After dehydration in acetone, cells were embedded in spur resin, and thin sections (90 nm) were cut using a Reichert Ultracut E microtome. The sectioned grids were stained with a saturated solution of uranyl acetate and lead citrate. Sections were examined at 80 kV using a JEOL 1200EX transmission electron microscope. Western blot analysis Cells were pelleted at 500 g for 5 min and lysed in cold lysis buffer [20 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton X-100, 2.5 mmol/L sodium Selleckchem CT99021 PPi,

1 mmol/L β-glycerolphosphate, 1 mmol/L Na3VO4, 1 μg/mL leupeptin, and 1 mmol/L phenylmethylsulfonyl fluoride]. After sonication for 5 s, lysates were clarified by centrifugation at 12,000 g for 30 min at 4°C. Identical amounts (25 μg of protein) of cell lysates were separated by 8% or 15% SDS-PAGE gel electrophoresis, and the proteins were transferred onto nitrocellulose or polyvinylidene difluoride membranes. Membranes were then incubated in a blocking solution consisting

of 5% www.selleckchem.com/products/pd-0332991-palbociclib-isethionate.html powered milk in TBST [10 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20] for 1 h, followed LDN-193189 concentration by immunoblotting with the respective antibodies. The proteins of interest were detected using enzyme-linked chemiluminescence, according to the manufacturer’s protocol. Transfection of siRNA The target sequence for the JNK1/2-specific siRNA was 5’-AAA AAG AAU GUC CUA CCU UCU-3’ (GeneBank accession number NM002750.2), the target sequence for the Beclin 1-specific siRNA was 5’-UGG AAU GGA AUG AGA UUA ATT-3’ (GeneBank accession number NM003766.2) and the target sequence for the Atg-5-specific siRNA was 5’-TGT GAT GTT CCA AGG AAG AGC-3’ (GeneBank accession number NM004849.2). The control siRNAs (no silencing) for these siRNAs were synthesized by GenePharma Co. (Shanghai, China). siRNAs were transfected into the cells using Lipofectamine 2000 (Invitrogen) according to the protocol provided

by the manufacturer. Determination of intracellular ROS production Production of intracellular ROS 4��8C was measured using the fluorescent dye 2,7-dichlorofluorescein diacetate (DCF-DA). The cells were plated at a density of 1 × 105 in 6-well plates, allowed to attach overnight, and exposed to the treatments described in the figure legends. The cells were then incubated with 10 M DCFHDA for 20 min at 37°C in a 5% CO2 incubator, washed and resuspended in PBS at 1 × 106 cells/ml. The cells were analyzed by FACS flow cytometry at an excitation wavelength of 514 nm, and the fluorescence intensity of DCF was measured at an emission wavelength of 525 nm. Untreated cells served as controls. The amount of intracellular ROS was expressed as the fold-increase of DCF fluorescence compared with the control. Analysis of autophagy by GFP-LC3 redistribution To monitor the formation of GFP-LC3 puncta, the cells were transiently transfected with 1.0 mg GFP-LC3 plasmid, and then treated as described in the figure legends.