Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: Canonical or non-canonical STAT1 pathway protein expression. Cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) for 1 h, 4 h or 16 h. IRF9 ( A , E , F ), phosphorylated JAK2 ( B , G – I ), phosphorylated STAT1 ( C , J – L ) and phosphorylated NFkB p65 ( D , M – O ) protein expression was determined. Representable blots of proteins of interest and β-actin control are shown in A-D. Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.001, medium control as compared to 2 mM butyrate control or to IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells in absence of butyrate. Activated cells were also compared to activated cells treated with butyrate.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of butyrate and histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) on mRNA expression of genes related to the STAT1 signaling cascade. Intestinal epithelial cells were activated with IFN-γ or IFN-γ+TNF-α and treated with 2 mM butyrate (green bars) or 10 μM TSA (pink bars) for 4 h. mRNA expression was measured in CXCL10 ( A , B ), IRF9 ( C , D ) JAK2 ( E , F ) and SOCS1 ( G , H ). Data are represented as mean ± SEM ( n = 4). Significant differences are shown as ** p < 0.01, *** p < 0.001, **** p < 0.001 Control compared to butyrate, TSA, IFN-γ ( A , C , E , G ) or IFNγ+TNFα ( B , D , F , H ) activated cells and activated cells compared to butyrate or TSA treated activated cells.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: Butyrate Prevents Induction of CXCL10 and Non-Canonical IRF9 Expression by Activated Human Intestinal Epithelial Cells via HDAC Inhibition

doi: 10.3390/ijms23073980

Figure Lengend Snippet: The effect of histone deacetylase inhibitor Trichostatin A (TSA) on downstream proteins of the STAT1 signaling cascade. Protein expression in IFN-γ or IFN-γ+TNF-α-activated intestinal epithelial cells (IECs) after 4 h incubation with 10 μM TSA (pink bars). Proteins measured were IRF9 ( A , E ), phosphorylated JAK2 ( B , F ), phosphorylated STAT1 ( C , G ), phosphorylated NFκB p65 ( D , H ). Data are represented as mean ± SEM ( n = 3). Significant differences are shown as * p < 0.05, ** p < 0.01, *** p < 0.001, control compared to IFN-γ-activated IECs, IFN-γ+TNF-α-activated IECs or 10 μM TSA control. Activated cells were compared to activated cells treated with TSA.

Article Snippet: As primary antibodies phosphorylated JAK2 (1:200, Thermo Fisher Scientific), phosphorylated STAT1 (1:200, R&D systems), phosphorylated NF-kappaB p65 (1:1000, Cell signaling, Danvers, MA, USA), IRF9 (1:1000, Cell signaling) and β-actin (1:1000, Cell signaling) were used.

Techniques: Histone Deacetylase Assay, Expressing, Incubation, Control

Journal: PLoS ONE

Article Title: Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

doi: 10.1371/journal.pone.0068318

Figure Lengend Snippet: Top three IPA-generated networks of differentially expressed molecules identified by DIGE and iTRAQ labeling between early and mock, late and mock, and late and early WNV-infected brain samples.

Article Snippet: As a positive control for the p-701-STAT1 antibody response, a lysate of murine bone marrow-derived macrophages stimulated for two hours with IFN-γ (PeproTech; 20 ng/ml) was used.

Techniques: Labeling, Cell Function Assay

Journal: PLoS ONE

Article Title: Altered Protein Networks and Cellular Pathways in Severe West Nile Disease in Mice

doi: 10.1371/journal.pone.0068318

Figure Lengend Snippet: (A) Protein samples from each group used for proteomic analysis were minimally labeled with cyanine-3 dye. At the top, a representative protein profile of three biological replicates from brain lysates of mock-, and WNV early- and late-infected mice, separated by 10% SDS-PAGE is shown. WB with fluorescence-based methods was used to detect an overlaid fluorescent scan of the general protein patterns (Cy3 dye (green)) and the specific immunoreactive proteins (FITC or Cy5 dye (red)). To better visualize protein detection signals observed with each specific antibody used, corresponding cropped WB images are presented in grey levels. (B) The graphs correspond to the mean ± S.D. of protein quantity measured by densitometry of the antigenic bands. Densitometry analyses were performed using TotalLab Quant v12.2 software (Nonlinear Dynamics), and data were normalized to levels of global protein pattern intensity. The values indicated under each graph correspond to fold changes from paired comparisons ( i.e., WNV-E/mock, WNV-L/mock, WNV-L/WNV-E). WNV-E/−L, biological replicates of brain samples infected by West Nile Virus and collected at early or late time-points. The significance of the differential protein expression are indicated *, p<0.05; **, p<0.01; ***, p<0.001. A.U., arbitrary units. α-, antibody anti-; ★, IFN-γ activated mice bone marrow derived macrophage (p-701-STAT1 positive control); #, no quantification for p-701-STAT1. CAPN9, calpain 9; CLTC, clathrin heavy chain; DNM1, dynamin 1; GFAP, glial fibrillary acidic protein; HUWE1; E3 ubiquitin-protein ligase; MAP1B, microtubule-associated protein 1B; MAP2, microtubule-associated protein 2; PRDX6, peroxiredoxin 6; STAT1/2, signal transducer and activator of transcription 1/2; VIM, vimentin.

