Extended Data Fig. 5: Related to NADK facilitates DHFR activity in low folate conditions.
From: Cytosolic NADK is conditionally essential for folate-dependent nucleotide synthesis
(a) Immunoblots for expression of DHFR and NADK in control and DHFR-knockout cells. RAPTOR served as the loading control. (b) (left) Relative growth of NADK-knockout and MTX-treated control cells versus vehicle-treated control cells (mean ± s.d., n = 3 biologically independent samples). Two-tailed Welch’s t-test comparing the respective mean ± s.d. (bar) versus mean ± s.d. (vehicle-treated control cells) between bars. (right) MTX is a potent DHFR inhibitor. MTX, methotrexate. (c) Relative levels of tetrahydrofolate (THF), 10-formyl-THF (10-fTHF), and 5-methyl-THF (5-mTHF) in MTX- versus vehicle-treated control cells (mean ± s.e.m., n = 3 biologically independent samples). Two-tailed Welch’s t-test versus vehicle-treated control cells. (d) Schematic for a method to evaluate DHFR activity by measuring THF production from reactions containing recombinant DHFR and different combinations of folic acid, DHF, and NAD(P)H. (e) Pseudocolor Coomassie-stained gel imaged using a LIC-OR Odyssey FC. 1: M.W. standards; 2: DHFR-3xFLAG. (f) Schematic for a method to evaluate MTHFD1 (CD) activity by measuring 5,10-meTHF production from reactions containing recombinant MTHFD1, 10-formyl-THF, and NAD(P)H. (g) Pseudocolor Coomassie-stained gel imaged using a LIC-OR Odyssey FC. 1: M.W. standards; 2: MTHFD1-3xFLAG. (h) 5,10-meTHF levels measured from reactions that contained recombinant MTHFD1 with indicated substrate combinations (mean ± s.d., n = 3 independent reactions). Value above brackets indicates fold-change between bars. Two-tailed Welch’s t-test. (i) Dependency phenotypes for NADK from conditional essentiality profiling of the indicated cell lines using a focused sgRNA library11.
