Fig. 4: The interaction between NONO and HIF-1/2 was enhanced under hypoxia.

A–E In situ proximity ligation assay (PLA) on Hep3B cells demonstrated the interaction between NONO and HIF-1ɑ (A), HIF-1β (B), HIF-2ɑ (C), CBP (D), and p300 (E) under normoxia or hypoxia. Positive PLA signals showed HIF-1ɑ/NONO complex (A), HIF-1β/NONO complex (B), HIF-2ɑ/NONO complex (C), CBP/NONO complex (D), or p300/NONO complex (E) which were shown as red clusters, and cell nuclei were counterstained with blue. Scale bar = 10 μm (×63). F HepG2 cells were transfected with HA-tagged NONO plasmid for 24 h, followed by hypoxia (1% O₂) or normoxia treatment for 24 h. Finally, cells were collected to immunoprecipitated with anti-HA antibody, loaded for western blotting with anti-HIF-1α, HIF-2ɑ, HIF-1β, and CBP/p300 antibodies. IgG as negative control. G Computational docking models for human NONO (green) and HIF-1ɑ (red), HIF-1β (blue), HIF-2ɑ (purple), CBP (yellow), p300 (cyan) were predicted using ZDOCK. H, J The interaction between DBHS proteins and HIF-1ɑ (H) or HIF-1β (I) or HIF-2ɑ (J) in HepG2 cells was also determined by performing PLA (upper panel) and Co-IP (lower panel). For PLA assays, protein complexes were presented as red clusters, and cell nuclei were showed as blue ovals. Scale bar = 10 μm (×63). For Co-IP assays, cells were co-transfected with HA-tagged DBHS plasmids and Flag-tagged HIF-1ɑ (H) or HIF-2ɑ (J) for 48 h. Then cells were collected, immunoprecipitated with anti-HA antibody, loaded for western blotting with indicated antibodies. IgG as negative control.