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. Author manuscript; available in PMC: 2016 May 18.
Published in final edited form as: Nat Genet. 2015 Oct 19;47(12):1475–1481. doi: 10.1038/ng.3421

Figure 3.

Figure 3

NRF2 regulates the expression of serine/glycine biosynthesis genes through ATF4. (a) ATF4 mRNA expression in A549 cells expressing scramble shRNA (SCR), or NRF2 shRNA #1. (b) Western blot of NRF2, ATF4 and ACTIN expression in cells from (a). (c) Western blot of NRF2, ATF4 and serine pathway enzyme expression in lysates from A549s expressing scramble (SCR), NRF2 shRNA #1, or ATF4 shRNAs #1 or #2. (d) mRNA expression in cells from (c). (e) ATF4 knockdown impairs serine biosynthesis. Cell lines from were grown in the presence of U-13C-glucose for the indicated time points, the metabolites extracted and the fractional 13C-labeling on serine analysed by LC/MS. (f) ATF4 rescues serine biosynthesis enzyme expression following NRF2 knockdown. A549 cells were infected with lentivirus encoding mATF4 prior to infection with scramble or NRF2-targeting lentivirus. (g) Western analysis of NRF2, ATF4, and ACTIN expression in the cells from (f). (h) ATF4 rescues the serine biosynthesis defect in shNRF2 A549 cells. Cells were assayed as in (e). (i) ATF4 rescues the growth of H1975 cells in serine deficient media. Cells expressing luciferase (LUC) or ATF4 were grown in the indicated media for 3 days and cell number normalized to cells grown in full media. (j) Chromatin immunoprecipitation of ATF4 to the PHGDH, PSAT1 and SHMT2 promoters. Samples were normalized to IgG control immunoprecipitations. Results are the average of 3 technical (a, d, f, j) or biological (e, h, i) replicates.