Supplementary Materialscells-09-02346-s001. to prevent 7KC-induced oxidative stress was characterized by a decrease in reactive oxygen species (ROS) production in whole cells and at the mitochondrial level; by an attenuation of the increase in the level and activity of catalase; by attenuating the decrease in the expression, level and activity of glutathione peroxidase 1 (GPx1); by normalizing the expression, level and activity of superoxide dismutases 1 and 2 (SOD1, SOD2); and by reducing the decrease in the expression of nuclear erythroid 2-like factor 2 (Nrf2) which regulates antioxidant L-Mimosine genes. QCT, RSV and API also prevented mitochondrial dysfunction in 7KC-treated cells by counteracting the loss of mitochondrial membrane potential (m) and attenuating the decreased gene expression and/or protein level of AMP-activated protein kinase (AMPK), sirtuin 1 (SIRT1) and peroxisome proliferator-activated receptor coactivator-1 (PGC-1) implicated in mitochondrial biogenesis. At the peroxisomal level, QCT, RSV and API prevented the impact of 7KC by counteracting the decrease in ATP binding cassette subfamily D member (ABCD)3 (a peroxisomal mass marker) at the protein and mRNA levels, as well as the decreased expresssion of genes associated with peroxisomal biogenesis (for 10 min. The absorbance was read by spectrophotometry at 412 nm on a Tecan Infinite M 200 Pro (Tecan, M?nnedorf, Switzerland). The activity of GPx is usually expressed in mol of GSH/min/mg of protein. Protein content was determined with the Pierce TM BCA protein assay kit (Ref: 23227; ThermoScientific). 2.13. Morphological Characterization of Apoptotic Cells: Evaluation Tmem178 of Nuclear Morphology with Hoechst 33342 Nuclear morphology of 7KC-treated N2a cells cultured without or with polyphenols was characterized by fluorescence microscopy after staining with Hoechst 33342 (2 g/mL) [80]. Normal cells have regular and round L-Mimosine nuclei whereas, apoptotic cells are characterized by condensed and/or fragmented nuclei. At the end of the treatment, cells were deposited onto glass slides by cytocentrifugation (5 min, 1500 rpm) with a cytospin 2 (Shandon, Sheschire, WA7 1PR, UK), then mounted in fluorescent mounting medium (DakoCytomation, Dako, Coppenhagen, Denmark) and stored in the dark at 4 C until observation. The slides were examined under an Axioskop right microscope (Zeiss, Jena, Germany) with ultraviolet light. A total of 300 cells per sample were counted to calculate the percentage of apoptotic cells. 2.14. Cytological Characterization of Autophagic Cells after Staining with Monodansylcadaverine The presence of cytoplasmic structures corresponding to autophagic vacuoles was examined by staining with monodansylcadaverine (MDC) [81]. MDC (Ex max 340 nm, Em max 530 nm) (Sigma) was prepared at 0.1 M in DMSO and added to the culture medium at a final concentration of 0.1 mM. After 15 min of incubation at 37 C, N2a cells seeded on glass slides in 6-well plates, were washed and stained cells were immediately examined under an Axioskop A1 light microscope (Zeiss) by using UV light excitation. Three hundred cells were examined for each sample. 2.15. Protein Analysis: Polyacrylamide Gel Electrophoresis and Western Blotting Protein analysis was realized as L-Mimosine previously described by polyacrylamide gel electrophoresis and Western blotting [68]. After 48 h of treatment, adherent and nonadherent cells L-Mimosine were collected, washed in PBS and lysed for 30 min on ice in a RIPA buffer (10 mM Tris-HCl, pH 7.2, 150 mM NaCl, 0.5% Nonidet NP40, 0.5% Na deoxycholate, 0.1% SDS, 2 mM EDTA and 50 mM NaF) containing a complete protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN, USA) diluted 1/25. Cell lysates were cleared by centrifugation at (20 min, 20,000 0.05 or less. Thus, for further experiments, 7KC was used at 50 M. So, among the concentrations of polyphenols chosen to assess cytoprotection, L-Mimosine concentrations below 12.5 M were used to avoid cytotoxicity (Determine 1). Of note, measurement of RSV, QCT and API uptake in N2a cells taking into account the fluorescence characteristics of these molecules showed concentration-dependent uptake (in the concentration range 3.125 to 25 M) at culture times 3, 24 and 48 h (Determine S1). 3.2. Effects of Resveratrol, Quercetin, Apigenin and -Tocopherol on 7-Ketocholesterol-Induced Plasma Membrane Damage Evaluated by Staining with Propidium Iodide The effects of RSV, QCT and API on 7KC-induced plasma membrane damage was evaluated with propridium iodide (PI). A wide range of polyphenol concentrations was used (1.5 to 25 M) in order to precisely identify the concentrations capable of preventing plasma membrane damages (increased permeability to PI). PI was chosen to evaluate plasma membrane permeability, due to its.