Figure 4: lncRNA LncHIFCAR associates with HIF-1α and functions as a HIF-1α co-activator.

(a) Representative (n=3) western blot analysis of HIF-1α protein levels in LncHIFCAR-overexpressing HeLa cells and controls (lower panel), or in siRNA-transfected SAS cells (upper panel) under 24 h of normoxia (N) or hypoxia (H) treatment. NC, negative control. (b) lncRNA LncHIFCAR enriched in HIF-1α immunoprecipitates under hypoxia. Sixteen hours normoxia- or hypoxia-treated SAS nuclear extracts were immunoprecipated using mouse IgG or anti-HIF-1α antibody. Immunoprecipitation of HIF-1α-associated RNA was validated by qRT–PCR and shown as the relative fold of the RNA enrichment. (c) Representative (n=3) immunoblot detection of HIF-1α retrieved by biotinylated LncHIFCAR RNA pull-down assay. Biotin-labelled lambda RNA, sense or antisense LncHIFCAR RNAs were incubated with 16 h hypoxia-treated HeLa nuclear extracts and then pulled down by streptavidin beads, followed by western blot analysis. (d,e) HIF-1α binding domain on LncHIFCAR. RNAs corresponding to indicated LncHIFCAR fragments were biotinylated and incubated with recombinant HIF-1α protein, followed by streptavidin pull-down as described above. Representative (n=3) immunoblot detection of the associated HIF-1α protein was shown. CB, Coomassie brilliant blue staining. (f) HIF-1 target gene expression in SAS cells overexpressing control vector, wild-type or mutant (500–1,500) LncHIFCAR RNA. Specific primers are designed for the quantification of wild-type (region A) or mutant (500–1,500) (region B) LncHIFCAR levels as shown in the schematic diagram and the primer sequences are provided in Supplementary Data 2. (g) LncHIFCAR binding domain within HIF‐1α. Schematic representation of HIF‐1α functional domains and GST-HIF-1α variants are shown at the top. The Coomassie Blue staining showed loading of the proteins and arrowheads mark the GST-HIF-1α truncates. LncHIFCAR RNA were pulled down by GST-fusion proteins pre-bound on glutathione-Sepharose beads, followed by qRT–PCR detection of LncHIFCAR RNA retrieved as presented as percentage relative to input RNA. bHLH, basic helix–loop–helix; PAS, Per-ARNT-Sim; TAD, transactivation domain. (h) LncHIFCAR facilitates HIF-1α complex formation. HIF-1α was immunoprecipitated from hypoxic vector control or LncHIFCAR knockdown SAS cell extracts. Co-immunoprecipitation of p300 and HIF-1β was reduced in LncHIFCAR knockdown cells. Graphs show mean±s.d. n, the number of independent experiments performed.