CYTSA Depletion in Colorectal Cancer Cells

One or two different siRNAs (5′-GAACUGAACAGCCAGGAUA [dT][dT]-3′ and 5′-ACAGAAGGCTATCAGAATA[dT][dT]-3′) that targeted human CYTSA were used to deplete CYTSA in CRC cells. A validated non-targeting siRNA (Sigma-Aldrich, St. Louis, MO, USA) was used as a control [15 (link)]. The jetPRIME siRNA transfection reagent (Polyplus, New York, NY, USA) was used according to the manufacturer’s instructions. In brief, the siRNA and jetPRIME reagent were mixed together, incubated for 15 min, and added to CRC cells grown in cell culture medium without antibiotics in six-well plates. After 24 h, cells were washed with phosphate-buffered saline (PBS) and cultured with regular cell culture media with antibiotics. Forty-eight hours from the start of transfection, cells were lysed with either (1) TRIzol^® to isolate RNA or (2) radioimmunoprecipitation assay (RIPA) buffer to extract proteins for further analyses.