Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces STAT1 activation in BV2 cells. BV2 cells were exposed to 20 ng/mL of IFNγ at different time points and activation of STAT1 was evaluated through different experiments. ( A ) Total protein extracts of IFNγ-treated BV2 cells at different time points were analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody, and after stripping, the same blot was analyzed with anti-STAT1 antibody. Densitometric analysis revealed a STAT1 phosphorylation upon IFNγ treatment, peaking at 15 min compared with untreated BV2 cells (CTR). ( B ) RT-qPCR of iNOS and COX2, two STAT1-dependent genes, revealed an increase in their mRNA levels after 18 and 24 h of IFNγ treatment in BV2 cells. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. untreated cells (CTR). Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Stripping Membranes, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment counteracts IFNγ-dependent STAT1 activation in BV2 cells. ( A ) Total protein extracts from BV2 cells pre-treated overnight with GEE and subsequently exposed to 20 ng/mL IFNγ for 18 h were analyzed by Western Blot using a phospho-Tyr701 STAT1 antibody. The same blot was analyzed with anti-Tubulin antibody to check the amount of loaded proteins. Densitometric analysis showed decreased STAT1 phosphorylation in GEE pre-treated cells compared to CTR. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). # p < 0.05 vs. IFNγ-treated cells. ( B ) RT-qPCR analysis revealed that iNOS and COX2 mRNA levels were reduced in BV2 cells pre-treated overnight with GEE and then exposed to 20 ng/mL IFNγ for 24 h. Data are expressed as mean ± SD (n = 3). ** p < 0.01 vs. CTR, ## p < 0.01 vs. IFNγ-treated cells. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Quantitative RT-PCR

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: GEE pre-treatment inhibits IFNγ-induced STAT1 phosphorylation and nuclear translocation in BV2 cells. The cells were immunostained with phospho-Tyr701 STAT1 (red) and analyzed by confocal microscopy (lens 60×). Nuclei were stained with DAPI (cyan). The orthogonal sections (ZX and ZY) of merge image reveal that pTYR 701 STAT1 protein is inside the nuclei. Scale bars indicate 10 μm. Images are representative of three separate experiments.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Phospho-proteomics, Translocation Assay, Confocal Microscopy, Staining

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: IFNγ treatment induces S-glutathionylation of STAT1. ( A ) Total protein extracts of BV2 cells left untreated (CTR) or treated with 20 ng/mL IFNγ for the indicated times were subjected to immunoprecipitation with anti-STAT1 antibody. Immunoprecipitated STAT1 (IP: STAT1) was analyzed by Western Blot under non-reducing conditions using anti-SSG antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Densitometric analysis revealed increased STAT1 S-glutathionylation after 5 and 15 min of IFNγ treatment compared to untreated BV2 cells. * p < 0.05, ** p < 0.01 vs. untreated BV2 cells (CTR). ( B ) Total protein lysates from the same untreated and treated BV2 cells were reserved before pull-down (input) and analyzed by Western Blot with anti-phospho-Tyr701 STAT1 antibody and, after membrane stripping, re-probed with anti-STAT1 antibody. Images are representative of four independent experiments. Single data points are shown as red dots.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Immunoprecipitation, Western Blot, Membrane, Stripping Membranes

Journal: Antioxidants

Article Title: ROS-Driven STAT1 S-Glutathionylation Sustains IFNγ Signaling and Pro-Inflammatory Microglial Polarization

doi: 10.3390/antiox14121395

Figure Lengend Snippet: FNγ induces M1 polarization in BV2 cells through oxidative stress and STAT1 activation. ( A ) The expression of the pro-inflammatory cytokines IL-6 and TNFα in the supernatant of BV2 cells was evaluated by ELISA after 18 h of IFNγ stimulation, with or without GEE pretreatment. GEE markedly reduced the IFNγ-induced secretion of both cytokines, confirming the involvement of oxidative stress in M1 polarization. Data are expressed as mean ± SD (n = 3). * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ. ( B ) Parental and STAT1 knockdown in BV2 cells were treated with 20 ng/mL of IFNγ for 18 h. Total protein extracts were analyzed by Western Blot with anti-iNOS and anti-Actin antibodies. Densitometric analysis revealed reduced iNOS induction in STAT1 knockdown in BV2 cells compared with parental cells upon IFNγ treatment. Parental and STAT1 knockdown in BV2 cells cultured without IFNγ were used as controls (CTR). Images are representative of three independent experiments. Single data points are shown as red dots. * p < 0.05, ** p < 0.01 vs. CTR. # p < 0.05 vs. IFNγ.

