Stabilization buffer

Phenylmethanesulfonyl fluoride (PMSF), McCoy’s 5A medium, Dulbecco’s Modified Eagle Medium (DMEM), tris-base, sodium chloride, bovine serum albumin (BSA), TRIzol^® reagent, tween 20, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), and sodium azide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Phosphate-buffered saline (PBS) was purchased from Unionward (Taipei, Taiwan). Trypsin-EDTA, penicillin/streptomycin and fetal bovine serum (FBS) were purchased from Gibco (Grand Island, NY, USA). Recombinant RNasin ribonuclease inhibitor, oligo d(T)21 and M-MLV reverse transcriptase were purchased from Promega (Madison Avenue, New York City, NY, USA). 4′,6-diamidino-2-phenylindole (DAPI), lysis buffer, stabilization buffer and Hoechst 33,342 were purchased from ChemoMetec A/S (Gydevang, Allerod, Denmark). CYTO-ID^® autophagy detection kit was purchased from Enzo Life Sciences (cat. no. ENZ-51031; Madison Avenue, NY, USA). Chemiluminescence (ECL) kit was purchased from Bio-Rad (cat. no. 1705061, Hercules, CA, USA).

To evaluate the cell cycle, hESCs were seeded at 5 × 10⁵ cells on a Matrigel-coated 60-mm dish and cultured for 24 h. Cells were harvested identically to the cell culture method, and a cycle analysis was performed using a NucleoCounter^® NC-250™ (Chemometec, Frederiksborg, Denmark) according to the manufacturer’s instructions. Cells were washed once with 1× PBS without Ca²⁺ and Mg²⁺ (Gibco™, USA), and the PBS was removed completely. The cells were gently pipetted using a lysis buffer (ChemoMetec) containing 10 μg/mL DAPI and incubated for 5 min in the same humidified atmosphere used for cell culture. Stabilization buffer (ChemoMetec, Denmark) was added to the lysed cells, loaded on NC-Slide A8™ (ChemoMetec, Denmark), and measured using NucleoView™ NC-250 software v1.11 (ChemoMetec, Denmark).

Cell cycle profiles of HCT116 cells were determined with an NC-3000 image cytometer (ChemoMetec, Allerod, Denmark) in accordance with manufacturer’s protocol. Briefly, cells were seeded at 200,000 cells/well on a 6-well plate for 24 h. Two ml of indicated compounds (5.0 μM for sunitinib and 7, 3.0 μM for 13–15) were added to cells. After incubation for 24 h, 100,000 cells were harvested and centrifuged at 400 g at room temperature for 5 min, washed once with PBS (50 μM), and resuspended in lysis buffer (50 μl) (ChemoMetec, Allerod, Denmark) containing 10 μg/ml of 2-(4-amidinophenyl)-1H-indole-6-carboxamidine (DAPI, ChemoMetec, Allerod, Denmark). The cells were incubated at 37 °C for 5 min and then stabilization buffer (50 μl) (ChemoMetec, Allerod, Denmark) was added to the mixture. The cellular fluorescence was measured with an NC-3000 image cytometer using NC-SlideA8 (ChemoMetec, Allerod, Denmark). The NC-3000 software (ChemoMetec, Allerod, Denmark) was used for image acquisition, image analysis and quantification, and data visualization.