Figure 5: LncHIFCAR physically binds to the target chromatin and enhances the recruitment of HIF-1α and p300.

(a) ChIRP-qPCR detection of LncHIFCAR occupancy on the indicated target loci under normoxic and hypoxic conditions. ChIRP assays were performed with hypoxia (H)- or normoxia (N)-treated SAS cell lysate. Specific tiling biotinylated oligonucleotides complementary to LncHIFCAR or control LacZ RNA were used to pull down the RNA-associated chromatin. The inset graph shows the retrieved RNA level in the streptavidin pulled down complex, quantified by qRT–PCR. GAPDH mRNA was used to evaluate nonspecific binding of the biotinylated probes. The LncHIFCAR-associated HIF-1 target promoters were detected by qPCR. The retrieval of DNA was estimated as percentage of input chromatin, whereas Actin promoter served as a negative control region. (b,c) ChIP analysis of HIF-1α (b) and p300 (c) association with the promoter regions of HIF-1 target genes. The chromatin was prepared from vector control or LncHIFCAR knockdown SAS cell lines treated with normoxia (N) or hypoxia (H). ChIP was performed with anti-HIF-1α, anti-p300 or IgG and the recovered DNA was analysed by qPCR. The fold enrichment, indicated by fold of IgG, was calculated by normalizing the levels against nonspecific IgG-bound DNA. Inset, protein levels of HIF-1α and p300 in control and LncHIFCAR knockdown cells under normoxia (N) and hypoxia (H). Graphs show mean±s.d. n, the number of independent experiments performed.