Figure 4.

Cezanne regulates proteasome-independent degradation of HIF-1α

A Decreased HIF-1α levels in Cezanne knockdown cells were complemented by co-depletion of p97.

B, C Reduced HIF-1α levels caused by Cezanne depletion can neither be complemented with the proteasome inhibitor MG132 (B) nor the hydroxylase inhibitor DMOG (C).

D Control and Cezanne-depleted cells were treated with or without hypoxia and DMOG. Loss of Cezanne reduced HIF-1α-dependent luciferase activity also when hydroxylases were inhibited.

E, F Knockdown of Cezanne in VHL-deficient RCC4 cells (E, lanes 1 and 2) and A498 cells (F) had no effect on HIF-1α levels. Expression of exogenous VHL sensitised RCC4 cells for Cezanne regulation (E, lanes 3–6).

G Cells were exposed to hypoxia and subsequently treated with cycloheximide to block protein translation. HIF-1α degradation under these conditions was followed over time.

H Cells were treated with hypoxia before cycloheximide and MG132 were added. HIF-1α degradation could be rescued with proteasome inhibition in control cells but not in Cezanne-depleted cells.

I Loss of Cezanne did not affect protein levels of HIF-1α with mutations in the KFERQ-like motif (GFP-HIF-1α (AA)).

J Model: Cezanne prevents proteasome-independent HIF-1α degradation.

Data information: All experiments were performed three times, experiment in panel (E) was repeated four times; bar graphs represent the mean plus standard deviation of these independent experiments. P-values were calculated using Student's t-test (***P < 0.001).