Apoptosis induction and telomerase loss in HepG2 cells are independent from ROS production.

<p><b>a)</b> Effect of MTBITC on ROS production in adherent growing cells. Cells were exposed to MTBITC for 1 to 6 h, washed thoroughly with PBS and subsequently exposed to 100 µM spin probe CMH in Krebs-Hepes buffer. ROS were then quantified using ESR spectrometry. Positive control: 100 µM menadione. The pictures display the middle peak of ESR spectra of CMH spin probe labelled samples. Bars are mean ± SD, n = 3. <b>b)</b> Representative immunoblot for HNE Lys-adducts after exposure to MTBITC or DMSO for different time points. Total lysate of HepG2 cells was subjected to immunoblotting using an anti-HNE Lys antibody. Exposure of cells to HNE for 80 min. was used as positive control. The blot was reprobed with anti-β-actin antibody to ensure equal protein loading. <b>c+d)</b> Effect of NAC pre-treatment. HepG2 cells were pre-treated with NAC for 1 h, washed with PBS and subsequently exposed to 25 µM MTBITC or 0.1% DMSO for another 24 h. Cells were then prepared for (c) TRAP analysis or (d) flow cytometric analysis of the mitochondrial membrane potential (MMP) as parameter for apoptosis; *p≤0.05. M: 50 bp DNA marker; 1) 0.1% DMSO 2) 25 µM MTBITC 3) 5 mM NAC +0.1% DMSO,4) 5 mM NAC +25 µM MTBITC 5) cell lytic buffer 6) destilled water 7) 0.1% DMSO, heat treated. <b>e)</b> Effect of 25 µM MTBITC on mtDNA damage after 1 or 24 h exposure of HepG2 cells. Agarose gel electrophoresis of amplified mtDNA multiplex PCR products is shown in representative image details. Each lane contains amplification products obtained with mtDNA from one single cell using specific PCR primers. As positive control for mtDNA damage, cells were exposed to 10 min UV irradiation. f<b>)</b> Representative pictures of beta-galactosidase staining of HCC cells after exposure to MTBITC for 72 h, captured by light microscopy. beta-galactosidase positive cells are reflected by dark color of the cells. SC: solvent control = 0.1% DMSO. Scale bar = 200 µm, all panels have the same magnification.