Article Snippet: Membranes were blocked with 5% BSA or 5% fat dry milk in Tris-buffered saline with 0.1% Tween 20 (TBST) at room temperature for 1 h and then incubated with primary antibodies specific for anti-phospho-Tyr701 STAT1 (sc-136229 Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-STAT1 (sc-346, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-Tubulin (Oncogene, CP06-100UG), and anti-iNOS (ab-15323, Abcam, Cambridge, UK).

Techniques: Activation Assay, Expressing, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Cell Culture

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Baicalin suppresses the progression of Type 2 diabetes-induced liver tumor through regulating METTL3/m 6 A/HKDC1 axis and downstream p-JAK2/STAT1/clevaged Capase3 pathway.

doi: 10.1016/j.phymed.2021.153823

Figure Lengend Snippet: Fig. 4. The HKDC1 (m6A 2854 site) and HKDC1 protein levels in the HepG2 cells after treatment with siRNA fragments derived from m6A-related genes. A, The normalized level of HKDC1 (m6A 2854 site) treated with METTL3 siRNA. B, The normalized level of HKDC1 (m6A 2854 site) treated with METTL14 siRNA. C, The normalized level of HKDC1 (m6A 2854 site) treated with FTO siRNA. D, The normalized level of HKDC1 (m6A 2854 site) treated with ALKBH5 siRNA. E, The normalized level of HKDC1 (m6A 2854 site) treated with YTHDF1 siRNA. F, The protein bands of HKDC1/(p-)JAK2/(p-)STAT1/(cleaved-)caspase-3. G, The p-JAK/ JAK, p-STAT1/STAT1, and (cleaved-)caspase-3/caspase-3 ratios. The data are presented as mean ± SD, * p < 0.05 compared with the normal group, and & p < 0.05 compared with the high glucose + gene siRNA group (n = 3).

Article Snippet: The primary antibodies used in this study included p-JAK2, JAK2, p-STAT1, STAT1, cleaved-caspase-3, caspase-3, and β-actin (Santa Cruz Biotechnology, California, USA), anti-human HKDC1 antibody was purchased from Novus Biologicals Inc. (Colorado, USA).

Techniques: Derivative Assay

Journal: Phytomedicine : international journal of phytotherapy and phytopharmacology

Article Title: Baicalin suppresses the progression of Type 2 diabetes-induced liver tumor through regulating METTL3/m 6 A/HKDC1 axis and downstream p-JAK2/STAT1/clevaged Capase3 pathway.

doi: 10.1016/j.phymed.2021.153823

Figure Lengend Snippet: Fig. 6. The protein levels of the p-JAK2/ STAT1/cleaved-caspase-3 pathway in the HepG2 cells at different glucose concentra tions. A, The western blot bands of the HKDC1 and p-JAK2/STAT1/cleaved-caspase-3 pathway. B, Statistical data of the protein expression levels. C, The p-JAK2/JAK2, p- STAT1/STAT1, and cleaved-caspase-3/caspase- 3 ratios. All data are presented as mean ± SD, * p < 0.05 compared with the normal group, and # p < 0.05 compared with the high glucose group (n = 3).

Article Snippet: The primary antibodies used in this study included p-JAK2, JAK2, p-STAT1, STAT1, cleaved-caspase-3, caspase-3, and β-actin (Santa Cruz Biotechnology, California, USA), anti-human HKDC1 antibody was purchased from Novus Biologicals Inc. (Colorado, USA).

