Fig. 3
STAT3 represses SOCS3 expression through recruitment of DNMT1 in pancreatic cancer. a Structure of the SOCS3 gene. Bottom line is a genomic sequence of SOCS3 gene containing Exon 1, Intron 1, and Exon 2. The translation start site ATG is defined as +1. The grey vertical bar represents the recognized STAT3-SOCS3 binding site (−1046 through −1038). The arrows indicate primers used for MSP. b The SOCS3 gene promoter methylation status was analyzed in four pancreatic cancer cell lines using MSP. M and U, PCR products of methylated and unmethylated alleles, 155bp and 158bp, respectively; Con(M), methylation positive control; Con(U), positive control for unmethylation. c The SOCS3 gene promoter methylation status was analyzed in Aspc1 and Panc1 cells after IL-6 (100 ng/ml) and Bxpc3 and Capan2 cells after S31-201 (a STAT3 inhibitor, 10μM) treatment for 24 h using MSP. d Bxpc3 cells were infected with lentiviral vectors carrying control vector (EV) or DNMT1-specific shRNAs (shDNMT1), then followed by puromycin selection. Protein expression levels of DNMT1 and SOCS3 were analyzed in Bxpc3/EV and Bxpc3/shDNMT1 cells in the absence or presence of IL-6 (100 ng/ml) for 24 h. e Protein expression levels of SOCS3 were analyzed in Bxpc3 cells after 5-Aza (5μM) treatment for 72 h (with medium changed every day). Protein expression levels of SOCS3 were also analyzed in Panc1 cells which were cultured in the presence of IL-6 (100 ng/ml) or 5-Aza (5μM) 72 h (with medium changed every day) using western blots. f Panc1 cells were co-transfected with the indicated plasmids (reporter plasmid EV alone or DNMT1 plasmid simultaneously). Then, 24 h after the transfection, the cells were treated with IL-6 (100 ng/ml) or 5-AZA (5μM) for 48 h, and the luciferase activity was measured. g Lysates of Bxpc3 cells were subjected to immunoprecipitation with rabbit IgG or pSTAT3 antibody, and then followed by western blot to detect the interaction of phosphorylated STAT3 with DNMT1. The effect of IL-6 on the interaction between phosphorylated STAT3 and DNMT1 in Panc1 cells were also analysed using western blots. * represents the location of DNMT1 (183 kd)