Figure 5
Inhibition of Miz-1, Arnt and Hifα expression reduces CDKN2B mRNA levels. A-C) U2OS cells were transfected with siRNA (10 nM) against Arnt, Miz-1, Hif1α and Hif2α as indicated and cultured at normoxia (21% O2) or hypoxia (1% O2) for 6 hours. A) qRT-PCR analyses of CDKN2B mRNA levels after administration of the different siRNAs are presented as relative fold changes compared to control siRNA at normoxia (column 1). B) qRT-PCR analysis of mRNA expression of Arnt, Miz-1, Hif1α and Hif2α after siRNA treatment. The mRNA levels after administration of the different siRNAs are presented as relative fold changes compared to control siRNA at normoxia for each specific mRNA species. C) Protein levels of Arnt (upper left panel), Miz-1 (lower left panel), Hif1α (upper right panel) and Hif2α (lower right panel) in response to siRNA treatment and hypoxia were determined by immunoblotting. Immunoblotting for beta-actin was included to control for protein loading. D) U2OS cells were cultured at normoxia (21% O2) or at hypoxia (1% O2) for the time points indicated, and qRT-PCR analyses for Miz-1 mRNA expression were performed. The qRT-PCR values in A, B and D were normalized to the housekeeping gene HPRT-1, and are shown as average of two or three independent experiments performed in duplicates (n = 4 for A and B, n= 6 for D). E) U2OS cells were transfected with siRNA (10 nM) against Miz-1, Hif1α and Hif2α or a negative control siRNA together with the −35CDKN2B/luc reporter (300 ng) in the presence or absence of an Arnt expression vector (Arnt/Flag/pCMV, 100 ng) as indicated. The luciferase activity is presented as average +/− stdev, n = 3. Statistical analyses: (A-B and E) Student’s T-test, (D) ANOVA (Bonferroni); ***p ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05, ns: non significant.