Journal: The Journal of Biological Chemistry

Article Title: Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells

doi: 10.1074/jbc.RA120.012943

Figure Lengend Snippet: Basal and inducible expression of IL23A expression in CRC lines. A and B, IL23A is widely expressed in diverse human colorectal carcinoma lines and organoids. A, IL23A mRNA levels in 21 CRC cell lines were evaluated by qRT-PCR using an IL23A-specific Taqman probe. IL23A expression is normalized with that of GAPDH and compared against that of AGS human gastric epithelial cells and THP-1 human monocytes, known producers of IL23A (14). The corresponding Ct values of each sample (with a cut-off limit of 35 cycles) are presented in gray bars. Normalised IL23A mRNA levels are expressed relative to that of THP-1. Data are presented as mean ± SEM of four replicates. B, the expression levels of four human CRC-derived organoids relative to that of SW480. Normalised IL23A mRNA levels are expressed relative to that of SW480. Data are presented as mean ± SEM of four replicates. C, TNFα-induced IL23A expression in colorectal cancer cell lines. SW620, SW480, COLO 205, and HCT 116 cells were treated with TNFα (50 ng/ml) for 10 h and harvested for qRT-PCR measurement of IL23A mRNA levels. Normalized IL23A mRNA levels are expressed relative to that of untreated controls. Data are presented as mean ± S.E. of four replicates. D, PMA strongly induced IL23A expression. Four CRC cell lines were treated with PMA (1 μm) for 10 h and harvested for qRT-PCR measurement of IL23A mRNA. Normalized IL23A mRNA levels are expressed relative to those of DMSO-treated control samples (n = 4; mean ± S.E.). E and F, correlation plot of Gene Ontology (GO) MAPK pathway enrichment score (y axis) and IL23A Log2 gene expression value (x axis) in TCGA COADREAD cohort (E) and CRC cell lines (F). Correlation Rho and p value are computed using Spearman's correlation coefficient rank test. G, activating effects of TNFα and PMA are mediated via the proximal promoter. A firefly luciferase reporter construct containing the −1200 to +105 region of the IL23A promoter and its deletion variants were transiently transfected into SW620 cells, which were treated with TNFα (50 ng/ml) or PMA (1 μm) for 8 h and harvested for luciferase assay. Normalized luciferase activities are expressed relative to the values of samples transfected with an empty control vector. Data presented are as mean ± S.E. from four replicates. The p values are indicated as follows: *, p < 0.05; **, p < 0.01; ***, p < 0.001; n.s., not significant.

Article Snippet: Data availability The gene expression data of colorectal cancer samples from TCGA COADREAD cohort are publicly available in Broad Institute GDAC ( 58 ).

Techniques: Expressing, Quantitative RT-PCR, Derivative Assay, Control, Gene Expression, Luciferase, Construct, Transfection, Plasmid Preparation

Journal: The Journal of Biological Chemistry

Article Title: Inflammatory and mitogenic signals drive interleukin 23 subunit alpha (IL23A) secretion independent of IL12B in intestinal epithelial cells

doi: 10.1074/jbc.RA120.012943

Figure Lengend Snippet: CRC cells secrete IL23A in the absence of IL12B. A, relative expression levels of IL23A and IL12B in selected cancer cell lines. Normalized RNA-Seq data derived from cancer cell lines of hematopoietic, gastric, and colorectal (large intestine) lineages were extracted from the CCLE database (35) and charted against copy number. B, dot plot of IL23A and IL12B gene expression levels (y axis; mean ± S.E.) in TCGA COADREAD cohort (left panel; n = 382) and CRC cell line (right panel; n = 13). C, IL12B mRNA levels were evaluated by qRT-PCR using a specific Taqman hydrolysis probe. IL12B expression is normalized against that of GAPDH and compared with that of AGS human gastric epithelial cells and THP-1 human monocytes, the latter is known for producing IL23A/IL12B. The corresponding Ct values of every sample (with a cut-off limit of 35 cycles) are presented in gray bars. D, immunoprecipitation of endogenous IL23A from the culture supernatants of activated SW620, COLO 201, and HCT 116 cells. CRC cells were treated with DMSO (mock) or PMA (100 ng/ml) for 36 h before collection for immunoprecipitation (IP) by a monoclonal IL23A-specific antibody. The immunoprecipitation of IL23A and co-immunoprecipitation of IL12B were measured by reducing (IL23A) and nonreducing (IL12B) Western blotting. Culture supernatant of HEK293T cells expressing exogenous IL23A and IL12B was used as a positive control for canonical IL-23. E, the same supernatants used in immunoprecipitation experiment (C) were analyzed by ELISA for the presence of IL-23 (IL23A/IL12B).

Article Snippet: Data availability The gene expression data of colorectal cancer samples from TCGA COADREAD cohort are publicly available in Broad Institute GDAC ( 58 ).

Techniques: Expressing, RNA Sequencing, Derivative Assay, Gene Expression, Quantitative RT-PCR, Immunoprecipitation, Western Blot, Positive Control, Enzyme-linked Immunosorbent Assay