Sa3800 2

DLD-1, HT29 and HCT116 cells were seeded to six-well plates at a density of 3×10⁵, 4×10⁵ and 3.5×10⁵ cells per well, respectively. Cells were starved in serum-free medium (RPMI-1640 or DMEM) for 48 h. A total of 24 h before the end of starvation, miR-NC or miR-1291 was transfected at a final concentration of 30 nM. Cells were collected at the indicated times (0, 12, 24 and 48 h) and fixed in 70% ethanol for 30 min at 4°C. After fixation, cells were washed twice with PBS and incubated with RNase (Sigma Aldrich; Merck KGaA) for 20 min at 37°C. Cells were treated with propidium iodide (PI; Dojindo Molecular Technologies, Inc.) for 20 min on ice and analyzed by flow cytometry (Spectral Analyzer SA3800; Sony Biotechnology, Inc.) with SA3800 2.0 software (Sony Biotechnology, Inc.).

Apoptotic cells were assessed using an Alexa Fluor 488. Annexin V/Dead Cell Apoptosis kit (Thermo Fisher Scientific, Inc.). A total of 2×10⁵ cells were diluted with 100 µl 1X annexin-binding buffer, after which 5 µl Alexa Flour 488 Annexin V and 1 µl 100 µg/ml PI was added. Samples were subsequently incubated for 15 min at room temperature. A total of 400 µl 1X annexin-binding buffer was added to each cell suspension and apoptotic cells were counted by flow cytometry using Spectral Analyzer SA3800 (Sony Biotechnology, Inc.) with SA3800 2.0 software (Sony Biotechnology, Inc.).

HCT116 cells were seeded in six-well plates at a density of 1×10⁵ cells per well, incubated at 37°C overnight and transfected with miR-NC or miR-1291 at a final concentration of 50 nM. For CD133 marker expression, after 48 h of transfection, cells were resuspended and one million cells were incubated with antibodies against human CD133 (1:50; APC-conjugated; cat. no. 130-113-106; Miltenyi Biotec GmbH) on ice for 20 min in the dark. Samples were then washed twice with PBS containing 2% FBS. For CD166 marker expression, after 72 and 96 h of transfection, the cells were resuspended and one million cells were incubated with PE Mouse Anti-Human CD166 antibody (1:6.7; cat. no. 559263; BD Biosciences on ice for 20 min in the dark. Samples were then washed twice with PBS containing 2% FBS. The Spectral Analyzer SA3800 (Sony Biotechnology, Inc.) with SA3800 2.0 software (Sony Biotechnology, Inc.) was used for flow cytometric analyses. Dead cells were excluded by utilizing forward and side scatter.