“CellR” software v. 2.8 was employed to capture individual images and the fluorescent signal was quantified using static cytometry software “ScanR” v. 2.03.2 (Olympus). Following treatment and IWR-1 order incubation with fluorochromes, cells were washed in Hank’s balanced salt solution (HBSS) and life-cell images were recorded. Nuclei were stained with the fluorochrome Hoechst 33342 (1 μM) (last 30 minutes of the treatment). Mitochondria were visualized and mitochondrial mass was monitored in Hep3B cells treated with EFV (6 hours) using the fluorescent dye 10-N-nonyl acridine orange (NAO) 0.5 μM, which specifically binds to cardiolipin
independent of ΔΨm.20 We also used stably transfected HeLa cells expressing the red fluorescent protein mtdsRed tagged for mitochondrial localization and specifically designed for the fluorescent labeling of these organelles (details in Supporting Material). LC3 expression and localization were studied using HeLa cells stably expressing LC3-GFP, treated with EFV (24 or 48 hours) (details in Supporting Material). Lysosomes were stained with the fluorescent dye Lysotracker Green 0.1 μM (last 30 minutes of the treatment) in EFV-treated HeLa cells (24 hours). For cell proliferation/survival
studies, Hep3B, primary hepatocytes, or HeLa cells stably expressing mtdsRed were allowed to proliferate exponentially (48-well plates) for 24 hours in the presence of EFV. To study the role Dabrafenib ic50 of autophagy, cells were cotreated with 2.5 mM 3-methyladenine (3MA), a specific inhibitor of autophagosome formation, for 1 hour prior to EFV treatment and during the entire treatment period (24 hours). Cells were counted according to Hoechst fluorescence (25 images/well).
Apoptosis was studied in Hep3B cells as bivariate Annexin V/PI analysis (apoptosis detection kit, Abcam). Following treatment (24 hours), the medium was replaced with HBSS containing 0.9 μL/well of AnnexinV-fluorescein (to detect phosphatidyl serine exteriorization) and incubated (30 minutes), after which 0.3 μL/well of the chromatin-detecting PAK5 dye propidium iodide (PI) was added (5 minutes) to label dead or damaged cells. The protein kinase inhibitor staurosporine (STS) was employed as a positive proapoptotic control. Hep3B (5 × 104/chamber), primary hepatocytes (105/chamber), or HeLa cells (3 × 104/chamber) were seeded in 4-well Lab-Tek chamber slides (Nalge Nunc International, Naperville, IL). After treatment, cells were fixed in 3.5% glutaraldehyde (1 hour, 37°C), postfixed in 2% OsO4 (1 hour, room temperature), and stained with 2% uranyl acetate in the dark (2 hours, 4°C). Finally, cells were rinsed in sodium phosphate buffer (0.1M, pH 7.2), dehydrated in ethanol, and infiltrated overnight in araldite (Durcupan, Fluka, Buchs, Switzerland). Following polymerization, embedded cultures were detached from the chamber slide and glued to araldite blocks. Serial semithin (1.