Techniques: Western Blot, Expressing

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 1 STAT1 promotes expression of PD-L1 and proliferative biomarkers in HNSCC cells. a, b Gene expression of Stat1 determined by RT- qPCR, STAT1 and GAPDH protein levels, determined by western blot, and phospho STAT1 (pSTAT1) protein levels after IFNγ stimulation determined by flow cytometry, in Stat1 siRNA and scrambled RNA treated a CAL27 and b UM-SCC22A cells. STAT1 and GAPDH protein levels were detected after 72 h of siRNA treatment, and western blot band intensity of STAT1:GAPDH were calculated in scramble and Stat1 siRNA-treated cells. Numbers in histogram plots represent percentage of phospho STAT1 positive cells. Controls are unstimulated cells. c, e PD-L1 expression by c CAL27 and e UM-SCC22A cells after 72 h of siRNA treatment, determined by flow cytometry. d, f Flow gating and frequencies of AnnexinV+PI− populations among d CAL27 and f UM-SCC22A cells after 72 h of siRNA treatment. h, i Gene expression of Ki67 and Mcm3, determined by RT-qPCR in h CAL27 and i UM-SCC22A cells after 48 or 72 h siRNA treatment. Data are presented as mean +/−SEM. *p value <0.05; **p value <0.01; ***p value <0.001, ****p value <0.0001 between groups.

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: Expressing, Gene Expression, Quantitative RT-PCR, Western Blot, Cytometry

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 2 Global STAT1 deficiency in a 4NQO model of HNSCC results in increased incidence of carcinogenesis. a 4NQO model of carcinogen induced oral carcinogenesis showing time period of 4NQO administration and terminal sacrifice. b Kaplan–Meier curve depicting survival of 4NQO-treated Stat1+/+ and Stat1−/−mice through the duration of the experiment. c Counts of lesions and tumours on the tongues of Stat1+/+ and Stat1−/−4NQO-treated mice determined at terminal sacrifice. d Representative images of mouse tongues containing tumours from 4NQO-treated Stat1+/+ and Stat1−/−at terminal sacrifice. e Tabular illustration of the incidence and multiplicity of lesions and tumours in experimental mice. f Representative H&E-stained images of dysplastic lesions in tongues from Stat1+/+ and Stat1−/−mice. g, h Representative immunohistochemistry images of oral lesions in tongues of Stat1+/+ and Stat1−/−mice stained with g KI-67 and h cleaved caspase 3. Bar graphs depict regions of positive staining from 10 representative fields each for n = 6 or 8 mice per group. i Fold induction of Ki67, Aurka, Casp3 and Bcl2 transcripts in the tongues of control and carcinogen-induced Stat1+/+ and Stat1−/−mice determined by RT-qPCR (n = 10 cancer-induced mice per group). Data are presented as mean +/−SEM. *p value <0.05; ***p value <0.005 between groups.

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: Staining, Immunohistochemistry, Control, Quantitative RT-PCR

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 3 STAT1 promotes PD-1/PD-L1 expression in the tumour microenvironment during HNSCC. a Gene and protein expression of Pd1 in the tongues of experimental mice determined by RT-qPCR and immunofluorescence microscopy. b, c Representative immunofluorescence staining of b PD1 (green), CD8 (red) and c PD1 (green), CD4 (red) in tongue tumours of carcinogen-induced Stat1+/+ and Stat1−/−mice. Bar graphs depict average numbers of PD1+CD8+ and PD1+CD4+ cells obtained from 10 representative fields each for n = 6 or 8 mice per group d, e PD-1 surface expression by d CD8+ cells and e CD4+ cells from the lymph nodes and spleens of experimental mice. Graph shows average percentage of PD1+CD8+ and PD1+CD4+ cells (n = 15 or 17 cancer induced mice per group). f Gene and protein expression of Pdl1 in the tongues of experimental mice determined by RT-qPCR and immunofluorescence microscopy. g Representative immunofluorescence staining of PDL1 (green) and CD11b (red) in tongue tumours of carcinogen-induced Stat1+/+ and Stat1−/−mice. Bar graph depicts average numbers of PDL1+CD11b+ cells obtained from 10 representative fields each for n = 6 or 8 mice per group h, i Comparative histograms of PD- L1 surface expression by h M-MDSCs and j M12 macrophages in carcinogen-induced Stat1+/+ and Stat1−/−mice. Graphs show mean fluorescence intensities (MFI) of PD-L1 surface expression by h M-MDSCs, i G-MDSCs, j M1 macrophages and k M2 macrophages in lymph nodes, spleens and bone marrow of experimental mice (n = 15 or 17 cancer-induced mice per group). Data are presented as mean +/−SEM. *p value <0.05; **p value <0.01; ***p value <0.001 between groups.

