Fig. 5: NONO stabilizes HIF-1 complex and HIF-2 complex.

A HepG2 cell lysates were incubated with GST-fusion proteins of the indicated fragments of HIF-1ɑ in GST pull-down assays. B, C The effect of NONO on the stabilization of HIF-1ɑ/β complex (B) and HIF-1ɑ/CBP complex (C) in Hep3B was determined by performing PLA in the presence or absence of NONO. Protein complexes were presented as red clusters, and cell nuclei were showed as blue ovals. Scale bar = 10 μm (×63). D Huh7 cells with NONO knockout or not were treated with 1% O₂ for 24 h, then was collected to be immunoprecipitated with anti-HIF-1ɑ antibody, finally loaded for western blotting with indicated antibodies. IgG as negative control. E Lysate from Huh7 cell with NONO knockout or not treated with 1% O₂ treatment for 24 h was subject to CUT&RUN assay. CUT&RUN assay products were quantified by qPCR with the indicated pairs of primers. F HepG2 cell lysates were incubated with GST-fusion proteins of the indicated fragments of HIF-2ɑ in GST pull-down assays. G, H The effect of NONO on the stabilization of HIF-2ɑ/β complex (G) and HIF-2ɑ/p300 complex (H) in SMMC-7721 was determined by performed PLA in the presence or absence of NONO. Protein complexes were presented as red clusters, and cell nuclei were showed as blue ovals. Scale bar = 10 μm (×63). I HepG2 cells were transfected with HA-tagged HIF-2ɑ for 48 h, then cell lysates were collected to incubate with HA-tagged beads. The presence of HA-tagged HIF-2ɑ or endogenous p300 proteins were analyzed by immunoblotting. IgG as negative control. J HepG2 cell was transfected with Flag-tagged HIF-1ɑ (left) or HIF-2ɑ (right) plasmids for 48 h, then cells were collected and incubated with indicated fragments of NONO in GST pull-down assays.> K HepG2 cells with NONO-knockout were transfected with empty vector, full-length of NONO or truncated mutant (aa1-274 or aa275-471) for 24 h, followed by hypoxia treatment (1% O₂) for 24 h. Finally, the relative expression of HIF-1/2 target genes were determined by RT-qPCR.