Fig. 2.

Overexpression of CK1δ impairs and inhibition of CK1δ by D4476 increases formation of HIF-1α/ARNT complexes.

(a) HeLa cells were co-transfected with pcDNA3.1 or pcDNA3.1-CK1δ and pEGFP plasmids. Twenty hours post-transfection cells were incubated under hypoxia (1% O₂) for 4 h and HIF-1α/ARNT complexes were detected by in situ PLA. (b) Detection of HIF-1α/ARNT complexes, in the absence or presence of D4476 (10 μΜ) under hypoxia (1% O₂) for 4 h, by in situ PLA. For (a) and (b): left panels: microscopical images. Right panels: quantification of results presenting the average number of nuclear dots per cell ± SEM (n = 50). (c) Determination of HIF-1 transcriptional activity in HeLa cells incubated for 16 h under normoxia or hypoxia (1% O₂) in the absence or presence of D4476 (10 μΜ). Results are shown as fold increase in relation to the corresponding normoxic conditions and represent the mean of three independent experiments performed in triplicate ± SEM. (d) Western blot analysis of HeLa cells incubated for 4 h under normoxia or hypoxia (1% O₂) in the absence or presence of D4476 (10 μΜ), for detection of HIF-1α and ARNT protein levels. (e) Histograms show the HIF-1α/actin (left) or ARNT/actin (right) protein levels ratio according to quantification of blots from three independent experiments performed as in (d).