Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

Journal: EMBO Molecular Medicine

Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

doi: 10.1038/s44321-024-00144-9

Figure Lengend Snippet: ( A ) Principal component analysis (PCA) plot for the sequenced samples of WT ( n = 6), KO ( n = 5), WT+NPCs ( n = 5) and KO+NPCs ( n = 6) cerebella. Percentage of variance is reported for both PC1 (first component) and PC2 (second component). ( B ) The number of deregulated genes (DEGs) for the different comparison is reported, considering a p.adj < 0.05. The number of DEGs with a LogFoldChange lower than −1 (down-regulated genes) or LogFoldChange greater than 1 (upregulated genes) is also indicated. ( C ) Dot plot of Gene Ontology (GO) enriched pathway analysis in the cerebellum, indicating the top 30 most enriched pathways of the comparison between KO+NPCs versus KO samples. For a description of the statistical method implemented in DESeq2 or ORA see Boyle et al and Yu et al , respectively. ( D ) The graph shows the LogFoldChange (LFC) of genes belonging to the GO pathway “Interferon-γ response” in the comparisons KO versus WT, WT + NPC versus WT, and KO+NPCs versus KO. ( E ) Gene set enrichment analysis (GSEA) of IFNγ response in cerebella, indicating a significant enrichment of the gene set in KO+NPCs vs KO comparison, as well as in KO+NPCs vs WT comparison. ( F ) The histogram reports the transcriptional levels of genes associated to IFNγ pathway. Data, expressed as percentage of WT, are shown as mean ± SEM. Parp9: * p = 0.0292 WT vs KO, § p = 0.0508 KO vs KO+NPCs; Iftm3: *** p = 0.0007 WT vs KO, *** p = 0.0008 KO vs KO+NPCs; Irf8: *** p = 0.0002 KO vs KO+NPCs; Bst2: * p = 0.0274 WT vs KO, *** p = 0.0010 KO vs KO+NPCs by two-way ANOVA followed by Sidak’s post-hoc test. n = 5 WT, n = 6 WT+NPCs, n = 5 KO, n = 6 KO+NPCs. ( G ) Western blot analysis of phosphorylated STAT1 (P-STAT1) in WT or KO neurons cultured with NPCs (for 14 days) ( n = 7 mouse embryos/genotype). Data are represented as mean ± SEM and expressed as percentage of WT neurons cultured alone. * p = 0.0242 by two-way ANOVA followed by Sidak’s post hoc test. Representative bands of P-STAT1 and the corresponding lanes of TGX-stain free gel are reported. .

Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

Techniques: Comparison, Western Blot, Cell Culture, Staining

Journal: EMBO Molecular Medicine

Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

doi: 10.1038/s44321-024-00144-9

Figure Lengend Snippet: ( A ) The histogram represents the cell survival (% untreated WT neurons) ± SEM, assessed by MTT assay. IFNγ was added for 24 h in DIV13 primary neurons and three doses were tested: 25, 75, and 100 ng/ml. ( B ) The histogram reports the mean ± SEM of the levels of phosphorylated STAT1 after IFNγ treatment. Data are normalized to total protein content, visualized by a TGX stain-free technology. Representative bands of phosphorylated STAT1, and the corresponding lanes of TGX-stain-free gel, in WT and KO neurons treated or not with IFNγ are depicted. § p = 0.0003 WT-UT vs WT 100 ng/ml and § p < 0.0001 for the other comparisons, by two-way ANOVA followed by Tukey’s post-hoc test. § denotes a significant difference respect to the corresponding untreated control of the same genotype. WT and KO neurons derived from 3 different mice/genotype.

Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

Techniques: MTT Assay, Staining, Control, Derivative Assay

Journal: EMBO Molecular Medicine

Article Title: Neural precursor cells rescue symptoms of Rett syndrome by activation of the Interferon γ pathway

doi: 10.1038/s44321-024-00144-9

Figure Lengend Snippet: Reagents and tools table

Article Snippet: Membranes were incubated for 1 h in blocking solution [5% BSA in 0.1% Tween-20 in Tris-buffered saline containing (TBST)], and incubated overnight at 4 °C with the following primary antibodies: rabbit anti-phosphorylated Tyr701 Stat1 (clone 58D6, 1:1000; #9167, Cell Signalling; RRID:AB_561284) or rabbit anti-Bdnf (1:1000; ab108319, Abcam; RRID:AB_10862052).

