Fig. 4: HIF1A-As2 directly interacts with DHX9.

A Silver staining of cell lysates upon HIF1A-As2 pull-down with biotinylated antisense probes after UV crosslinking in H1299 cells. Samples pre-treated with RNase A or pull-down with housekeeping gene UBC were used as negative controls. The yellow arrow indicates the band for DHX9. B The HIF1A-As2 pull-down cell lysate were performed with Mass Spectrometry. Mass Spectrometry profiling showing the peptide sequences of DHX9 listed on the top of the graph. Data are representative of two independent biological replicates. C Immunoblotting showing the DHX9 interacts with HIF1A-As2. D Schematic representation of Cross-linking RNA Immunoprecipitation (CLIP) assay. E Immunoblotting confirming the immunoprecipitation of DHX9. F CLIP assay indicating the enrichment of HIF1A-As2 in the DHX9 antibody-bound complex. UBC and lncRNA CCDST were used as negative and positive controls, respectively. G Sequential immunofluorescence (IF) and smFISH representative images showing the co-localization of HIF1A-As2 and DHX9 in CALU6 and H1299 cells. DAPI, blue; HIF1A-As2, red; DHX9, green. Scale bar, 75 μm. H Construct mapping of the binding region of HIF1A-As2 to DHX9. Bottom, Diagrams of full-length and different truncated HIF1A-As2 constructs. Middle, In vitro transcribed HIF1A-As2 constructs with according sizes. Top, Immunoblotting analysis for DHX9 upon pull-down by different HIF1A-As2 truncated constructs in H1299 cells. I CLIP assay showing the direct interaction between HIF1A-As2 and DHX9. Flag-tagged DHX9 FL, DHX9 dsRBDs Del or empty vector (NT) was co-transfected with pCDH-HIF1A-As2 plasmid in BEAS2B cells for 48 h. CLIP was performed with Flag-specific antibody (Top) and HIF1A-As2 level was examined by RT-qPCR (Bottom). Data show mean ± S.D (n = 3). **p value < 0.001, *p value < 0.05 by two-tailed Student’s t test.