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. 2016 Jun 16;5:e10575. doi: 10.7554/eLife.10575

Figure 4. Respiratory chain dysfunction impairs mitochondrial 1C metabolism.

(a) Confirmation of altered serine levels in spent media with EtBr-induced mtDNA depletion. n = 3. (b) Tracing serine metabolism using 13C-labeled glucose. Serine synthesis rates are inferred by labeling cells for 30 min with 13C6-glucose and measuring the amount of 13C3-serine that emerges. Simultaneously, serine consumption rates can be inferred by the amount of unlabeled serine that disappears during the labeling. n = 3. (c) Testing impairment of mitochondrial 1C metabolism using serine isotope scrambing in SHMT1 KO cells. Impairments in mitochondrial 1C metabolism downstream of 13CH2-THF are reflected in increased generation of 13C1-serine (see Figure 4—figure supplement 1). n = 2. (d) Testing impairment of mitochondrial 1C metabolism by assaying formate production from serine using isolated mitochondria with acute RC inhibition. n = 3.

DOI: http://dx.doi.org/10.7554/eLife.10575.013

Figure 4.

Figure 4—figure supplement 1. Rationale of serine isotope scrambling assay.

Figure 4—figure supplement 1.

Fully labeled serine (left) is broken down to glycine and methylene-THF by SHMT2. The labeled methylene-THF has two fates: oxidation by MTHFD2 to formyl-THF and recombination with unlabeled glycine, which is present in excess, to form singly labeled serine. Decreased MTHFD2 turnover is expected to give rise to increased synthesis of singly labeled serine.
DOI: http://dx.doi.org/10.7554/eLife.10575.014
Figure 4—figure supplement 2. Alterations in cellular NAD+/NADH ratio elicited by mitochondrial manipulations.

Figure 4—figure supplement 2.

Cellular NAD+ and NADH were determined using a commercially available assay kit (Sullivan et al., 2015). n = 3.
DOI: http://dx.doi.org/10.7554/eLife.10575.015
Figure 4—figure supplement 3. Serine isotope scrambling in other cell types. *, different from 0 day EtBr with p<0.05 (n = 3).

Figure 4—figure supplement 3.

DOI: http://dx.doi.org/10.7554/eLife.10575.016
Figure 4—figure supplement 4. Determination of homocysteine remethylation.

Figure 4—figure supplement 4.

Deuterium label was traced from labeled formate to labeled methionine, to determine the amount of homocysteine that is consumed by remethylation. n = 3.
DOI: http://dx.doi.org/10.7554/eLife.10575.017