Figure 2. Increased glycolysis and depletion of aspartate in early hypoxia are independent of HIF1α.

(A) Western blot to assess levels of HIF1α and a panel of HIF1α targets in wild-type (wt) MCF7 and HIF1α^mut MCF7 cells incubated in 21% O₂ or at 1% O₂ for the indicated lengths of time. The asterisk marks a HIF1α immunoreactive band of smaller molecular weight than HIF1α that increases upon hypoxia in HIF1α^mut MCF7 cells, indicative of a truncated HIF1α that likely lacks the transactivation domain where the sgRNA sequence is targeted at. See also Appendix Fig. S2A. (B) Log₂ fold changes in mRNA expression levels of a panel of HIF1α targets in MCF7 cells exposed to 1% O₂ for 3 or 24 h, compared to control cells at 21% O₂. (C) Heatmap showing log₂ fold changes in mRNA expression levels of a panel of HIF1α targets in wild-type (wt) and HIF1α^mut MCF7 cells exposed to 1% O₂ for 3 or 24 h, compared to control cells in normoxia. (D) Fraction labelled (left) and absolute abundances of the M + 2 isotopologue (right) of citrate from [U-¹³C]-glucose in wild-type (wt) and HIF1α^mut MCF7 cells after incubation with the tracer at 21% O₂ or 1% O₂ for the indicated lengths of time. Time points indicate both the duration of hypoxia treatment and incubation with the tracer. See also Appendix Fig. S2C. (E) Changes in lactate and aspartate abundance in wild-type (wt) and HIF1α^mut MCF7 cells incubated in 21% O₂ or 1% O₂ for the indicated lengths of time, compared to control cells in normoxia. Data information: Data are representative of experiments with similar conditions performed independently N times as follows: N ≥ 2 (A, D, E), N = 1 (B, C). Datapoints in (D, E) represent mean ± s.d. n = 3 (B, C) and n = 4 (D, E) cultures for each time point and condition. Statistical errors in (D, left and E) were propagated to calculate variance of the change in isotopic labelling between normoxia and hypoxia for each cell line. FDRs in (B, C) were calculated using the ’exactTest’ function of the edgeR package (see 'Methods') with a cut-off set at 1%; only changes with FDR < 0.01 are shown. The P values shown were calculated by two-way ANOVA Sidak’s test (D, left and E) or two-way ANOVA Tukey’s test (D, right). ns non-significant, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Source data are available online for this figure.