Silencing of Choline kinase A or B reduced Akt(ser 473) phosphorylation in MDA-MB 468 cells. A, The screen was set up using MDA-MB 468 cells as described in the method. The percentage pAkt(ser473) signal is calculated by obtaining total intensity (from fluorohore-Alexa 488) of the signal divided by total number of cells imaged. The reading is compared to the non-targeting siRNA transfected cells (scr, set to be 100%). B, Cells transfected with 50 nM pool (P) or deconvoluted (D1-4) ChoK A or B siRNA for 3 days and 30 μg whole cell lysate were subjected to western blot with the indicated antibodies. The signals obtained from the western blot analysis were quantified with Image J program and Akt(ser473) phosphorylation were normalized to total Akt. The ratios were compared to scr control (set as 1) indicated by the values below the blots. C, siRNA were transfected to cells as (B) for 2 days. 500 ng total RNA were reverse transcribed to cDNA subjected to real time PCR (ABI7500) with the appropriate primer sets. Transcript levels were normalized to the scrambled control (scr). The data is an average of triplicate experiments.