Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 3. Effect of IKK inhibitors on the enhancement of IFN-c1 Dex–induced TLR2 expression by IL-1b. (A) A549 cells containing the NF-jB reporter 6jBtk.luc were either not treated or pretreated with 1% DMSO or indicated concentrations of PS-1145, ML120B, or TPCA-1 for 90 minutes prior to stimulation with IL-1b (1 ng/ml). Cells were harvested for luciferase assay. Data from N independent experiments was expressed as fold (bars) and percent IL-1b (lines) and plotted as mean ± S.D. Scatterplots showing individual data points appear as Supplemental Fig. 4A. (B) A549 cells were pretreated with PS-1145, ML120B, or TPCA-1, each at 30 mM, for 1.5 hours prior to stimulation with IFN-c (10 ng/ml) plus Dex (1 mM) and IL-1b (1 ng/ml) as indicated. Cells were harvested at 6 hours for RNA extraction and quantitative polymerase chain reaction analysis of TLR2 and GAPDH mRNA. (C) Cells were treated as in (B) and harvested at 24 hours for western blot analysis of TLR2 and GAPDH protein. (B and C) TLR2 data, from N independent experiments, were normalized to GAPDH and are ex- pressed as mean ± S.D. and plotted as bar graphs overlaid with scatter plots. Significance was assessed by ANOVA with a Tukey multiple comparison test. Differences from untreated: *P # 0.05; **P # 0.01; ***P # 0.001; from IL-1b1IFN-c1Dex: #P # 0.05; ##P # 0.01; ###P # 0.001.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Expressing, Luciferase, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 4. Effect of Org34517 (Org) on the enhancement of IL-1b1IFN- c–induced TLR2 expression by dexamethasone. A549 cells were pre- treated with Org34517 (1 mM) for 1 hour prior to stimulation with IL- 1b (1 ng/ml) plus IFN-c (10 ng/ml) in the absence or presence of various concentrations of Dex. (A) After 6 hours, cells were harvested for RNA extraction and quantitative polymerase chain reaction analysis of TLR2 and GAPDH mRNA. (B) At 24 hours, cells were harvested for western blot analysis of TLR2 and GAPDH. In each case, TLR2 data, from N independent experiments, were normalized to GAPDH and are plotted as means ± S.D. Significance between treatments without and with Org was assessed by paired t test (two tailed). Note: unpaired t test was used at 0.1 mM Dex due to one sample failure in the western blotting. *P # 0.05; **P # 0.01; ***P # 0.001. For illustration, curves were fitted following Schild analysis that assumed a common maximal effect (Emax) and a Schild slope factor of 1, with the lower asymptote fixed as the response to IL-1b1IFN-c. This produced pA2 values of 8.07 (± 0.16) and 7.85 (± 0.29) for TLR2 mRNA and protein, respectively. Scatterplots for all data in (A) and (B) are shown as Supplemental Fig. 5, A and B.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Expressing, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot, Two Tailed Test, Produced

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 5. Effect of IL-1b, glucocorticoid, and IFN-c on STAT1 phosphorylation. A549 cells were either untreated or treated with IFN-c (10 ng/ml) prior to harvesting for western blot analysis at the indicated times. Representative blots of (A) phospho-STAT1 (P-STAT1 Y701) and GAPDH and (B) P-STAT1 (S727), total STAT1, and GAPDH from N independent experiments are shown. In each case, densitometric data normalized to GAPDH are plotted as means ± S.D. (error bars). Significance relative to untreated at 15 minutes was assessed by ANOVA with a Dunnett’s mul- tiple comparison test. (C) A549 cells were either not treated or treated with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 (Y701 and S727) and GAPDH. Representative blots of P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for P-STAT1 (Y701) (upper graph) and P-STAT1 (S727) (lower graph) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to un- treated (*) or IL-1b1IFN-c (#) was tested using ANOVA with a Tukey post hoc test. *, #P # 0.05; **, ##P # 0.01; ***, ###P # 0.001. Scatterplots for all data in (A) and (B) are shown as Supplemental Fig. 6.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Western Blot, Comparison

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 6. Effect of JAK inhibitors on STAT1 phosphorylation. (A) A549 cells were pretreated with AZD1480, barcitinib and ruxolitinib at the indi- cated concentrations for 30 minutes, followed by stimulation with IFN-c (10 ng/ml) for 1 hour. Cells were harvested for western blot analysis of phospho-STAT1 (P-STAT1) and GAPDH. Representative blots for P-STAT1 (Y701), P-STAT1 (S727), and GAPDH from five independent experi- ments are shown. (B) Densitometric data for Y701 P-STAT1 and S727 P-STAT1 from western blots shown in (A) were normalized to GAPDH and plotted as mean ± S.D. (C) Data were expressed as percent IFN-c effect for each of AZD1480, barcitinib, and ruxolitinib and plotted as means ± S.D. Log EC50 of each compound for the inhibition of P-STAT1 (Y701) and P-STAT1 (S727) is expressed as mean ± S.D. and shown as scatter plots in the inserts within each graph of (C). Note: one dataset was excluded for ruxolitinib, and the full data are shown as Supplemental Fig. 8. (D and E) A549 cells were either not treated or pretreated with AZD1480, barcitinib, and ruxolitinib, each at 1 mM, for 1 hour prior to stimulation with IL-1b (1 ng/ml) and IFN-c (10 ng/ml) as indicated. Cells were harvested at 1 hour for western blot analysis of P-STAT1 and GAPDH. Representative blots for P-STAT1 (Y701 & S727) and GAPDH from N independent experiments are shown. Densitometric data for (D) P-STAT (S727) and (E) P-STAT1 (Y701) were normalized to GAPDH, expressed as means ± S.D., and plotted as bar graphs overlaid with scatter plots. Significance relative to untreated (*), IFN-c (#), or IL-1b 1 IFN-c ($) was tested using ANOVA with a Tukey post hoc test. *, #, $P # 0.05; **, ##, $$P # 0.01; ***, ###, $$$P # 0.001.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Phospho-proteomics, Western Blot, Inhibition