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: Expressing, Quantitative RT-PCR, Microscopy, Staining

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 4 STAT1 is required for anti-tumour function by T-lymphocytes during HNSCC carcinogenesis. a Fold induction of Ifng, Gzmb and Cxcl9 transcripts in the tongues of experimental mice determined by RT-qPCR. b IFNγ protein expression in tongue lysates of carcinogen-induced Stat1+/+ and Stat1−/−mice as determined by ELISA. c, d Representative immunohistochemistry images of oral tumours in tongues of Stat1+/+

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 5 TRIM24 is selectively expressed in HNSCC cells and negatively regulates STAT1. a Volcano plot showing top 100 upstream regulators based on Ingenuity pathway analysis of RNA sequencing data from oral tumour bearing Stat1+/+ and Stat1−/−mice (n = 4 per group). b Predicted mechanistic network analysis showing transcriptional targets of TRIM24 based on Ingenuity pathway analysis of RNA sequencing data from oral tumour-bearing Stat1+/+ and Stat1−/−mice. Genes in red are upregulated and blue are downregulated in the RNA sequencing data set. c Correlation of TRIM24 expression levels with STAT1 expression in tumours from HNSCC patients samples acquired from The Cancer Genome Atlas. Significance determined by Pearson correlation coefficient. d Gene expression of Trim24 determined by RT-qPCR in CAL27 cells treated with scramble or Stat1 siRNA for 72 h. e TRIM24 protein expression by CAL27 cells treated with scramble or Stat1 siRNA for 72 h, determined by flow cytometry. f Gene expression of TRIM24 in tumours of HNSCC tumour-bearing mice orthotopically injected with the LY2 cell line. g TRIM24 protein expression, determined by flow cytometry, in HNSCC cells of mice orthotopically injected with the LY2 cell line. h, j Gene expression of Trim24 and Stat1 determined by RT-qPCR in (H) CAL27 and (J) UMSCC22A cells treated with scramble or Trim24 siRNA for 48 or 72 h. i, k Protein expression of TRIM24, STAT1, pSTAT1 and GAPDH, determined by western blot, in i CAL27 and k UMSCC22A cells accompanied by the protein band intensities relative to GAPDH in scramble and Trim24 siRNA-treated cells after 48 and 72 h (n = 3 samples per group). Data are presented as mean +/−SEM. #p value <0.1, *p value <0.05; **p value <0.01; ***p value <0.001 between groups.

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: RNA Sequencing, Expressing, Gene Expression, Quantitative RT-PCR, Cytometry, Injection, Western Blot

Journal: British journal of cancer

Article Title: STAT1 is regulated by TRIM24 and promotes immunosuppression in head and neck squamous carcinoma cells, but enhances T cell antitumour immunity in the tumour microenvironment.

doi: 10.1038/s41416-022-01853-z

Figure Lengend Snippet: Fig. 6 Effects of TRIM24 on HNSCC cells are mediated by STAT1-dependent and independent mechanisms. a, b Gene expression of a Stat1 and b Trim24 determined by RT-qPCR in CAL27 cells at 72 h post-siRNA treatment of cells treated with scramble, Stat1, Trim24 and Stat1/Trim24 siRNA. c PD-L1 protein expression by siRNA-treated CAL27 cells, determined by flow cytometry. d Gene and e protein expression of Ki67, Mcm3, Bcl2, and Casp8, determined by RT-qPCR and Western blot in siRNA-treated CAL27 cells at 48 or 72 h. f Graphical model demonstrating the influence of STAT1 and TRIM24 on gene/protein expression and cell function in myeloid cells, T cells and HNSCC cells. Statistical significance (p < 0.05) in comparison to a +Stat1+Trim24, b −Stat1+Trim24 and c +Stat1−Trim24 is noted above each group (n = 5 biological replicates per group for RT-PCR and 3 biological replicates per group for Western blot). Data are presented as mean +/−SEM.

Article Snippet: Antibodies used included STAT1 (14994T), phospho STAT1 (9167S), TRIM24 (79030S), MCM3 (4012S), cleaved CASP8 (8592T), GAPDH (2118S) (Cell Signaling), BCL2 (IMG80093, IMGENEX) and KI67 (M7240, Agilent).

