Journal: Scientific Reports

Article Title: STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes

doi: 10.1038/s41598-018-37294-z

Figure Lengend Snippet: Activation of STAT3 and induction of IRF9 and IRDS genes in MCS. ( a , b ) HCT116 cells cultured as MCS (3D) for 6 days compared to cells in monolayer (2D) harvested at indicated time points was subjected to ( a ) Western blot analysis of tyrosine-phosphorylation and protein levels of STAT3 and IRF9 and ( b ) qRT-PCR analysis of mRNA levels of the indicated IRDS genes (n = 3). The heatmap is showing the mean mRNA expression of the individual genes (left) and the box plot display mRNA levels of the IRDS panel as a group (right). ( c ) Representative immunohistochemical staining of IRF9, p-STAT3 and total STAT3 in paraffin embedded HCT116 cells cultured as MCS for 6 days. ( d ) Representative images of HCT116 and DLD1 cells grown as MCS for 6 days. ( e ) DLD1 cells cultured as MCS or in 2D and harvested at indicated time points. Protein levels and phosphorylation of STAT1, STAT3 and IRF9 were analysed by Western blot. ( f , g ) Cells were transfected with siRNA targeting IRF9 and cultured as MCS for 48 h. ( f ) Western blot analysis showing knockdown of IRF9 and protein and phosphorylation levels of STAT1 and STAT3. The blots in this figure have been cropped; full-length blots are shown in Supplementary Fig. . ( g ) mRNA expression of the panel of IRDS genes were analysed using qRT-PCR (n = 3). Quantification data of Western blots are available in Supplementary Table .

Article Snippet: The following antibodies were from Cell Signaling Technology: IRF9 (#76684), STAT1 (#9172), P-Y701-STAT1 (#9171), STAT2 (#72604), P-Y690-STAT2 (#88410), STAT3 (#4904), P-Y705-STAT3 (#9145).

Techniques: Activation Assay, Cell Culture, Western Blot, Phospho-proteomics, Quantitative RT-PCR, Expressing, Immunohistochemical staining, Staining, Transfection, Knockdown

Journal: Scientific Reports

Article Title: STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes

doi: 10.1038/s41598-018-37294-z

Figure Lengend Snippet: STAT1 and STAT3 activation and IRF9/IRDS genes induction is dependent on gp130/JAK signalling. ( a–c ) HCT116 cells were cultured as MCSs for 6 days in the presence of either DMSO or JAK inhibitors Pyridone 6 (Pyr6) 5 µM or Ruxolitinib (Ruxo) 1 µM. Protein phosphorylation and total levels of STAT1, STAT3 and IRF9 in samples treated with ( a ) Pyr6 or ( b ) Ruxo were analysed by Western blot. ( c ) mRNA expression of the IRDS panel was evaluated by qRT-PCR (n = 3). Mean expression of the individual genes is displayed in the heatmap (left) and mRNA levels of the IRDS panel as a group is shown in the box plot (right). ( d , e ) HCT116 cells were cultured as MCSs for 6 days in the presence of DMSO or gp130 inhibitors SC144 (1 µM) or Atovaquone (AQ) (10 µM). ( d ) Protein phosphorylation and total levels of STAT1, STAT3 and IRF9 were analysed by Western blot and ( e ) mRNA expression of the indicated IRDS genes was evaluated by qRT-PCR (n = 2). Heatmap showing the mean mRNA expression of individual genes (left), and the box plot show expression of the IRDS panel as a group (right). ( f ) HCT116 cells seeded at low density were treated with CM and either JAK or gp130 inhibitors for 24 h. Protein phosphorylation and total leves of STAT1, STAT3 and IRF9 in cells treated with JAK inhibitors or gp130 inhibitors were analysed by Western blot. Quantification data of Western blots are available in Supplementary Table .

Article Snippet: The following antibodies were from Cell Signaling Technology: IRF9 (#76684), STAT1 (#9172), P-Y701-STAT1 (#9171), STAT2 (#72604), P-Y690-STAT2 (#88410), STAT3 (#4904), P-Y705-STAT3 (#9145).

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

Journal: Scientific Reports

Article Title: STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes

doi: 10.1038/s41598-018-37294-z

Figure Lengend Snippet: STAT3 is required for IRF9 and IRDS genes induction in MCS. ( a–d ) HCT116 cells were transfected with siRNA and cultured as MCS (3D) for 2 days. ( a ) Western blot analysis showing knockdown of STAT1 and protein expression of STAT3 and IRF9 in the same protein lysates. ( b ) mRNA levels of the IRDS genes, analysed by qRT-PCR (n = 3). mRNA levels of the individual genes are available in Supplementary Fig. . ( c ) Western blot analysis of showing knockdown of STAT3 and protein expression of STAT1 and IRF9 in the same protein lysates. ( d ) mRNA levels of the panel of IRDS genes, analysed by qRT-PCR (n = 3). mRNA expression of the individual genes is displayed in Supplementary Fig. . ( e ) Representative images of HCT116 cells transfected with siRNA and cultured as spheres for 48 h. ( f ) DLD1, A4 and A4wt cells were cultured in 2D or 3D and harvested at the indicated time points. Phosphorylation and expression of STAT1, STAT3 and IRF9 was analysed by Western blot in the same protein lysates. Quantification data of Western blots are available in Supplementary Table .

Article Snippet: The following antibodies were from Cell Signaling Technology: IRF9 (#76684), STAT1 (#9172), P-Y701-STAT1 (#9171), STAT2 (#72604), P-Y690-STAT2 (#88410), STAT3 (#4904), P-Y705-STAT3 (#9145).

Techniques: Transfection, Cell Culture, Western Blot, Knockdown, Expressing, Quantitative RT-PCR, Phospho-proteomics

Journal: Scientific Reports

Article Title: STAT3 is activated in multicellular spheroids of colon carcinoma cells and mediates expression of IRF9 and interferon stimulated genes

doi: 10.1038/s41598-018-37294-z

Figure Lengend Snippet: STAT3 is recruited to the IRF9 promoter in cells grown in 3D. ( a ) A schematic depiction of CHIP-seq data from the UCSC genome browser showing enrichment of STAT3 at the IRF9 promoter region (UCSC genome browser assembly February 2009 GRch37/hg19). ( b ) The position and sequence of the STAT binding site in the IRF9 promoter region (RefSeq: NM_006084.4). ( c , d ) CHIP assay results evaluating the binding of STAT3 to the IRF9 promoter in HCT116 cells cultured in 2D and 3D. Chromatin was immunoprecipitated with an anti-STAT3 antibody followed by qRT-PCR (n = 3) ( c ) or semi-quantitative PCR ( d ). Location of the primers used are shown in figure ( a ). Error bars represent S.E.M. ( e ) Correlation analysis between STAT3 and IRF9 protein abundance in primary colorectal cancer tumour samples (n = 95) and cell lines (n = 44). The x-axis is binned in four groups (n = 34–35 per group) according to abundance of STAT3. Scatter plot is shown with Pearson correlation coefficient and corresponding p-value.

Article Snippet: The following antibodies were from Cell Signaling Technology: IRF9 (#76684), STAT1 (#9172), P-Y701-STAT1 (#9171), STAT2 (#72604), P-Y690-STAT2 (#88410), STAT3 (#4904), P-Y705-STAT3 (#9145).

Techniques: ChIP-sequencing, Sequencing, Binding Assay, Cell Culture, Immunoprecipitation, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Quantitative Proteomics