Fig 2: PITX1 controls HIF-1β levels at the JMJD2A, JMJD2B and JMJD2C promoters. (A) HeLa cells were transfected with control or PITX1 siRNA, prior to treatment with 1% O2 for 24 hours. Cells crosslinked and processed for chromatin immunoprecipitation, using HIF-1α and HIF-1β antibodies. Levels of these transcription factors were analyzed at the JMJD2B promoter by qPCR. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (B) HeLa cells were treated and processed as in A, but levels of HIF-1β present at the JMJD2A, JMJD2C and CA9 promoters were analyzed. Levels of HIF-1α are also presented for JMJD2A and JMJD2C. Graph depicts mean and standard deviation of a minimum of 3 independent experiments, Figure 5 (See previous page). except for JMJD2A, which presents the mean and standard deviation of 2 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001. (C) HeLa cells were co-transfected with HIF-1β and empty vector or PITX1 expression constructs for 48 hours prior to crosslinked and lysed. Chromatin immunoprecipitation was performed using the indicated antibodies. Levels of HIF-1β were analyzed at the indicated promoters by qPCR. Graph depicts mean and standard deviation of a minimum of 3 independent experiments. Student's t-test was performed to calculate p values, and levels of significance are denoted as follows: *P < 0.05, **P < 0.01, and ***P < 0.001.