Techniques: Modification, Isolation, Amplification, SYBR Green Assay, Recombinant, Plasmid Preparation, Purification, Sequencing, Software, Cell Culture, Membrane, Staining

Journal: PLoS ONE

Article Title: SLFN11 can sensitize tumor cells towards IFN-γ-mediated T cell killing

doi: 10.1371/journal.pone.0212053

Figure Lengend Snippet: A) Schematic overview of experimental design in B. B) Growth kinetics of cells left untreated or treated with 10 ng/ml of IFN-γ for 24 h, in the presence or absence of Q-VD-OPh. C). Western blot analysis of phosphorylated STAT1 in HAP1 cells transduced with a control lentiviral vector or with lentiviral vectors encoding independent SLFN11-targeting shRNA, either left untreated or exposed to 10 ng/ml of IFN-γ for 24h. D) IRF1 transcript levels following exposure to 10 ng/ml of IFN-γ for the indicated times in parental, IFNGR1 KO, and SLFN11 KO cells.

Article Snippet: The following antibodies were used for western blot analysis: anti-HSP90: H114 (SantaCruz); anti-TUBA1A: 2144s (Cell Signaling Technology); anti-phosphorylated STAT1 7649s (Cell Signaling Technology); anti γ-H2AX 2577s (Cell Signaling Technology); anti-phosphorylated CHK1 12302s (Cell Signaling Technology); anti-phosphorylated ATM 10H11.E12 (Millipore); anti-SLFN11 HPA023030 (Atlas).

Techniques: Western Blot, Transduction, Control, Plasmid Preparation, shRNA

Journal: Journal of Advanced Research

Article Title: Rational design of synthetically tractable HDAC6/HSP90 dual inhibitors to destroy immune-suppressive tumor microenvironment

doi: 10.1016/j.jare.2022.06.009

Figure Lengend Snippet: Effect of compound 17 on expression of PD-L1 and IDO in colorectal cancer cell lines induced by IFN-γ. The cells were treated with IFN-γ alone or in combination with Compound 17 at the indicated concentration for 48 h. (A) PD-L1, IDO and STAT1 pathway inhibition analyzed by Western blot analysis. β-Actin was used as a loading control. (B and C) PD-L1 cell surface expression analyzed by flow cytometry. The MFI ratio= (PD-L1 MFI/isotype MFI). The data are presented as mean ± S.D. obtained from three independent experiments. *P < 0.05；**P < 0.01；***P < 0.001 compared with the IFN-γ alone group.

Article Snippet: Nitrocellulose membranes were then incubated with primary antibodies against the following antigens: HSP90, Src, AKT, Rb, phosphorylated Rb (p-Rb), FAK, HSP70, α-tubulin acetylation, acetyl histone H3, phosphorylated STAT1 (p-STAT1), STAT1 (Cell signaling, Danvers, MA, USA), α-tubulin, Histone H3 (Genetex, Irvine, USA), IDO (Santa Cruz Biotechnology Dallas, TX, USA) and PD-L1 (Novus, Littleton, CO, USA) at 4°C for overnight.

Techniques: Expressing, Concentration Assay, Inhibition, Western Blot, Control, Flow Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus-enhanced Adhesive Hydrogels.

doi: 10.1002/advs.202410769

Figure Lengend Snippet: Scheme 1. Schematic representation of siRNA@FA-EVs/GS hydrogel inhibited M1 macrophage polarization and pro-inflammatory cytokines production through the inhibition of STAT1 phosphorylation, which reduced the transformation of endometrial stromal cells into myofibroblasts and collagen deposition, and inhibited the formation of intrauterine adhesion.

Article Snippet: The anti-phosphorylated STAT1 (pSTAT1) and anti-CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Inhibition, Phospho-proteomics, Transformation Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus-enhanced Adhesive Hydrogels.

doi: 10.1002/advs.202410769

Figure Lengend Snippet: Figure 1. A, B) Heatmap analysis of proteomics and transcriptomic dataset. C) Volcano plot exhibited DEGs and DEPs in the transcriptomic dataset and proteomics dataset. D) Gene set enrichment analysis (GSEA) of genes altered GO and KEGG pathway analysis. E) Venn diagram analysis of the intersection of transcriptomic data and proteomics data. F) Immunofluorescence co-localization of pSTAT1 in M1 (CD86-labeled) and M2 (Arg1-labeled) macrophages in IUA. G) STAT1 binding sites in the promoter region of TNF-𝛼and IL-1𝛽. H) The concentration of TNF-𝛼and IL-1𝛽in the disease course (0, 1, 3, 7, 14, 28 days after surgery), Data are presented as mean ± SD, n = 3.

