Inhibition of COT1 mRNA and protein expression by silencing COT1 RNA (siCOT1). A: siRNAs were transfected into cells using Amaxa nucleofector and incubated for 48 h, then 1 nM 1,25D was added for 48 h. The cells were harvested to determine efficiency of knock down of COT1 mRNA. siControl is a non-specific siRNA used here as a control. “C” represents vehicle controls; “D,” cells treated with 1,25D. Asterisks () indicate significant (P < 0.05) difference from cells transfected with control RNA. B: Immunoblots for the target protein after transfection with the silencing RNAs showed that siCOT1 effectively (~50% HL60, ~90% U937 cells) knocked down COT1 protein. OD = optical density signals for COT1 relative to the internal control, Crk-L protein.