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. Author manuscript; available in PMC: 2021 Jul 5.
Published in final edited form as: Sci Signal. 2021 Jan 5;14(664):eabc4436. doi: 10.1126/scisignal.abc4436

Figure 6. Fumarate covalently modified the core components of mitochondrial nucleoids in FH-deficient cell lines and primary tumors, leading to loss of mtDNA and increased mutational loads in mtDNA genes.

Figure 6.

(A-E) Schematic representation of the mutations in the mtDNA genome identified in the five HLRCC FH−/− cell lines. In C and D, the mtDNA of UOK271 and UOK350 cells is shadowed because these cells have entirely or nearly entirely lost their mitochondrial genome, respectively. (F) Silver staining of nucleoid core components purified by immunoprecipitation of TFAM in control cells (RPTEC) and in the FH−/− HLRCC UOK262 and UOK268 cell lines before and after re-expression of FH (UOK262_FH; UOK268_FH). (G) Immunoblots to core nucleoid components POLG, POLG2, TWINKLE, TFAM and SSBP1 on nucleoids isolated by differential centrifugation and purified by TFAM immunoprecipitations. (H) Samples as in (F) were immunoblotted for 2SC. Data in F-H are representative of five independent experiments. (I) mtDNA copy number relative to control RPTEC cells in FH−/− HLRCC cell lines before and after re-expression of WT FH (n=3-5 biological replicates per cell line); this experiment was repeated three times. (J and K) POLG activity assay of processive and exonuclease activities of POLG present in purified nucleoids from UOK262 (J) or UOK268 (K) cells. Data are representative of four independent experiments. (L) Silver staining of nucleoid core components purified by differential centrifugation steps and TFAM immunoprecipitations in two FH+/+ normal kidney cortex samples, in two HLRCC normal kidney cortex samples FH+/− (N4 and N3), in two primary tumors FH−/− (T4 and T3), and in two metastatic tumors FH−/− (M4 and M3). (M) Immunoblots to the core nucleoid components POLG, POLG2, TWINKLE, TFAM and SSBP1 isolated from tissue samples as in (L). (N) Samples as in (L) were immunoblotted for 2SC. Data in L-N are representative of four independent experiments. (O) Fumarate levels in HLRCC FH+/− normal kidney cortex samples (N4, N3), in two HLRCC FH−/− primary tumors (T4 and T3) and in two metastases (M4 and M3).