Figure 2 from Hypoxia-Induced Long Noncoding RNA HIF1A-AS2 Regulates Stability of MHC Class I Protein in Head and Neck Cancer

Direct regulation of HIF1A-AS2 by HIF1α. A, ChIP-seq analysis of the HIF1α binding on the regulatory region of HIF1A-AS2 in human umbilical vein cell line (EA.hy926), human prostatic adenocarcinoma cell (PC3), human renal cell carcinoma cell line (RCC4), and human squamous cell carcinoma of the hypopharynx (FaDu). ChIP-seq data were extracted from GEO: GSM3402530, GSM3145502, GSM3417826, and GSM5224574 and analyzed using ChIP-Atlas. B, Top: Schematic representation of the organization of the regulatory region of human HIF1A-AS2 and the luciferase reporter constructs used in the experiment. The HREs are indicated. Bottom: Luciferase reporter assay. Bottom left, 293T cells were co-transfected with HIF1α(ΔODD) /control vector and the reporter plasmid. Lower right, 293T cells were co-transfected with HIF1α(ΔODD) and the indicated reporter plasmid. Data represent the mean ± SD. n = 3 independent experiments (each experiment contains two technical replicates). C, Left top: Western blots of HA for indicating the establishment of the HA-HIF1α(ΔODD) constitutive expression in SAS [HIF1α(ΔODD) versus SAS-vector control (Vec)] cells. Left bottom: RT-qPCR for analyzing the expression of HIF1A-AS2 in SAS-HIF1α(ΔODD) versus SAS-vector control (Vec) cells. Data represent the mean ± S.D. n = 3 independent experiments (each experiment contained two technical replicates). Right top: schema showing the regulatory regions of HIF1A-AS2 and the ChIP/control primers for the experiment. Right bottom: quantitative ChIP for analyzing the enrichment of HIF1α at the HIF1A-AS2 regulatory region in SAS-HIF1α(ΔODD) versus SAS-vector control (Vec) cells. One representative experiment of three independent experiments is shown. The replicates are presented in Supplementary Fig. S2. *, P < 0.05; **, P < 0.01; ***, P < 0.001.