PMC

(A) SB3 expression in human hepatoma HepG2 cells stably transfected with a mock construct or with a SB3 plasmid. TOM20 was used as a loading control of the Western immunoblot. (B-E) MTT analysis of cell viability after treatment with the reported concentrations of the chemotherapeutics 5-fluorouracil (B), etoposide (C), cisplatin (D) and doxorubicin (E). Cells were treated for 48 hours with 5-fluorouracil and etoposide, and for 24 hours with cisplatin and doxorubicin. Bars are mean values ±S.D. of tetrazolium salt absorbance for 2×10⁴ recorded cells (n=6, *, p<0.005 with a Student's t test).

(A, B) SB3 and Complex I and TRAP1 immunoprecipitations (IPs) on mitochondria from HepG2 cells (A) and on wild-type and SB3-transgenic mouse liver mitochondria (B). The interaction between SB3 and Complex I is shown by co-IP; to detect Complex I, two different subunits (GRIM19 and NDUFB8) were probed. (C, D) Spectrophotometric analysis of the NADH:ubiquinone oxidoreductase enzymatic activity of complex I in mitochondria from HepG2 cells (C) or from mouse liver (D); both in (C) and in (D), n=6, *, p<0.005 with a Student's t test). (E) PTP opening of HepG2 cells treated with EM20-25 is measured with the whole-cell CRC assay. Where indicated, cells were pretreated with N-acetyl-cysteine (NAC). Experiments were carried out in a glutamate/malate buffer to maximize Complex I activity. Bars indicate the ratio between the CRC detected in the different experimental conditions (CRC) and in untreated cells (CRC0; n=6, *, p<0.005 with a Student's t test). A CRC/CRC0 lower than 1 indicates PTP induction.

(A) Representative traces of cytofluorimetric cell death analysis by Annexin-V and propidium iodide (PI) staining. In black, double negative, viable cells; in green, Annexin-V positive, apoptotic cells; in blue, Annexin-V/PI double positive, late apoptotic cells; in red, PI positive, necrotic cells. Numbers indicate the percentages of cells in each condition. Cells were treated for 2h30min with EM 500 μM; where indicated, 500 μM N-acetyl-cysteine (NAC) was added 1 hour before starting chemotherapeutic treatment. (B, C) Fluorimetric analysis of ROS levels in HepG2 cells (B) and in wild-type and SB3-transgenic mouse liver mitochondria (C) treated for 2 hours with the reported EM20-25 concentrations; where indicated, N-acetyl-cysteine (NAC) was added 1 hour before starting the experiment. Bars are mean values ±S.D. of CM-H2DCFDA fluorescence (arbitrary units) for 2×10⁴ recorded cells or 200 μg mitochondria (n=6, *, p<0.005 with a Student's t test).

(A) Fluorimetric analysis of ROS levels in HepG2 cells treated for 5 hours with the reported cisplatin concentrations with or without N-acetyl-cysteine (NAC). Bars are mean values ±S.D. of CM-H2DCFDA fluorescence (arbitrary units) for 2×10⁴ recorded cells (n=6, *, p<0.005 with a Student's t test). (B) Representative traces of cytofluorimetric cell death analysis by Annexin-V and propidium iodide (PI) staining. In black, double negative, viable cells; in green, Annexin-V positive, apoptotic cells; in blue, Annexin-V/PI double positive, late apoptotic cells; in red, PI positive, necrotic cells. Numbers indicate the percentages of cells in each condition. Cells were treated for 24 hours with 50 μM cisplatin; where indicated, NAC was added 1 hour before starting chemotherapeutic treatment. (C) Subcellular fractionation, partial trypsin digestion and Western immunoblot of the mitochondrial fractions of HepG2 cells. TRAP1 was used as a marker of mitochondrial matrix; Bax as a marker of outer mitochondrial membrane; calnexin (CNX) was used as an endoplasmic reticulum marker to check for purity of mitochondrial fractions. 50 μg of mitochondrial proteins were loaded per lane. (D) Partial trypsin digestion and Western immunoblot of wild-type and SB3-transgenic mouse liver mitochondria. Cyclophilin D (CyP-D) was used as a marker of mitochondrial matrix, CNX was used as an endoplasmic reticulum marker to check for purity of mitochondrial fractions. 50 μg of mitochondrial proteins were loaded per lane. (E) PTP opening of HepG2 cells treated with cisplatin is measured with the whole-cell CRC assay. Where indicated, cells were pretreated with N-acetyl-cysteine (NAC). Experiments were performed after 16 hours of drug treatment, i.e. before the beginning of the cell death process, and were carried out in a glutamate/malate buffer to maximize Complex I activity. Bars indicate the ratio between the CRC detected in the different experimental conditions (CRC) and in untreated cells (CRC₀; n=6, *, p<0.005 with a Student's t test). A CRC/CRC₀ lower than 1 indicates PTP induction.