Fig. 2.

Hypoxia transcriptionally regulates nuclear-encoded mitochondrial gene expression through NRF1. a, b Analysis of protein levels determined by immunoblotting using the indicated antibodies (a), and of mRNA levels, determined by real-time PCR (qRT-PCR) for the indicated genes (b), at the indicated time points in MDA-MB-231 cells cultured under hypoxia. qRT-PCR results were normalized to the housekeeping gene B2M. Detailed statistical data of (a) are shown in Supplementary Fig. 1a. c MDA-MB-231 cells were pretreated with 25 µg ml⁻¹ cycloheximide (CHX) for 2 h under normoxia, then cultured under hypoxic conditions for the indicated time points. Cells were harvested and analyzed by immunoblotting using the indicated antibodies. Detailed statistical data are shown in Supplementary Fig. 2b. d Stable NRF1-knockdown MDA-MB-231 cells cultured under hypoxia were analyzed by immunoblotting using the indicated antibodies at the indicated time points. Detailed statistical data are shown in Supplementary Fig. 2c. e NRF1 siRNA was transfected into indicated cell lines and cultured under hypoxia for 36 h and then cell lysates were analyzed by immunoblotting using the indicated antibodies. 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). One-way ANOVA was used to compare data