Fig. 3: HIFAL nuclear translocation upon hypoxia drives PKM2/PHD3 complex into the nucleus.

a–c Knockdown of HIFAL prevents the nuclear translocation of PKM2 and PHD3. Cells were transiently transfected with HIFAL-LANs for 48 h, and then cultured under hypoxia for 24 h. HIFAL (green) was detected by digoxin labeled probes. PKM2 (a) or PHD3 (b) (red) were immunostained with antibodies. The cell lysates were fractionated and the cytoplasmic or nuclear expression of PKM2 and PHD3 was evaluated by western blotting (c). Scale bars, 10 μM. d Exogenous HIFAL translocates into the nucleus upon hypoxia. The HIFAL null MDA-MB-231 cells were transiently transfected with exogenous HIFAL RNA for 48 h and then hybridized with digoxin labeled HIFAL probes (lower panel). The level of endogenous or exogenous (EX) HIFAL in MDA-MB-231WT or HIFAL null cells was detected by qRT-PCR. Scale bars, 10 μM. e, f HIFAL knockout prevents the nuclear translocation of PKM2 and PHD3 under hypoxia. The HIFAL null MDA-MB-231 cells were cultured under normoxia or hypoxia and then subjected to immunofluorescence staining of PKM2 (e) and PHD3 (f) and ISH of HIFAL. Scale bars, 10 μm. g, h Exogenous HIFAL induces the translocation of PKM2 (g) and PHD3 (h) into nucleus upon hypoxia. Scale bars, 10 μM. i Nuclear HIFAL expression correlates with nuclear PKM2 and PHD3 expression in breast cancer tissues. Representative image showed HIFAL expression detected by ISH and PKM2 and PHD3 expression detected by immunohistochemistry. Scale bars, 50 μM. j, k, Knockdown of HIFAL reduces PKM2 binding with HIF-1α and p300. Cells were transiently transfected with HIFAL-LANs for 48 h, and then cultured under hypoxia. Knockdown of HIFAL reduces PKM2 binding with HIF-1α (j) and p300 (k). The right panel shows the relative HIFAL level in the MDA-MB-231 cells. For d (upper), j (right), k (right), bar graphs represent means ± SD of experimental triplicates, and p values were determined by two-sided unpaired t-test. Source data are provided as a Source Data file.