AA and thrombin-induced platelet HETE production in patient (■, mean of 2 visits) and control subjects (□, mean ± SE).

Luciferase reporter studies on ALOX12 promoter in PMA-treated HEL cells. (A) Schema showing 5′ upstream region of ALOX12 gene with 4 RUNX1 consensus sites. (B) Effect of 5′ sequential truncation of ALOX12 promoter region on expression of luciferase reporter in untreated (■) and PMA-treated (□) HEL cells. Fold increase in luciferase activity was measured as ratio of control vector to luciferase vectors carrying various lengths of promoter and normalized to Renilla luciferase constructs. Data are mean ± SEM of 3 experiments. (C) Effect of disruption of individual RUNX1 sites on ALOX12 expression. Each RUNX1 site was disrupted sequentially by introducing point mutations (□ boxes) in RUNX1-binding sites. Data are mean of 3 experiments.

EMSA using oligonucleotide probes with RUNX1-binding sites 1A, 1B, 2, and 3 and nuclear extracts from PMA-treated HEL cells. (A) EMSA using wild-type oligonucleotide probe with sites 1A (−1498/−1493) and 1B (−1491/−1486), and nuclear extract from PMA-treated HEL cells. Lane 1 indicates probe alone; lane 2, probe with nuclear extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled site 1A mutant probe; lane 5, competition with unlabeled site 1B mutant probe; lane 6, effect of RUNX1 antibody; lane 7, effect of nonspecific IgG; and lane 8, competition with unlabeled probe with sites 1A and 1B mutated. (B) EMSA using oligonucleotide probes with site 1A mutated (mutant probe 1A lanes 1-5) or site 1B mutated (mutant probe 1B lanes 6-10). Lanes 1 and 6 indicate probe alone; lanes 2 and 7, protein binding with nuclear extract; lanes 3 and 8, competition with excess respective unlabeled probe; lanes 4 and 9, effect of nonspecific IgG; and lanes 5 and 10, inhibition of binding with RUNX1 antibody. (C) EMSA using oligonucleotide probe with site 2 (−708/−703). Lane 1 indicates free probe; lane 2, protein binding with extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled probe with site 2 mutated; lane 5, inhibition of binding with RUNX1 antibody; and lane 6, effect of nonspecific IgG. (D) EMSA using oligonucleotide probe with site 3 (−526/−521). Lane 1 indicates free probe; lane 2, protein binding with extract; lane 3, competition with excess unlabeled probe; lane 4, competition with unlabeled probe with site 3 mutated; lane 5, inhibition of binding with RUNX1 antibody: and lane 6, effect of nonspecific IgG.

EMSA using oligonucleotide probes with RUNX1-binding sites 1A, 1B, 2, and 3 and recombinant RUNX1. (A) EMSA using oligonucleotide probe with sites 1A and 1B and recombinant RUNX1. Lane 1 indicates probe alone; lane 2, probe with recombinant RUNX1; lane 3, competition with excess unlabeled probe; lane 4, effect of RUNX1 antibody (arrow, supershift); and lane 5, effect of nonspecific IgG. (B) EMSA using recombinant RUNX1 and oligonucleotide probes with site 2 (−708/−703; lanes 1-5) and with site 3 (−526/−521; lanes 6-10). Lane 1 indicates free probe with site 2; lane 2, protein binding with recombinant RUNX1; lane 3, competition with excess unlabeled probe; lane 4, effect of RUNX1 antibody (, supershift); lane 5, effect of nonspecific IgG; lane 6, free probe with site 3; lane 7, protein binding with recombinant RUNX1; lanes 8, competition with excess unlabeled probe; lane 9, effect of RUNX1 antibody (, supershift): and lane 10, effect of nonspecific IgG.

Binding of RUNX1 to ALOX12 promoter region in vivo. ChIP using control IgG (column 1) and RUNX1 antibody (column 2). Column 3 shows PCR of total input DNA. Data are PCR amplification of ALOX12 promoter regions encompassing sites 1A and 1B (−1679/−1376 nt), site 2 (−840/−600 nt), site 3 (−597/−360 nt), and glyceraldehyde-3-phosphate dehydrogenase.

Effect of siRNA RUNX1 knock down on ALOX12 promoter activity and protein. (A) Effect on ALOX12 promoter activity. HEL cells were cotransfected with RUNX1 or mock siRNA and ALOX12 luciferase-reporter construct (−1593/−1). Bar graphs represent activity as mean ± SD of 3 experiments. (B) Western blotting analysis of RUNX1, 12-LO, and actin in HEL cells transfected with mock or RUNX1 siRNA. Data are representative of 3 experiments.

Effect of transient overexpression of RUNX1 on ALOX12 promoter activity. (A) Effect on ALOX12 promoter activity. HEL cells were cotransfected with RUNX1-pCMV6 expression vector (■), empty pCMV6 vector (), or neither (□), along with ALOX12 luciferase-reporter construct (−1593/−1), wild-type, or with mutations in sites 1A, 1B, 2, or 3. Reporter activity was measured at 48 hours. Bar graphs represent activity as mean ± SD of 3 experiments. (B) Western blotting analysis of RUNX1 and actin (control) in HEL cells transfected with pCMV6 vector alone or with RUNX1-pCMV6 vector.