Figure 2. FH-deficient tumors exhibited altered mitochondrial ultrastructure and respiratory chain dysfunction.

(A-C) Electron microscopy of HLRCC primary tumor (A, FH^−/−), metastasis (B, FH^−/−), and non-tumor renal cortex from HLRCC patients (C, FH^+/−). A total of 10 tissue specimens were obtained from three patients. 100-500 cells per specimen grid were evaluated before representative images were obtained. (D) In-gel assay of NADH dehydrogenase activity of Complex I (CI) in mitochondrial lysates from primary HLRCC tumors and renal cortex samples. (E) Native immunoblot (IB) for the nuclear-encoded Complex I subunit NDUFS1 in normal renal cortical and HLRCC tumor mitochondria (T, FH^−/−). (F) Native IB for the mitochondrial-encoded Complex IV (CIV) subunit MT-CO1 in normal renal cortical and HLRCC tumor mitochondria to assess Complex IV levels and incorporation of Complex IV into supercomplexes containing Complex I, dimeric complex III (CIII₂) and variable number of copies (from 1 to 4) of Complex IV (CIV_n). (G) Native IB for the nuclear-encoded subunit of Complex III UQCRC2 in normal renal cortical and HLRCC tumor mitochondria to assess the assembly of Complex III-containing supercomplexes. (H) SDS-PAGE immunoblots for Complex I NDUFS1, Complex III UQCRC2 and Rieske protein UQCRFS1, and Complex IV MT-CO1 in normal renal cortical and tumor mitochondria. TOM20 served as the loading control for mitochondrial proteins. In (D) to (H), lane 1 consisted of renal cortex mitochondria from a non-HLRCC patient (FH^+/+, N stands for normal), lane 2 was renal cortex mitochondria from HLRCC patient 10 (FH^+/−, N stands for normal), and lanes 3 and 4 were primary tumor mitochondria from patients 4 and 3, respectively (FH^−/− ,T stands for tumor).