Fig. 2
Identification of ETV5 promoter element responding to ERK1/2 activity. (A) Human genomic sequence retrieved from GRCh38/hg38 between − 2458 and + 25 from the transcription start site of ETV5. Underlines indicate restriction enzyme sites. The double underline indicates exon 1 of ETV5. Boxes indicate putative binding sites of ETS transcription factors in the most probable responsive region to ERK1/2 (the hatched box in #2 of C). (B) Reporter assays using PGV-P2 reporter vectors replaced SV40 promoter region with the human genomic sequences between − 2458 and + 25 from the transcription start site of ETV5 (Full) and no genomic sequences (None) in human pancreatic cancer cells, AsPC-1, MIAPaCa-2, and PCI-35, showed strong promoter activities of Full. (C) Reporter activities of various truncated promoter sequences using AsPC-1 and MIAPaCa-2, The significance level was corrected by Bonferroni method to 0.0125. *p < 0.0125, **p < 0.001, ***p < 0.001. (D) Reporter activities of Full and #2 in AsPC-1 and MIAPaCa-2 treated with U0126. (E) Site directed mutagenesis of consensus binding sites of ETS transcription factors in #2 promoter sequence (M1-M8; Table 2) on reporter activities in AsPC-1 and MIAPaCa-2. Black bars indicate less activities compared to the non-mutated #2 promoter sequence. The significance level was corrected by Bonferroni method to 0.00625. *p < 0.00625, **p < 0.001, ***p < 0.001. (F) Chromatin immunoprecipitation assay using anti-ETS1 antibody for candidate ETS1 binding site (M8). PCR products shown were input control (lane 1), immunoprecipitants with anti-ETS1 antibody (lane 2), immunoprecipitants with nonspecific immunoglobulin (lane 3), and negative control (lane 4). Unless otherwise noted, *p < 0.05, **p < 0.01, ***p < 0.001. All data are means and standard errors of du-plicates.