Techniques: Gene Expression, Quantitative RT-PCR, Expressing, Cytometry, Western Blot, Cell Function Assay, Comparison, Reverse Transcription Polymerase Chain Reaction

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: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 1. CCN1 gene expression is upregulated by virus but not by chemotherapy or radiation therapy. Real-time PCR analysis of CCN1 gene expression in LN229 glioma cells treated with rHSVQ1 at multiplicity of infection (MOI) ¼ 1 (A), 10 gy radiation or 0.5 mmol/L temozolomide (B), VSV (ts 45) at MOI ¼ 0.1 (C), adenovirus (type 5) at MOI ¼ 100, NDV at MOI ¼ 2, or WtHSV-1 at MOI ¼ 1, 24 hours after treatment. Data shown are the mean CCN1 gene expression relative to endogenous GAPDH and error bars are SEM of at least 3 replicates and represent at least 3 independent experiments. ns, not significant; , P < 0.05; , P < 0.001.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Gene Expression, Virus, Real-time Polymerase Chain Reaction, Infection

Journal: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 2. Extracellular CCN1 expression inhibits viral transgene expression, replication, and cell killing. A, U251T2 glioma cells and Gli36DEGFR-H2B-RFP glioma cells transiently transfected with pcDNA3.1myc-hisBþCCN1 (CCN1) or pcDNA3.1myc-hisBþempty (control), 24 hours before being infected with rHsvQ1-IE4/5-Luc [multiplicity of infection (MOI) ¼ 1]. Twenty-four hours postinfection, virus-encoded luciferase activity (relative light units, RLU) was measured in infected cell lysates. Data shown are % RLU/mg SEM relative to control. B, OV-encoded luciferase activity (RLU) of Cy-1 tetracycline-inducible glioma cells treated doxycycline for 24 hours, before infection with rHsvQ1-IE4/5-Luc (MOI ¼ 1). Results presented are % RLU/mg SEM relative to uninduced cells, 6 and 24 hours postinfection. C and D, confocal fluorescent and bright field images of GFP-positive infected U251T2 (C) and LN229 (D) glioma cells seeded on plates coated with CCN1/BSA (5 mg/mL) infected with rHsvQ1-IE4/5-Luc. E, inhibition of endogenous CCN1 increases OV transgene expression in3 different glioma cell lines. Quantification of OV-encoded luciferase activity of U251T3, LN229, and Gli36DEGFR-H2B-RFP glioma cells infected with rHsvQ1-IE4/5-Luc CCN1 mAb measured 24 hours postinfection. Data shown are % RLU/mg SEM relative to control. F, rescue of CCN1-mediated viral inhibition by CCN1-neutralizing monoclonal antibody. Cy-1 glioma cells treated doxycycline were infected with rHsvQ1-IE4/5-LucCCN1 mAb. Virus- encoded luciferase activity was quantified 24 hours postinfection. Results presented are the % RLU/mg SEM relative to control of at least 3 different experiments. Scale bar, 100 mm; ns, not significant; , P < 0.05; , P < 0.01; , P < 0.001. IgG, immunoglobulin G.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Expressing, Transfection, Control, Infection, Virus, Luciferase, Activity Assay, Inhibition

Journal: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 3. CCN1 in the ECM limits OV replication and cytotoxicity. A, total infectious virus particles obtained 24 hours postinfection of Cy-1 or controlLN229cellsdoxycyclinefor24hourspriorinfectionwithrHSVQ1 [multiplicity of infection (MOI) ¼ 2.5]. Data shown are fold change in numberofvirusparticlesSEMbetweencontrolanddoxycycline-treated cells. B, percentage surviving cells in infected Cy-1 cells (doxycycline) relative to uninfected (doxycycline) cells on days 1, 2, and 3 postinfection with rHSVQ1 (MOI ¼ 1). Data shown are percentage cell survivalininfectedCy-1cellsdoxycyclineatdifferenttimepointsrelative touninfectedcells(day0).C,reducedviralreplicationintumorsinducedto express CCN1. Mice implanted with Cy-1 or LN229 cells fed sucrose doxycycline were infected intratumorally with rHSVQ1 as described in Materials and Methods. Forty-eight hours postinfection, the number of virus particles in each tumor was measured by a standard plaque assay. Data shown are fold change in number virus particles SEM between control and doxycycline-treated cells. ns, not significant; , P < 0.05; , P < 0.001.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Virus, Infection, Plaque Assay, Control

