Rnases

The effect of recombinant SLURP1 on cell cycle arrest was investigated by flow cytometry analysis. Caco2, Colo320DM, and H508 cells cultured in 96-well plates were treated with rSLURP1 protein at 200, 1000, and 2000 nM/well for 24 h in a humidified, 5% CO₂ atmosphere. After treatment, cells were stained with propidium iodide and prepared for flow cytometer analysis. Briefly, cells were fixed with 70% ethanol for 30 min at 4 °C followed by PBS wash. Cells were treated with RNases (Qiagen, Hilden, Germany), and the cell populations in the G1, S, and G2/M phases were quantified. The experiment was repeated twice, and the average values were considered for the analysis.

Cells were reverse transfected with siRNAs 96 h before analysis at a confluence of 40 %. MSCs were collected by trypsination and stained with 200 μg/ml of propidium iodide (VWR), 0.1 % (w/v) sodium azide (Sigma Aldrich), 0.1 % (v/v) Triton-X100 (Sigma Aldrich) and 10 μg/ml RNAses (Qiagen) for 2–4 h at 4 °C. Single cells were analyzed for fragmented DNA, sub G1, S and G2/M peaks by fluorescence flow cytometry array (BD Biosciences). Analysis was performed with the FlowJo software 887 (Tree Star Software).