Article Snippet: The anti-phosphorylated STAT1 (pSTAT1) and anti-CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Labeling, Binding Assay, Concentration Assay

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus-enhanced Adhesive Hydrogels.

doi: 10.1002/advs.202410769

Figure Lengend Snippet: Figure 5. A,B) The phosphorylation level of STAT1 assessed by fluorescence microscopy and flow cytometric assays. C) The fluorescence intensity ratio of pSTAT1 in the nucleus of macrophages. D) The percentage of pSTAT1-positive macrophages was detected by flow cytometry. E) Schematic representation of siRNA@FA-EVs/GS inhibited M1 macrophage polarization by inhibiting STAT1 phosphorylation. Data are presented as mean ± SD. *p < 0.05 compared to the control group, **p < 0.05 compared to the LPS+GS group, ***p < 0.05 compared to the LPS+siRNA@EVs/GS group, n = 6.

Article Snippet: The anti-phosphorylated STAT1 (pSTAT1) and anti-CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Phospho-proteomics, Microscopy, Cytometry, Control

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Gene Therapy for Inflammatory Cascade in Intrauterine Injury with Engineered Extracellular Vesicles Hybrid Snail Mucus-enhanced Adhesive Hydrogels.

doi: 10.1002/advs.202410769

Figure Lengend Snippet: Figure 10. A,B) Heatmap and volcano plots of DEGs. C) GSEA analysis was applied to perform function annotation. D) The normalized count values of the STAT1, IL-1𝛽, and TNF-𝛼E) The protein bands of pSTAT1 and CD86. F) The immunofluorescence images of pSTAT1/CD86 in IUA tissues. G) The percentage of pSTAT1/CD86 positive cells evaluated by immunofluorescence staining in each group. H, I) The protein expression of pSTAT1 and CD86 was detected. Data are presented as mean ± SD. *p < 0.05 compared with IUA+GS; **p < 0.05 compared with IUA+siRNA@EVs/GS group, ns, no significance between two groups, n = 6.

Article Snippet: The anti-phosphorylated STAT1 (pSTAT1) and anti-CD86 were bought from HUABIO (Hangzhou, China).

Techniques: Staining, Expressing

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular bacterial infection-induced IFN-gamma is critically but not solely dependent on Toll-like receptor 4-myeloid differentiation factor 88-IFN-alpha beta-STAT1 signaling.

doi: 10.4049/jimmunol.172.10.6345

Figure Lengend Snippet: FIGURE 4. IFN-R and IFN-R signaling completely account for STAT1 phosphorylation in C. pneumoniae-infected BMM. WT, IFN- R/, and IFN-R/ BMM were infected with C. pneumoniae. Protein extracts were pre- pared at the indicated time points after infection and separated by SDS-PAGE, electroblotted onto nitrocellulose membranes, and immunoblotted with Abs that specifically recognize actin, total STAT1, and phosphorylated STAT1. Abs were detected with HRP-conjugated anti-IgG, followed by ECL detection. A representative from three in- dependent experiments is shown.

Article Snippet: Samples were then transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V, 250 mA for 1 h. Immunostaining was performed using polyclonal rabbit anti-I- B- , monoclonal mouse antiphosphorylated (Ser32/36) I- B- , polyclonal rabbit anti-phosphorylated (Tyr701) STAT1, polyclonal rabbit anti-STAT1 (all 1/1000 dilution; Cell Signaling, Beverly, MA), and polyclonal rabbit anti-actin (1/500 dilution; Sigma-Aldrich).

Techniques: Phospho-proteomics, Infection, SDS Page

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular bacterial infection-induced IFN-gamma is critically but not solely dependent on Toll-like receptor 4-myeloid differentiation factor 88-IFN-alpha beta-STAT1 signaling.

doi: 10.4049/jimmunol.172.10.6345

Figure Lengend Snippet: FIGURE 5. STAT1 is necessary for enhanced IFN- mRNA levels and controls growth of C. pneumoniae in BMM. A and B, WT and STAT1/

Article Snippet: Samples were then transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V, 250 mA for 1 h. Immunostaining was performed using polyclonal rabbit anti-I- B- , monoclonal mouse antiphosphorylated (Ser32/36) I- B- , polyclonal rabbit anti-phosphorylated (Tyr701) STAT1, polyclonal rabbit anti-STAT1 (all 1/1000 dilution; Cell Signaling, Beverly, MA), and polyclonal rabbit anti-actin (1/500 dilution; Sigma-Aldrich).