Journal: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 4. Transcript profiling of Cy-1 cells induced to express CCN1. A, heat map representing hierarchic clustering of a subset of the differentially regulated genes, plotted using the log2 values of the genes with P < 0.05 (unpaired t test) that are involved in the type-I IFN response. Each column represents a sample plotted in triplicate, and each row in the heat map represents a gene that is differentially regulated in that particular comparison of samples. The color scale represents the degree of expression of the gene, green being the lowly expressed (below 3.0) and red being the highly expressed (above þ3.0) genes in the sample sets, with black as the center of the scale at "0." B, biological functions associated with genes significantly changed by the induction of CCN1. The significance of each canonical pathway is determined based upon the P values determined using right tailed Fisher exact test and with a threshold less than 0.05. The top 9 possible canonical pathways of the genes induced by CCN1 induction are shown. The ratio of number of genes in a given pathway satisfying the cutoff and total number of genes present in that pathway was determined by IPA. C, IPA-generated pathway associated with type-I interferon-responsive genes expressed upon induction of CCN1. Solid lines represent a direct interaction; dotted lines represent an indirect interaction.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Comparison, Expressing, Generated

Journal: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 6. CCN1-mediated OV inhibition is dependent on its interaction with cell surface a6b1 integrin independently of its ability to bind to avb3 and avb5. A and B, doxycycline-induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc [multiplicity of infection (MOI) ¼ 0.1], in the presence of cilengitide (A; cRGD, 50 mg/mL), an avb3 antagonist, or LM609 (B), a function blocking antibody against anti-avb3 (50 mg/mL). Viral transgene expression was determined by measuring luciferase activity. Data shown are % RLU/mg relative to control-treated cells. C, LN229 glioma cells were seeded on fibronection (a known avb3 agonist)-coated plates (5 mg/mL) or control plates and infected with rHsvQ1-IE4/5-Luc at MOI ¼ 0.1 for 24 hours. Viral transgene expression was determined by luciferase quantification, normalized to mg protein and represented as % RLU/mg relative to control cells. D, doxycycline-induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc (MOI ¼ 0.1), in the presence of P1F6, a function blocking antibody against anti-avb5 (50 mg/mL). Viral transgene expression was determined by luciferase quantification, normalized to mg protein and represented as % RLU/mg relative to control cells. E, LN229 glioma cells seeded on control or vitronectin (a known agonist for integrin avb5)-coated plates (5 mg/mL) were infected with rHsvQ1-IE4/5-Luc at MOI ¼ 0.1 for 24 hours. Viral-expressed luciferase activity was measured and is represented as % RLU/mg relative to control cells. F, doxycycline-induced Cy-1 cells were infected with rHsvQ1-IE4/5-Luc (MOI ¼ 0.1), in the presence of function blocking antibody GoH3 against integrin a6 or P5D2 against integrin b1 (50 mg/mL). Viral-expressed luciferase activity was measured and is expressed as % RLU/mg relative to uninduced cells. G, LN229, Gli36DEGFR-H2B-RFP, and U251T2 glioma cells were seeded on laminin (a known agonist for a6b1)-coated plates (5 mg/mL) or noncoated plates and infected with rHsvQ1-IE4/5-Luc at MOI ¼ 0.1 for 24hours. Viral-expressed luciferase activity was measuredand is expressed as % RLU/mg relative to control cells. H, LN229 glioma cells seeded on laminin-coated plates (5 mg/mL) were infected with rHsvQ1-IE4/5-Luc in the presence or absence of function blocking antibody GoH3 against integrin a6. Viral-expressed luciferase activity was quantified and represented as % RLU/mg relative to control cells. Cy-1 cells were incubated in the presence of doxycycline and harvested at the indicated time points.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Inhibition, Infection, Blocking Assay, Expressing, Luciferase, Activity Assay, Control, Incubation

