Zen version 2012

Cells (75,000 cells/ml) were planted into a 35 mm glass bottom dish (NEST, Jiangsu, China) and incubated overnight in complete medium. After drug treatment, cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min, permeabilized with 0.5% Triton X-100 (Amresco) for 20 min, and blocked with normal goat serum (ZSJB-BIO, Beijing, China) for 30 min at room temperature. Cells were probed with primary antibody against LC3B (CST, 1:200) at 4 °C overnight and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, 1:500) in the dark for 1 h at room temperature, whereas nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; Beyotime, 1:3000) in the dark for 5 min at 4 °C. All reagents were diluted in phosphate-buffered saline (PBS) and all steps were followed by PBS-washing for three times. Images were captured with the ZEN version 2012 software (Zeiss, Gottingen, Germany) using a laser scanning confocal microscope LSM 780 (Zeiss). Dots of specific green signals within cells were considered to be LC3-positive puncta.

The SKBR3/PR cells were grown on surface-treated coverslips in 24 well plates (approximately 50,000 cells per well) for 24 h until between 50 and 60% confluence. The cells were later exposed to different formulations of Resveratrol, Res-SLNs, and TPGS-Res-SLNs (equivalent dose of free resveratrol 30 μM). After a 12-h period of drug treatment, the cells were fixed with 4% paraformaldehyde (Solarbio, Beijing, China) for 18 min, permeabilized for 8 min with 0.2% Triton X-100, and blocked for 55 min with 1% bull serum albumin (Amresco) at room temperature. The cells were probed overnight with a primary antibody against E-Cadherin (Sigma; 1:100 dilution) at 4°C and Alexa Fluor 488-conjugated goat anti rabbit IgG (Molecular Probes, Eugene, OR, 1:100) in the dark for 1 h at room temperature. After three further washes, the cells were counterstained using 4, 6-diamidine-2-phenylindole (Beyotime, 1:1000) in the dark for 5 min at room temperature. All reagents were diluted in phosphate buffered saline (PBS) with all the steps followed by three-times-5 min PBS washing. Images were captured using ZEN version 2012 software (Zeiss, Gottingen, Germany) under a laser scanning confocal microscope LSM 780 (Zeiss).

Cells (300,000 cells/ml) were seeded on a 35 mm glass bottom dish (NEST, Jiangsu, China) and incubated overnight in complete medium. After drug treatments for 12 h, we fixed cells with 4% paraformaldehyde (Solarbio, Beijing, China) for 10 min, permeabilized with 0.5% Triton X-100 (Amresco, Solon, OH) for 20 min, and blocked with 5% bull serum albumin (Amresco) for 30 min at room temperature. Cells were probed with primary antibody against β-tubulin III (1:50) at 4 °C overnight and Alexa Fluor 488-conjugated goat anti-rabbit IgG (Molecular Probes, Eugene, OR, 1:500) in the dark for 1 h at room temperature. We counterstained cell nuclei with diamidino-phenyl-indole (DAPI) (Beyotime, 1:3000) in the dark for 5 min at room temperature. All reagents were diluted in phosphate-buffered saline (PBS) and all steps were followed by 5-min PBS washing three times. We captured images with ZEN version 2012 software (Zeiss, Gottingen, Germany) using a laser scanning confocal microscope LSM 780 (Zeiss). We applied the same exposure settings for all images of identical proteins.