Figure 4.

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).