Data from N independent experiments were normalized to GAPDH and expressed as log 2 fold of untreated or as a % of IL-1β-treated. Data are plotted as box-and-whisker plots or mean ± SE. Using normalized gene/GAPDH values, significance was tested by one-way ANOVA with a Tukey’s post-hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 indicates significance relative to the no-inhibitor (naïve) control within each treatment group (NS, IL-1β or TNFα).

Article Snippet: Fluorescent antibodies were: GAPDH hFABTM Rhodamine (12004167, Bio-rad), Goat ANTI-RABBIT IgG StarBrightTM Blue (12004161, Bio-rad), and Goat anti Mouse IgG (H/L):DyLight®800 (SA5–10176, Bio-rad).

Techniques: Incubation, Concentration Assay, Western Blot, Whisker Assay, Control