Fig. 4.

CPAP promotes the activity of the IL-6/STAT3 pathway. a, b The promoter activity (a) and gene expression level (b) of IL-8, ICAM-1, IL-6, and HIF1-α were determined in Hep3B cells expressing GFP or GFP-CPAP by reporter assay and RT-qPCR, respectively. Cells were serum starved, treated with (■) or without (□) IL-6 for 24 h, and then harvested for analysis. c GFP- or GFP-CPAP-expressing Hep3B cells were treated with IL-6 for 24 h; the medium was then replaced by fresh culture medium, and the cells were cultured for an additional 24 h. The conditioned media were collected to determine the expression of IL-8 by enzyme-linked immunosorbent assay (ELISA). d Sera from HCC patients (n = 31) were used to perform IL-8 ELISA. HCC tissues (T) and paired nontumorous tissues (NT) were used to analyze the CPAP mRNA expression level by RT-qPCR. The serum expression level of IL-8 is positively correlated with CPAP mRNA overexpression (T/NT) in HCC patients; Pearson correlation coefficient, R = 0.5162, p = 0.003. Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001. e GFP-CPAP-expressing Huh7 cells were treated with (+) or without (−) IL-6 for 4 min, and then total cell lysates were collected to perform a coimmunoprecipitation assay using anti-STAT3 antibodies. The immunoprecipitated complexes were subjected to Western blot analysis using the indicated antibodies. f (i) In vivo PLA using anti-STAT3 and anti-CPAP antibodies in IL-6-treated (+) or untreated (−) GFP- or GFP-CPAP-expressing Huh7 cells. (ii) Huh7 cells were cotransfected with Myc-STAT3/C1 and GFP-PN1 or GFP-A5C and then treated with (+) or without (−) IL-6 for 4 min. The interaction between PN1 or A5C and Myc-STAT3/C1 was detected by in situ PLA using anti-GFP and anti-Myc antibodies. The red signals indicate positive interactions. DAPI is a DNA-specific dye. The quantitative results of PLA red signals are shown