Journal: Cancer Research

Article Title: Extracellular Matrix Protein CCN1 Limits Oncolytic Efficacy in Glioma

doi: 10.1158/0008-5472.can-11-2526

Figure Lengend Snippet: Figure 6. (Continued) I, representative Western blot of Cy-1 cells treated doxycycline for 2 minutes indicating CCN1 protein induction at a very early time point. GAPDH protein level was used as a control. J, supernatants were concentrated and analyzed for changes in IFNa secretion relative to nontreated control cells by ELISA. Data are shown as the mean SEM of at least 3 replicates and represent at least 3 different experiments. K, Cy-1 cells doxycycline were incubated with an antibody against the IFNa receptor chain 2 (50 mg/mL) before infection with rHsvQ1-IE4/5-Luc (MOI ¼ 1). Virus-expressed luciferase activity (RLU) was quantified. Data shown are % RLU/mg SEM in doxycycline-treated cells relative to uninduced cells. L, schematic of experimental setup for 6M-N, showing the culture of JiEGFR cells with infected or uninfected LN229 cells. M, Western blot for Stat1 phosphorylation of JiEGFR cells, cultured in the presence of secreted medium from infected or uninfected LN229 cells. Total Stat1 and GAPDH were used as controls. N, Western blot for Stat1 phosphorylation of JiEGFR cells, cultured in the presence of secreted medium from infected LN229 cells, cultured in the presence or absence of CCN1-neutralizing antibodies. Total Stat1 and GAPDH were used as controls. ns, not significant; , P < 0.05; , P < 0.01; , P < 0.001.

Article Snippet: Antibodies were obtained from the following sources: CCN1 (Novus Biologicals); glyceraldehydes-3-phosphate dehydrogenase (GAPDH) and ITGA6 (Abcam); STAT1 and PSTAT1 (Cell Signaling); STAT2, PSTAT2, LM609, P1F6, GoH3, P5D2, and IFNaR2 (Millipore); sheep antimouse HRP (GE Healthcare); goat anti-rabbit HRP; immunoglobulin G negative control (DAKO).

Techniques: Western Blot, Control, Enzyme-linked Immunosorbent Assay, Incubation, Infection, Virus, Luciferase, Activity Assay, Phospho-proteomics, Cell Culture

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

Journal: International Journal of Molecular Sciences

Article Title: Human Interleukin 23 Receptor Induces Cell Apoptosis in Mammalian Cells by Intrinsic Mitochondrial Pathway Associated with the Down-Regulation of RAS/Mitogen-Activated Protein Kinase and Signal Transducers and Activators of Transcription factor 3 Signaling Pathways

doi: 10.3390/ijms141224656

Figure Lengend Snippet: The mitogenic signaling pathways are repressed by IL-23R in 293ET cells. ( a ) Western blot analysis was performed using anti ERK, anti-phosphorylated ERK, anti STAT1, anti-phosphorylated STAT1, anti Akt, and anti-phosphoryled Akt antibodies. The tranfected Myc-IL-23R after 48 h transfection was visualized by anti Myc antibody. Beta-actin gene expression was served as internal control; and ( b ) Western blot analysis was performed using anti STAT3 and anti-phosphorylated STAT3 antibodies. β-actin gene expression was served as internal control. In both ( a ) and ( b ), the relative intensity of individual band was calculated based on the expression of β-actin by using the Quantity One program.

Article Snippet: Rabbit anti-STAT1 and Rabbit anti-p-STAT1 were purchased from Bioworld Technology Inc. (Bioworld, CA, USA).

Techniques: Western Blot, Transfection, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Human Interleukin 23 Receptor Induces Cell Apoptosis in Mammalian Cells by Intrinsic Mitochondrial Pathway Associated with the Down-Regulation of RAS/Mitogen-Activated Protein Kinase and Signal Transducers and Activators of Transcription factor 3 Signaling Pathways

doi: 10.3390/ijms141224656

Figure Lengend Snippet: The intracellular signaling pathways are altered upon the overexpression of IL-23R in HeLa cells. ( a ) Western blot analysis was performed anti-phosphorylated extracellular regulated protein kinases (p-ERK), anti ERK, anti STAT3 and anti-phosphorylated STAT3 antibodies. β-actin gene expression was served as internal control; and ( b ) Western blot analysis was performed using using anti-phosphoryled Akt, anti Akt, anti-phosphorylated STAT1 and anti STAT1 antibodies. β-actin gene expression was served as internal control. In both ( a ) and ( b ), the relative intensity of the individual bands were calculated based on the expression of β-actin by using the Quantity One program.

Article Snippet: Rabbit anti-STAT1 and Rabbit anti-p-STAT1 were purchased from Bioworld Technology Inc. (Bioworld, CA, USA).

Techniques: Over Expression, Western Blot, Expressing

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