Fig 1: Asparagine levels regulate serine uptake and serine metabolism gene expression.(a) Asparagine levels in the media, measured by LC-MS, from serum- and amino acid-starved LPS2 cells pre-loaded with asparagine (Pre N) and unstimulated (No AA) or stimulated with different amino acid sub-categories: non-polar, basic or Ser/Thr. Non-polar amino acids include leucine, isoleucine, methionine, tryptophan and phenylalanine; basic amino acids include lysine, arginine and histidine; Ser/Thr includes serine and threonine. (b) Relative intracellular asparagine levels in amino acid-starved glutamine-independent LPS2 cells as measured by LC-MS following 1-, 5- or 120-min stimulation with complete glutamine-free medium. Prior to stimulation, cells were either pre-loaded with asparagine (Pre N) or starve medium (No pre). Declining intracellular asparagine levels over time indicates exchange with extracellular amino acids. (c) Relative intracellular amino acid levels in amino acid-starved glutamine-independent LPS2 cells as measured by LC-MS following 5-min stimulation with complete glutamine-free medium. Prior to stimulation, cells were either pre-loaded with asparagine (Pre N) or starve medium (No pre). (d) Immunoblot showing serine synthesis pathway protein levels in HeLa cells, 48 h post doxycycline induction of scrambled shRNA or ASNS shRNA expression in the presence or absence of 0.1 mM exogenous asparagine. (e) Levels of ATF4 binding to the indicated gene by ChIP–qPCR in HeLa cells 48 h post doxycycline induction of scrambled shRNA or ASNS shRNA expression in the presence or absence of 0.1 mM exogenous asparagine. Chromatin was immunoprecipitated using anti-ATF4 antibody or IgG as a negative control. Values indicate amount of immunoprecipitated DNA as a percentage of input chromatin. (f) Immunoblot showing ASNS, ATF4, PSAT1 and PSPH protein levels with or without a 48-h doxycycline induction of ASNS shRNA in HeLa cells stably expressing scrambled or ATF4 shRNA. Error bars denote s.d. of the mean (n=3). P values were calculated by the Student's t-test: *P<0.05; **P<0.01; ***P<0.001. NS, not significant.