A2058

Cells were fixed in 4% formalin, permeabilized with 0.1% Triton X-100, and blocked with 5% BSA (A2058, Sigma-Aldrich) in PBS for 30 min. Then cells were stained with Alexa Fluor 488 Phalloidin (A12379, Thermo Fisher Scientific) to visualize filamentous (F)-actin. The nuclei were stained with 4′,6-diamidino-2-fenylindool (DAPI, 62248, Thermo Fisher Scientific). Images were taken by confocal microscopy (SP8, Leica Microsystems).

LNCaP cells were seeded at 1.5 × 10⁶ cells per 10 cm dish and transfected with siRNA as described. At 72 and 144 h post-transfection, the cells were treated with 10 μM EdU (Sigma-Aldrich, 900584) for 30 min. Remaining EdU was washed off the cells with PBS before harvesting cells, and then, 1 × 10⁶ cells were fixed in 70% ethanol and frozen at − 20 °C. Cells were then diluted 1 in 4 with PBS then pelleted and re-suspended in 1 ml of PBS containing 1% BSA (Sigma-Aldrich, A2058). Cells were again pelleted, re-suspended in 500 μl of click reaction mix (10 μM carboxyfluorescein TEG-azide, 10 mM Sodium L-ascorbate and 2 mM Copper-II-sulphate diluted in PBS), and incubated in the dark at room temperature for 30 min. Samples were then diluted with 5 ml of PBS containing 1% BSA and 0.1% Tween-20. Cells were again pelleted, washed with PBS and then resuspended in 500 μl of PBS containing 1% BSA, 0.1 mg/ml of RNase and 1 μg/ml of DAPI. Samples were analysed on the Canto II (BD Biosciences). Forward and side scatter were used to select a population of cells free of cell debris and doublets. Cells were analysed using B450 (FTIC – EdU positive) and B510 (DAPI) lasers. 50,000 single-cell events were recorded for each ample. FlowJo software v10.5 was used to analyse the data. Data were collected in biological triplicate.

HCT116 cells (human colon cancer cells, p53 wildtype), HT29 cells (human colon cancer cells, p53 mutant), SW480 cells (human colon cancer cells, p53 mutant), MDA-MB-231 cells (human breast cancer cells), MDA-MB-468 cells (human breast cancer cells), A375 (human skin cancer), and A2058 (human skin cancer) were purchased from ATCC (American Type Culture Collection, Virginia, USA). NCM460 cells (normal human colon mucosa cells) were obtained by a cell licensing agreement with INCELL Corporation (USA). HS756T (human stomach cancer), SNU1 (human stomach cancer), and SNU216 (human stomach cancer) cells were purchased from Korea Cell Line Bank (Korea). HCT116 cells were cultured in McCoy’s 5a medium (Sigma-Aldrich) supplemented with 2 mM glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS) at 37 °C and 5% CO₂. HT29 and SW480 cells were cultured in RPMI medium (Sigma-Aldrich) supplemented with 2 mM glutamine, 1% penicillin-streptomycin, and 10% FBS at 37 °C and 5% CO₂. MDA-MB-231, MDA-MB-468, A375, A2058, HS-746T, SNU1, and SNU216 cells were supplemented with 2 mM glutamine, 1% penicillin-streptomycin and 10% FBS in DMEM medium (Sigma-Aldrich) at 37 °C and 5% CO₂.

Adherent cells were trypsinized and washed twice with 1% bovine serum albumin (BSA) (A2058, Sigma-Aldrich) in phosphate-buffered saline (PBS). The cells were then incubated with fluorescein isothiocyanate (FITC)-conjugated anti-human CD44 (347943, BD Biosciences) and R-phycoerythrin (PE)-conjugated anti-human CD24 (555428, BD Biosciences) antibodies (1:400 dilution) for 30 min at 37 °C in the dark. Fluorescein isothiocyanate (FITC)/PE-conjugated IgG isotypes (560952/560951, BD Biosciences) were used as a control. Cells were washed twice with 1% BSA in PBS and resuspended in 500 ml of PBS prior to analysis on a FACS Canto flow cytometer (BD Biosciences).