Article Snippet: As a positive control for the p-701-STAT1 antibody response, a lysate of murine bone marrow-derived macrophages stimulated for two hours with IFN-γ (PeproTech; 20 ng/ml) was used.

Techniques: Labeling, Infection, SDS Page, Fluorescence, Software, Expressing, Derivative Assay, Positive Control

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Upregulation of IFN -inducible genes and STAT1 phosphorylation in residual tumour foci after AC treatment. ( A ) In vivo response of HBCx-17 to AC (2/100 mg kg −1 ) (mean tumour volume±s.d.) n =11 per group. ( B ) H&E analysis and in situ hybridisation of Alu probes of one residual tumour foci after chemotherapy response (× 25). ( C ) Type I and II interferon distribution by ISG Database. ( D ) qRT–PCR analysis of STAT1 and eight other IFN -inducible genes in untreated samples, residual and regrowing tumours. HBCx-6, HBCx-8 HBCx-10 and HBCx-17 PDX were treated with AC, mean±s.d., n =5 per group. Comparative analysis of all samples from the four experiments in different experimental conditions (Rel: relapse; Rem: remission; Untr: untreated); fold-change refers to mean intensity value ratio between the two experimental condition indicated. P -value was calculated by t -test except for results indicated with *= t -test with Welch's correction or **=Mann–Whitney test. ( E ) Western blotting of P-STAT1 Tyr701/Ser727 , STAT1, IF44, LCN2 and OAS1 in three PDX models responding to AC treatment. Tumours were analysed at the indicated phases: untreated (U), residual (N=nodule) and relapse tumour (R=Relapse).

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: In Vivo, In Situ, Hybridization, Quantitative RT-PCR, MANN-WHITNEY, Western Blot

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: List of interferon-inducible genes differentially expressed ( P <0.05) between HBCx-6, HBCx-8 and HBCx-17 in residual tumour cells (Nodule) and untreated tumours (CTRL)

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Histone Deacetylase Assay

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Analysis of IFN/STAT1 pathway activity in chemo-responder and chemo-resistant PDX at early stage after chemotherapy. ( A ) mRNA expression of a 21-gene IFN/STAT1 signature analysed by qPCR at days 0, 3 and 7 post-AC treatment in 9 responders and 5 resistant models. Each curve represents the expression of one gene. ( B ) In vivo response to AC and irinotecan in the HER2+ HBCx13 model and analysis of IFN-inducible genes ( BST2 , CLDN1 , DDX60 , IFI44 , IFI44L , IFI6 , IFIT1 , IFIT3 , IFITM1 , IRF9 , MX1 , OAS1 , OAS2 , PARP12 , PARP9 , SAMD9 , STAT1 , STAT2 , UBE2L6 , ZNFX1 ) at D7 and D14.

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Activity Assay, Expressing, In Vivo

Journal: British Journal of Cancer

Article Title: Activation of IFN/STAT1 signalling predicts response to chemotherapy in oestrogen receptor-negative breast cancer

doi: 10.1038/bjc.2015.398

Figure Lengend Snippet: Activation of JAK/STAT1 pathway and expression of IFN- γ in the early response to AC. ( A ) Western blotting analysis of total STAT1 or P-STAT1 Tyr701/Ser727 in four responders and four resistant models at D0, D3 and D7 post-AC treatment. ( B ) Human and murine soluble IFN- γ expression determined by cytokine array in HBCx-10 tumours at D3, D7 and D14 after AC and during residual and regrowing phases. ( C ) Effect of RUX alone on HBCx-10 tumour growth. Mean RTV±s.d., n =10. ( D ) P-STAT1 Tyr701 foci detected by IHC in one untreated xenograft and the number of P-STAT1 Tyr701 -positive tumour cells in untreated and RUX-treated tumour samples. * P ⩽0.05 (Student's t -test). ( E ) Kaplan–Meier survival analysis of mice treated with chemotherapy alone and with chemotherapy and RUX ( P <0.0001, log-rank (Mantel–Cox) test). ( F ) Expression of IFN- inducible genes by qPCR in untreated, AC, RUX alone or RUX/AC groups at 8 days (D8) posttreatment.

Article Snippet: Lysates were resolved on 4–12% TGX gels (Bio-Rad, Marnes-la-Coquette, France), transferred into nitrocellulose membranes (Bio-Rad) and immunoblotted overnight at 4 °C or 1 h at room temperature with the following antibodies: P-STAT1 Tyr701 , CASP-3 (1/750–1000; Cell Signaling Ozyme), Total STAT1 (1/1000; Santa Cruz antibodies, Nanterre, France), P-STAT1 Ser727 , P- γ H2AX Ser139 (1/1000; Merck Millipore), OAS1, IFI44, LCN2, or Actin, as an internal control loading (1/1000; Sigma-Aldrich, St Louis, MO, USA).

Techniques: Activation Assay, Expressing, Western Blot