Fig. 5

Phosphorylation of SP1 was responsible for the up-regulation of MUC1 induced by CCL21-CCR7. a, b Silencing Sp1 could remarkably suppressed the up-regulation of MUC1 in KYSE410 and Eca9706 induced by CCL21. The starved siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 were treated with CCL21(100 ng/mL) for 24 h, then cells were harvested for immunoblotting for MUC1-C; c Silencing Sp1/Mutant of Sp1 binding site at −99/−97 could remarkably suppressed MUC1 promoter activity induced by CCL21. siSp1-Eca9706, siNC-Eca9706, siSp1-KYSE410 and siNC-KYSE410 transfected by MUC1-pGL2b luciferase reporter plasmid/KYSE410 and Eca9706 cells transfected by MUC1-pGL2b or MUC1 mutant-pGL2b -firefly luciferase reporter plasmid were treated with PBS or CCL21(100 ng/mL) for 24 h, then cells were harvested for detecting the luciferase activity; d Increasing phosphorylation of Sp1 induced by CCL21. Starved KYSE410 and Eca9706 cells were treated with CCL21(100 ng/mL) for 0, 0.5, 1,2 and 6 h, then cells were harvested for the immunoblot of p-Sp1; e The expression of p-Sp1 in KYSE410 treated with PBS or CCL21(100 ng/ml) for 6 h detected by immunofluorescence; f Blocking CCR7 could suppress the phosphorylation of Sp1 induced by CCL21. The starved KYSE410 and Eca9706 cells pretreated with the CCR7 antibody or IgG as control, were treated with PBS or CCL21 for 6 h, then cells were harvested for the immunoblot of p-Sp1; g Inhibiting ERK1/2 suppressed phosphorylation of SP1 induced by CCL21. Starved KYSE410 and Eca9706 cells were pretreated with DMSO as control or U0126 for 30 min. After treated with PBS or CCL21(100 ng/mL) for 6 h, the cells were harvested for immunoblot; h Inhibiting ERK1/2 suppressed Sp1 binding to MUC1 promoter at −99/−97. Starved KYSE410 cells were pretreated with DMSO as control or U0126 for 0.5. After treated with PBS or CCL21(100 ng/mL) for 12 h, the cells were harvested for the ChIP assay. The targeted DNA was amplified using MUC1 primers with 40 cycles of PCR. Each datapoint represents the mean ± SD of three repeated experiments. *P < 0.05