Ultracut-prepared ultrathin (0.07 μm) sections were stained with lead citrate. Finally, photomicrographs were obtained with a TEM (FEI Tecnai Spirit G2) using a digital camera (Morada, Soft Imaging System, Olympus). Stably transfected HeLa LC3-GFP and mtdsRed cells were treated with EFV (24 hours) and Lysotracker Green or Red 0.1 μM (Molecular Probes, Invitrogen, Eugene, OR) added for the last 30 minutes of the treatment to stain the lysosomes. After washing with HBSS, life-cell images were acquired with a Leica TCS-SP2 confocal laser scanning unit with argon and helium-neon

laser beams and attached to a Leica DM-IRBE inverted microscope. Images were captured at 63× magnification with HCX PL APO 63.0 × 1.32 oil UV objective. The excitation wavelength used for mtdsRed and Lysotracker Red was 543 nm, 488 nm in the case of LC3-GFP and Lysotracker Green, and the emission apertures HDAC inhibitor for fluorescence detection were 560-700 nm and 502-539 nm, respectively. Images were analyzed with LCS Lite software and overlapping

of the red and the green fluorescent signal was quantified with the program ImageJ. The Colocalization Colormap Plugin was used to calculate the Correlation Index (Icorr). Data were analyzed using BAY 73-4506 cost GraphPad Prism v. 3 software with one-way analysis of variance (ANOVA), followed by Newman-Keuls multiple comparison test or by Student’s t test. All values are mean ± standard error of the mean (SEM) and statistical significance was: *P < 0.05, **P < 0.01, and ***P < 0.001. Taking into consideration recently published evidence concerning EFV-induced mitochondrial Endonuclease dysfunction in hepatic cells, we delved more deeply by assessing mitochondrial mass and morphology. Fluorescence microscopy in NAO-stained

Hep3B and primary hepatocytes treated with EFV revealed considerable alterations of the mitochondrial signal, which were concentration-dependent and visible as early as 6 hours after EFV 50 μM treatment. Although the mitochondrial net spread over the entire cytoplasm in control (untreated) cells, EFV 50 μM treatment produced a localized and compacted mitochondrial signal (Figs. 1A, 8B). Similar modifications were obtained in Hep3B cells stained with another mitochondrial stain Mitotracker Green (data not shown). To further analyze these effects, we treated HeLa cells stably expressing mtdsRed with increasing concentrations of EFV for periods of up to 48 hours. Alterations of mitochondrial size and shape similar to those appearing in hepatic cells were detected (results not shown). Moreover, quantification of the red mitochondrial signal (mtdsRed) using static cytometry revealed a concentration-dependent increase in the relative mitochondrial mass (Fig. 1B) that was statistically significant as early as at 6 hours treatment with EFV 50 μM.