Role of Sp1 in CD39 hypoxia inducibility. (A) Confluent HMEC-1 monolayers were transiently transfected with plasmids expressing sequence corresponding to truncations at the 5′ end (CD39-300, −217 to +83 bp) or plasmids encoding Sp1 or GATA3 mutations, as indicated. Twelve hours later, cells were exposed to hypoxia or normoxia for 48 hours and assessed for luciferase activity. All transfections were normalized to cotransfected Renilla plasmid. Data are mean plus or minus SEM from 3 separate experiments (*P < .025 compared with the corresponding nonmutated control). (B) ChIP assay was used to examine Sp1 binding to the CD39 promoter in normoxic and hypoxic HMEC-1 cells. Reaction controls included immunoprecipitations using a nonspecific IgG monoclonal antibody and PCR performed using HMEC-1 DNA (Input). A representative experiment of 3 is shown. (C) Confluent HMEC-1 monolayers were exposed to mock treatment (C), Sp1 sense oligonucleotides (S), or Sp1 antisense (AS) oligonucleotides for 48 hours. Total protein was solubilized, and Sp1 expression was examined by Western blot. As additional control, the transcription factor cAMP-response element-binding protein was probed. (D) Confluent HMEC-1 monolayers were transiently transfected with plasmids expressing sequence corresponding to truncations at the 5′ end (CD39-300, −217 to +83 bp) in the presence or absence of SP1 sense or antisense oligonucleotides. Twelve hours later, cells were exposed to hypoxia for 48 hours and assessed for luciferase activity. Data are mean plus or minus SEM from 3 separate experiments (*P < .01).