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 7. Characterization of a STAT1-driven luciferase reporter. (A) A549 cells stably transfected with a luciferase reporter containing three copies of a consensus STAT1-binding motif [shown in (B)] were either not stimulated (NS) or stimulated with the indicated concentrations of IFN-c prior to harvesting for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. (B) (Upper panel) reporter construct showing three copies of the consensus/wild-type (WT; bold blue) or mutant (Mut; bold blue 1 red) STAT1-binding motif is shown. KpnI and XhoI restriction sites used to clone the construct in pGL3.TATA.neo vector are underlined, with cut sites shown as black arrowheads. (Lower panel) A549 cells were stably transfected with the WT or Mut STAT1 reporter constructs and either NS or treated with IFN-c (10 ng/ml) for 6 hours followed by harvesting for luciferase assay. Luciferase activity from N independent experiments was expressed as fold of NS, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Significance relative to untreated cells was deter- mined using paired t test. (C) A549 cells were either NS or incubated with lipid control (lip) alone or with the indicated concentrations of either control siRNA pool (C si-pool), or STAT1 siRNA pool (STAT1 si-pool) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of STAT1 and GAPDH from N independent experiments are shown. Data ob- tained from densitometry analysis were normalized to GAPDH, and mean ± S.D. was plotted as bar graphs overlaid with scatter plots. Signifi- cance relative to C si-pool at each siRNA concentration was tested by paired t test. *P # 0.05; **P # 0.01; ***P # 0.001. (D) A549 cells stably transfected with the WT STAT1 reporter construct were treated with lipid or siRNAs as described in (C) prior to stimulation with IFN-c (10 ng/ml) for 6 hours. Cells were harvested for luciferase assay, and raw luciferase units (RLU) from N independent experiments were expressed as mean ± S.D. and plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool at each siRNA concentration was tested by paired t test. (E) A549 cells stably transfected with the WT STAT1 reporter construct were either NS or pretreated with the indicated concentra- tions of AZD1480, barcitinib, or ruxolitinib for 30 minutes followed by stimulation with IFN-c (10 ng/ml). Cells were harvested for luciferase assay after 6 hours. Luciferase activity from N independent experiments was expressed as fold of NS and plotted as mean ± S.D. Scatterplots for all data in (A) and e are shown as Supplemental Fig. 9.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Luciferase, Stable Transfection, Transfection, Binding Assay, Activity Assay, Construct, Mutagenesis, Plasmid Preparation, Incubation, Control, Western Blot, Concentration Assay

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 8. Effect of JAK inhibitors on the enhancement of IL-1b1 Dex–induced TLR2 expression by IFN-c. A549 cells were either not stimulated or pretreated with AZD1480, barcitinib, or ruxolitinib, each at 1 mM, for 1 hour prior to stimulation with IL-1b (1 ng/ml) plus Dex (1 mM) and IFN-c (10 ng/ml) as indicated. (A) Cells were harvested at 6 hours for RNA extraction and quantitative polymerase chain reaction analysis of TLR2 and GAPDH mRNA. (B) Cells were harvested at 24 hours for western blot analysis of TLR2 and GAPDH protein. In each case, TLR2 data, from N independent experiments, were normalized to GAPDH, and mean ± S.D. were plotted as bar graphs overlaid with scatter plots. Significance was assessed by ANOVA with a Tukey multiple comparison test. Differences from untreated: **P # 0.01; ***P # 0.001; from IL-1b1IFN-c1Dex: ##P # 0.01; ###P # 0.001.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Expressing, RNA Extraction, Real-time Polymerase Chain Reaction, Western Blot, Comparison

Journal: Molecular pharmacology

Article Title: Synergy between Interleukin-1 β , Interferon- γ , and Glucocorticoids to Induce TLR2 Expression Involves NF- κ B, STAT1, and the Glucocorticoid Receptor.

doi: 10.1124/molpharm.123.000740

Figure Lengend Snippet: Fig. 9. Effect of p65, GR, and STAT1 knockdown on IL-1b, IFN-c, and dexamethasone-induced TLR2 expression in A549 cells. (A) A549 cells were incubated with pools of either control siRNAs (C si-pool), p65 siRNAs (p65 si-pool), GR siRNAs (GR si-pool), or STAT1 siRNAs (STAT1 si-pool) (1 nM each) for 48 hours. Cells were then incubated overnight with serum-free media prior to harvesting for western blot analysis. Representative blots of p65, GR, STAT1, and GAPDH from five independent experiments are shown. (B–E) A549 cells were incubated with siRNA pools as in (A), followed by treatment with IL-1b (1 ng/ml), IFN-c (10 ng/ml), and/or Dex (1 mM), as indicated, for 24 hours. Cells were harvested for western blot analysis. Representative blots of TLR2 and GAPDH from N independent experiments are shown. Densitometric data were normalized to GAPDH, expressed as fold of untreated, and mean ± S.D. were plotted as bar graphs overlaid with scatter plots. Significance relative to C si-pool in each case was tested using ANOVA with a Dunnett’s post hoc test. *P # 0.05; **P # 0.01; ***P # 0.001.

Article Snippet: A549 human pulmonary type II epithelial cells (American Type Culture Collection CCL-185; Research Resource Identifier: CVC_0023L) were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FBS and 2 mM L-glutamine (all Thermo Fisher Scientific).

Techniques: Knockdown, Expressing, Incubation, Control, Western Blot