On targetplus smartpool control sirna

HCT116 (ATCC) CRC cells were plated at 4500 cells per well in a 96-well plate, let attach for 16 h and transfected with SMARTpool ON-TARGETplus control siRNA or siRNAs targeting NAT2 using the DharmaFECT 2 transfection reagent (Dharmacon) in serum- and antibiotics-free McCoy’s 5A medium. After 5 h, the cells were supplemented with FBS to 10% final concentration and incubated for 24 h. Finally, cells were treated with APA or 5-FU in different concentrations and cell viability was evaluated in an MTT assay after 68 h.

The RKO and DLD-1 CRC cells were plated at 10,000 or 5000 cells, respectively, per well in a 96-well plate. After 16 h, the cells were exposed to serum- and antibiotics-free McCoy’s 5A medium and transfected with SMARTpool ON-TARGETplus control siRNA or siRNAs targeting DYRK1A, AURKA, or CDK7 using the DharmaFECT 2 transfection reagent (Dharmacon). After 5 h the cells were supplemented with FBS to 10% final concentration and incubated for 72 h. The cell viability was evaluated in an MTT assay as described above.

siRNA transfection was carried out using ON-TARGETplus SMARTpool control siRNA and CAMKK2 siRNA (Dharmacon, Lafayette, United States). AGS cells were transfected as previously described by using RNAiMAX (Invitrogen, Grand Island, NY) (Najar et al., 2021b (link)). They were plated at a density of 60,000 cells/well in six-well plates followed by transfection with 20 nM of CAMKK2 siRNA and control siRNA in Opti-MEM media (Gibco, CA, United States) using RNAiMAX. The cells were transfected with CAMKK2 siRNA and control siRNA in Opti-MEM media using Lipofectamine RNAiMAX transfection reagent. After transfection, the media was changed to complete media (DMEM +10% FBS) and the cells were allowed to grow for 48 h. Finally, the cells were harvested using lysis buffer, and lysates were used for immunoblotting experiments to check protein expression.