Techniques:

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular bacterial infection-induced IFN-gamma is critically but not solely dependent on Toll-like receptor 4-myeloid differentiation factor 88-IFN-alpha beta-STAT1 signaling.

doi: 10.4049/jimmunol.172.10.6345

Figure Lengend Snippet: FIGURE 6. Increased IL-15 mRNA accumulation in C. pneumoniae-infected BMM is dependent on signaling by IFN-R and STAT1. A, Total RNA was extracted from WT, IFN-R/, IFN-R/, and STAT1/ BMM at the indicated time points after infection with C. pneumoniae. The accu- mulation of IL-15 was visualized by RT-PCR. For the sake of clarity, only the C57BL/6 WT control is represented, although similar results were obtained with 129Sv/Ev WT BMM (data not shown). A representative from two independent experiments is shown. B–E, Increased IFN- mRNA accumulation in C. pneumoniae-infected BMM is cR dependent. RAG-1/ and RAG-1//cR/ BMM were infected with C. pneumoniae and lysed in SPG buffer at the indicated time points after infection. C. pneumoniae IFU in BMM lysates were quantified by HEp-2 infectivity assay. For each time point shown, bacterial levels were found to be at least 5 times higher in RAG-1//cR/ BMM compared with RAG-1/ BMM (B). Total RNA was extracted from RAG-1/ and RAG-1//cR/ BMM at the indicated time points after infection with C. pneumoniae (C–E). The accumulation of IFN- (C) and IFN- (D) mRNA was measured by a competitive PCR, while IL-1, IL-6, and TNF- were visualized by RT-PCR (E). A representative from three independent experiments is shown.

Article Snippet: Samples were then transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V, 250 mA for 1 h. Immunostaining was performed using polyclonal rabbit anti-I- B- , monoclonal mouse antiphosphorylated (Ser32/36) I- B- , polyclonal rabbit anti-phosphorylated (Tyr701) STAT1, polyclonal rabbit anti-STAT1 (all 1/1000 dilution; Cell Signaling, Beverly, MA), and polyclonal rabbit anti-actin (1/500 dilution; Sigma-Aldrich).

Techniques: Infection, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Intracellular bacterial infection-induced IFN-gamma is critically but not solely dependent on Toll-like receptor 4-myeloid differentiation factor 88-IFN-alpha beta-STAT1 signaling.

doi: 10.4049/jimmunol.172.10.6345

Figure Lengend Snippet: FIGURE 10. Molecular pathways controlling macrophage secretion of IFN- after infection with C. pneumoniae. BMM infection with C. pneu- moniae activates a TLR4-MyD88-dependent IFN- expression. IFN-- dependent, STAT1-mediated signaling is necessary for expression of IFN-. Activation of NF-B, which can occur in a TLR4-MyD88- and IFN--independent way, is also critical for IFN- induction.

Article Snippet: Samples were then transferred onto nitrocellulose membranes (Bio-Rad) by electroblotting at 100 V, 250 mA for 1 h. Immunostaining was performed using polyclonal rabbit anti-I- B- , monoclonal mouse antiphosphorylated (Ser32/36) I- B- , polyclonal rabbit anti-phosphorylated (Tyr701) STAT1, polyclonal rabbit anti-STAT1 (all 1/1000 dilution; Cell Signaling, Beverly, MA), and polyclonal rabbit anti-actin (1/500 dilution; Sigma-Aldrich).

Techniques: Infection, Expressing, Activation 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. 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: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

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: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

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: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

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. 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: After blocking, membranes were probed with primary antibodies against TLR2 (12276, Cell Signaling; RRID: AB_2797867 and ab108998, Abcam; RRID: AB_10861644), IjBa (sc371, SantaCruz; RRID: AB_10861644), Ser32/Ser36 phosphorylated IjBa (9246, Cell Signaling; RRID: AB_2267145), p65 (sc-8008, SantaCruz RRID: AB_628017), Ser536 phosphorylated p65 (3036, Cell Signaling; RRID: AB_331281), GR (PA1-511A, Thermo Fisher Scientific; RRID: AB_2236340), Tyr701 phosphorylated STAT1 (9167, Cell Signaling; RRID: AB_561284), Ser727 phosphorylated STAT1 (9177, Cell Signaling; RRID: AB_2197983), STAT1 (9172, Cell Signaling; RRID: AB_2198300), or GAPDH (MCA4739, Bio-Rad; RRID: AB_1720065).

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