Anti cytochrome c

MitoTracker Green FM, DAPI, and Alexa Fluor 488– phalloidin were purchased from Life Technologies (Carlsbad, CA). Digitonin and etoposide were from Wako Pure Chemical Industries (Osaka, Japan). Mdivi-1 was from Enzo Life Sciences (Farmingdale, NY). Z-VAD-FMK was purchased from Peptide Institute (Osaka, Japan). NU6140 and BI2536 were from Santa Cruz Biotechnology (Santa Cruz, CA). The following antibodies were used for Western blotting and immunostaining: anti-Drp1, anti–cytochrome c (BD Biosciences, Billerica, MA), anti–γ-tubulin (Sigma-Aldrich, St. Louis, MO), anti-Smac (ProSci, Poway, CA), anti–phospho-Plk1 (Thr-210), anti-Plk1 (Abcam, Cambridge, United Kingdom), anti–phospho-CDK2 (Thr-160), anti-γ-H2AX, anti–phospho-histone H3 (Ser-10; Cell Signaling Technology, Beverly, MA), Alexa Fluor 488 anti-mouse immunoglobulin G (Life Technologies), anti–voltage-dependent anion-selective channel protein 1 (VDAC1), anti–glyceraldehyde 3-phosphate dehydrogenase (GAPDH), anti-CDK2, anti–cyclin A, anti–cyclin E, anti–cyclin B1, anti-actin, and horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology). The Western Lightning Plus-ECL chemiluminescence detection kit was purchased from PerkinElmer-Cetus (Boston, MA).

Protein samples were prepared by lysing the serum small EVs in buffer containing 150 mM NaCl, 10 mM Tris-HCl (pH = 7.6), 10% glycerol, 0.5% Tween, and 10 mM mercaptoethanol, supplemented with 2× EDTA-free Protease Inhibitor Cocktail. The lysate was sonicated and then centrifuged at 4°C. Protein extracts of 40–60 μg were separated on a 12% SDS-PAGE, transferred on a membrane, and then incubated overnight at 4°C with primary antibodies specific for anti-CD63 (Abcam, Cambridge, UK), anti-CD81 (Abcam, Cambridge, UK), anti-TSG101 (Santa Cruz Biotechnology, Dallas, TX, USA), anti-Nucleoporin p62 (BD Biosciences, Franklin Lakes, NJ, USA), and anti-Cytochrome C (BD Biosciences, Franklin Lakes, NJ, USA). Subsequently, the membranes were incubated for 2 h at room temperature with anti-mouse immunoglobulin G (IgG) or anti-rabbit IgG horseradish peroxidase (HRP)-conjugated secondary antibodies (Santa Cruz Biotechnology, Dallas, TX, USA). Proteins were visualized with the UVP BioSpectrum 810 Imaging System.

A total of 20,000 cells/well were plated in a 24-well plate on cover slips (13 mm) and were allowed to adhere for 24 hours. All treatments were performed in a final volume of 500 μl. Following treatment, cells were first washed twice with ice-cold PBS, fixed in 500 μl paraformaldehyde (4% PFA) for 10 minutes, and permeabilized for 3 minutes with ice-cold 0.1% Triton X-100 in PBS. After subsequent washing steps, unspecific binding was blocked by 5% horse serum and 0.3% Triton X-100. Anti–cytochrome c, (BD Pharmingen) was applied in 1% horse serum and 0.3% Triton X-100 in PBS. Afterwards, cells were washed twice with ice-cold PBS. For secondary antibody incubation. Cy3-conjugated Donkey Anti-Mouse IgG (Dianova, Hamburg, Germany) was diluted in 1% horse serum and 0.3% Triton X-100 in PBS, and samples were incubated while gently shaking at room temperature in the dark and washed with ice-cold PBS. Cellular nuclei were stained with the monomeric cyanine nucleic acid stain TO-PRO-3 (Life Technolgies, Darmstadt, Germany) at a final concentration of 1 μmol/l in PBS for 10 minutes. Images were acquired with a Nikon Eclipse TE2000-S fluorescence microscope coupled to a DS-5Mc cooled color digital camera (Nikon, Düsseldorf, Germany) and NIS Elements AR (version 3.22) software from Nikon (TO-PRO-3: excitation 642 nm, emission 662 nm; Cy3: excitation 554 nm, emission 568 nm).