Figure 7: Asparagine levels regulate nucleotide synthesis.
From: Asparagine promotes cancer cell proliferation through use as an amino acid exchange factor
(a) Immunoblot of HeLa lysates 48 h post doxycycline-induced expression of scrambled shRNA or ASNS shRNA in asparagine-free DMEM (−Asn) or DMEM supplemented with 0.1 mM asparagine (+Asn). Lysates were immunoblotted for mTOR activity marker pS6K, phospho-CAD (S1859), total CAD and tubulin. (b) Immunoblot of HeLa lysates with (shASNS+Dox) and without (shASNS−Dox) a 48-h induction of ASNS shRNA expression and HeLa lysates 48 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA (shASNS). Lysates were immunoblotted for ASNS, PRPS1, PRPS2 and tubulin. (c) Relative levels of asparagine and PRPP extracted from HeLa cells 48 and 72 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA expression as measured by LC-MS. (d) Percentages of the indicated metabolites labelled with 15 N extracted from HeLa cells labelled with exogenous 15 N-serine (50:50 15 N:14 N) for 24 h at 24 h post induction of scrambled shRNA (Scr) or ASNS shRNA expression. (e) Relative levels of the indicated intracellular metabolites extracted from HeLa cells 72 h after doxycycline induction of scrambled shRNA (Scr) or ASNS shRNA expression as measured by LC-MS. 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.
