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. Author manuscript; available in PMC: 2024 Jun 6.
Published in final edited form as: Cell Metab. 2023 Apr 25;35(6):1057–1071.e12. doi: 10.1016/j.cmet.2023.04.003

Figure 5. FLVCR1-mediated choline uptake is required for mitochondrial homeostasis.

Figure 5.

a) Principal component analysis (PCA) of RNA-Seq from FLVCR1-knockout HeLa cells expressing a vector control or FLVCR1 cDNA cultured in choline depleted or choline replete media for 24 hours. b) Heat map of differentially expressed genes from RNA-Seq of FLVCR1-knockout HeLa cells expressing a vector control or FLVCR1 cDNA cultured in choline depleted or choline replete media for 24 hours. c) Gene set enrichment analysis (GSEA) of significantly upregulated transcripts in FLVCR1-knockout HeLa cells expressing an empty vector control in choline depleted media vs. FLVCR1-knockout HeLa cells + FLVCR1 cDNA in choline depleted media. d) Immunoblotting of indicated proteins in HEK293T cells expressing an empty vector control (pLV2 EV) or sgRNA targeting GCN2, HRI, PKR, or PERK and treated with CHKA inhibitor RSM932A (1μM) or vehicle for 24 hours. e) Immunoblotting of indicated proteins in HEK293T cells expressing an empty vector control (pLV2 EV) or two different sgRNA targeting DELE1 and treated with CHKA inhibitor RSM932A (1μM) or vehicle for 24 hours. f) Schematic for proteomic and polar metabolomic/lipidomic profiling of mitochondria from FLVCR1-knockout HeLa cells expressing an empty vector control or FLVCR1 cDNA and after extended culture in choline depleted and replete conditions. g) Principal component analysis (PCA) of mitochondrial lipidomics from FLVCR1-knockout HeLa cells expressing a vector control or FLVCR1 cDNA. Heatmap of mitochondrial lipid species. h) Heatmap of most differentially expressed mitochondrial proteins organized by ontology from FLVCR1-knockout HeLa cells expressing a vector control or FLVCR1 cDNA cultured in choline depleted or choline replete media. Statistical significance was determined by an ANOVA test with a permutation-based FDR q<1% considered significant. i) Flow cytometry of mitochondrial membrane potential (ΔΨm) as measured by TMRM in FLVCR1-knockout HeLa cells expressing an empty vector control or FLVCR1 cDNA and after extended culture in choline depleted and replete conditions. j) Representative electron micrographs of the mitochondria of FLVCR1-knockout HeLa cells expressing an empty vector control or FLVCR1 cDNA after extended culture in choline depleted conditions (Left). Scale bar = 0.5μm. Violin plots of mitochondrial surface area, outer mitochondrial membrane (OMM) perimeter, cristae density, cristae width, and cristae junction width calculated from electron micrographs. Statistical significance was determined by two-tailed unpaired t-test. n = 107 mitochondria, n = 444 cristae, n = 255 cristae junctions FLVCR1 KO cells; n = 68 mitochondria, n = 303 cristae, n = 183 cristae junctions FLVCR1 KO +FLVCR1 cDNA cells.