Fig. 5.

SIAH2 regulates nuclear-encoded mitochondrial gene expression through NRF1. a Wild-type or SIAH2^−/− MDA-MB-231 cells were transiently transfected with scramble or NRF1-targeted siRNA then cultured under normoxia or hypoxia for 36 h. Cells were harvested and analyzed by western blotting. Bottom: densitometric quantification of the indicated proteins. b Statistical analysis of qRT-PCR data from cells treated as in (a). qRT-PCR results were normalized to the housekeeping gene B2M. c Stable NRF1-knockdown MDA-MB-231 cells reconstituted with wild-type NRF1 or the NRF1-K230R mutant, together with mock and NRF1-knockdown MDA-MB-231 cells, were cultured under normoxia or hypoxia for 36 h and analyzed by immunoblotting. Bottom: densitometric quantification of the indicated proteins. d Cells treated as in (c) were analyzed by qRT-PCR and the data were statistically compared. qRT-PCR results were normalized to the housekeeping gene B2M. e MDA-MB-231 cells stably expressing K230R-NRF1 were cultured under hypoxia and then analyzed by immunoblotting using the indicated antibodies at the indicated time points. Detailed statistical data are shown in Supplementary Fig. 2d. For all panels, error bars indicate s.d., n = 3 biological replicates, average of n = 3 technical replicates for each biological replicate was used in (b) and (d). The two-tailed paired ratio t-test was used to compare data in (a-d) and one-way ANOVA was used to compare data in (e)