Figure 5. Serine dependence in cells with compromised mitochondrial function.
(a) Growth of T-REx-293 cells treated with OXPHOS inhibitors, with and without serine, and with serine replaced by formate or hypoxanthine. (b) Same, but of T-REx-293 cells depleted of mtDNA using EtBr. (c) Growth of T-REx-293 cells with knockouts of SHMT1 or SHMT2, with and without serine, and with serine replaced by formate. (d) Growth of S. cerevisiae in nonfermentable media with 5 nM antimycin (see Materials and methods), in the presence or absence of serine and adenine. n = 3 for panels a-c; n = 4 for panel d.
Figure 5—figure supplement 1. Additional data on RC inhibition-induced serine dependency in T-REx-293 cells.
(a) Quantitated growth rates, with and without media serine, for T-REx-293 cells treated with different mitochondrial inhibitors. n = 3. (b) Suppression of cellular growth in galactose, a nonfermentable sugar (Gohil et al., 2010), by a dose of CCCP (5 μM) that does not elicit serine dependency. n = 3. (c) Serine dose response curve of T-REx-293 cell growth in the presence of antimycin. n = 2.
Figure 5—figure supplement 2. SHMT1 and SHMT2 single and double knockouts.
(a) Western blots (top) and growth rates (bottom) for fourteen single cell clones derived from simultaneous CRISPR transfection for SHMT1 and SHMT2 knockout. All clones were expanded in the presence of hypoxanthine and thymidine (HT). (b) Western blots showing SHMT1 and SHMT2 single and double knockouts. (c) Formate synthesis from mitochondria isolated from SHMT1 and SHMT2 knockout cell lines. (d) Replicate serine dependence data from single knockouts. (e) Growth of SHMT1/SHMT2 double KO cells (two independent clones) with different 1C-related supplements. (f) 1C-rescued double knockouts do not show serine dependence, even when treated with RC inhibitor. n = 1 for panel a; n = 3 for panels c–f.
Figure 5—figure supplement 3. RC inhibition-induced serine dependence in other cell types.
n = 3.