Article Figures & Data
Figures
- Figure 1. Effects on glycolysis by the deletion of serine catabolic enzymes in 293A cells.
(A) Protein levels of SHMT1, SHMT2, hexokinase 1 (HK1), phosphofrutokinase (platelet type, PFKP), pyruvate kinase (muscle type ½, PFKM1/2), lactate dehydrogenase A (LDHA), and β-actin were measured in the WT, ΔSHMT1, or ΔSHMT2 293A cells by Western blotting. Two independent cell clones of each genotype were examined. Densitometry quantification of the glycolytic proteins followed by normalization to β-actin is plotted on the right. **P < 0.01 (t test). Data are presented as mean ± SD for three independent experiments. (B) Proliferation rates of the WT, ΔSHMT1, and ΔSHMT2 293A cells in the DMEM with 4.5 g/liter glucose. Data are presented as mean ± SD (n = 5). (C) Measurement of the lactic acid production from the WT, ΔSHMT1, and ΔSHMT2 293A cells that were grown in DMEM for 48 h. Data are presented as mean ± SD (n = 4) **P < 0.01 (t test).
- Figure 2. Effects on mitochondrial respiration by the deletion of serine catabolic enzymes in 293A and AML12 cells.
(A) Measurement of the cell proliferation of the WT, ΔSHMT1, and ΔSHMT2 293A cells in the DMEM-based galactose media. (B, C) Measurement of the intracellular ATP levels in the WT, ΔSHMT1, and ΔSHMT2 293A cells that were grown in the galactose (B) or glucose (C) media for 24 h. (D) Measurement of the basal oxygen consumption rates of the WT and ΔSHMT2 293A cells that were grown in the glucose media. (E) Phase-contrast images illustrating the WT and ΔSHMT2 AML12 cells that were grown in the galactose media for 72 h. The percentages of the live cells were plotted on the right. (F) Measurement of the intracellular ATP levels of the WT and ΔSHMT2 AML12 cells that were grown in the galactose media for 72 h. ***P < 0.001 (t test). Data are presented as mean ± SD. n = 5 for (A); n = 4 for (B–E); and n = 6 for (F).
- Figure 3. Effects on the content of mtDNA and the expression of the OXPHOS genes by deletion of SHMT2 in 293A cells.
(A) Measurement of the levels of the respiratory chain complexes (RCCs) in the WT, ΔSHMT1, and ΔSHMT2 293A cells by Western blotting, using the Total OXPHOS antibody cocktail to simultaneously detect the representative components of each individual RCCs: CI-NADH:ubiquinone oxidoreductase subunit B8; CII-succinate dehydrogenase complex subunit B; CIII-ubiquinol-cytochrome C reductase core protein 2; CIV-cytochrome C oxidase II; and CV-ATP synthase F1 subunit alpha. NDUFS1, an additional CI marker, and β-actin, the loading control, were also measured. (B) Bar graph illustrating the mtDNA copy numbers of the WT, ΔSHMT1, and ΔSHMT2 293A cells. Data are presented as mean ± SD (n = 4). (C) Scatter plot illustrating the log2-transformed relative mRNA levels of the nuclear-encoded OXPHOS genes in the ΔSHMT2 293A cells to the WT control, as determined by next-generation sequencing; t test (n = 3). (D) Bar graph illustrating the relative mRNA levels of the mitochondria-encoded OXPHOS genes in the ΔSHMT2 293A cells to the WT control, as determined by qRT-PCR analysis. Data are presented as mean ± SD (n = 3). (E) Metabolic radiolabeling to determine the synthetic rates of the mitochondria-encoded proteins in the WT and ΔSHMT2 293A cells. Left, autoradiography to visualize the mitochondria-encoded OXPHOS proteins; right, Coomassie blue staining of total cellular proteins.
- Figure 4. Effects on the assembly of mature respiratory chain complexes by the deletion of SHMT2.
(A–C) BNGE to detect individual mature respiratory complexes in the mitochondrial preparation from the WT and ΔSHMT2 293A cells, followed by visualization with Coomassie blue staining (A), Western blotting with the antibody against a Complex I (CI) marker NDUFS3 (B), or Complex I in-gel activity assay (C). CI, Complex I; CIII2/IV, CIII2/IV supercomplex; HSP60, heat shock protein 60.
- Figure S1. Effects on mitochondrial respiration by reconstitution of SHMT2.
(A) Measurement of OXPHOS protein levels by Western blotting in WT, ΔSHMT2, and SHMT2-reconstituted 293A cells using the Total OXPHOS antibody cocktail. (B) BNGE analysis of Complex I (CI) followed by Coomassie blue staining (top) and in-activity assay (bottom). (C) Cell growth of WT, ΔSHMT2, and SHMT2-reconstituted 293A cells in the galactose media. Data are presented as mean ± SD (n = 5).
- Figure 5. Effects of glycine and formate on the assembly of Complex I.
(A, B) Time- and dose-dependent effects on the levels of Complex I core subunit NDUFS1 by the supplementation of the ΔSHMT2 293A cells with glycine (A) or formate (B). Densitometry quantification of NDUFS1 followed by normalization to β-actin is plotted on the top. Data are presented as mean ± SD for three independent experiments. (C, D) BNGE resolution of the mitochondrial preparation from the WT and ΔSHMT2 293A cells that were treated with or without 2 mM formate for 72 h, followed by visualization of the individual mature respiratory chain complexes by the Western blotting probed with the Total OXPHOS antibody cocktail (C) and the Complex I (CI) in-gel activity assay (D). CI, Complex I. (E) Proliferation rates of the WT and ΔSHMT2 293A cells in the galactose media supplemented with or without 2 mM formate. Data are presented as mean ± SD (n = 4).
Supplementary Materials
Table S1 sgRNA for editing SHMT.
Table S2 qPCR primer for human gene.
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