Figure 1. The WWTR1 gene encoding TAZ is a HIF-1 target gene.

(A) Reverse transcription and quantitative real-time PCR (RT-qPCR) were performed to quantify TAZ mRNA levels in 5 breast cell lines following exposure to 20% or 1% O₂ for 24 h. For each sample, the expression of TAZ mRNA was quantified relative to 18S rRNA and then normalized to lane 1 (mean ± SEM; n = 3). ^**P < 0.01, ^***P < 0.001 versus MCF-10A at 20% O₂; ^###P < 0.001 versus MCF-10A at 1% O₂. (B) Immunoblot assays were performed to analyze TAZ protein expression in breast cell lines following exposure to 20% (N) or 1% (H) O₂ for 48 h. (C) TAZ mRNA expression was analyzed by RT-qPCR in the MDA-MB-231 (left), MDA-MB-435 (middle) and MCF-7 (right) non-targeting control (NTC) or empty vector (EV) subclone and subclones expressing shRNA targeting HIF-1α (sh1α) or HIF-2α (sh2α), which were exposed to 20% or 1% O₂ for 24 h. Data were normalized to lane 1 in each bar graph (mean ± SEM; n = 3). ** P < 0.01 versus NTC or EV at 20% O₂; ^## P < 0.01 versus NTC or EV at 1% O₂. (D) Immunoblot assays were performed to analyze HIF-1α, HIF-2α, TAZ, and actin protein expression using lysates prepared from MDA-MB-231 subclones exposed to 20% or 1% O₂ for 48 h. (E) The nucleotide sequence (non-coding strand) of a hypoxia response element (HRE; 5′-GCGTG-3′ HIF-1 binding site and 5′-CACA-3′ accessory sequence are shown in red) within intron 2 of the WWTR1 gene, located 26 kb from the transcription start site, is shown. Exons and intron are not drawn to scale. (F) HCC-1954 cells were exposed to 20% or 1% O₂ for 16 h and chromatin immunoprecipitation (ChIP) assays were performed using IgG or antibodies against HIF-1α (upper panel), HIF-1β (middle panel), or HIF-2α (lower panel). Primers flanking the HRE were used for qPCR and results were normalized to lane 1 (mean ± SEM; n = 3). ^*P < 0.05 versus 20% O₂ (Student's t test). (G) The WWTR1 HRE containing a wild type (WT: 5′-GCGTG-3′) or mutant (MUT; 5′-GAAAG-3′) HIF-1 binding site was inserted into pGL2-Promoter (encoding firefly luciferase) and co-transfected with pSV-Renilla (encoding Renilla luciferase) into HCC-1954 cells, which were incubated at 20% or 1% O₂ for 24 h. The firefly:Renilla luciferase ratio (Luciferase activity) was normalized to lane 1 (mean ± SEM; n = 3). ^***P < 0.001 versus WT at 20% O₂; ^###P < 0.001 versus WT at 1% O₂ (Student's t test).