PIK3R3 depletion results in inhibition of clonogenic and anchorage-independent growth of Ewing Sarcoma cells.

(A) Stable depletion of PIK3R3 protein in Ewing Sarcoma SK-ES-1 cells was achieved using lentiviral delivery of two different targeting shRNAs (PIK3R3-sh1 and 2, described in Methods; shControl: cells expressing non-targeting scrambled sequence RNA, also described in Methods). PIK3R3 RNA levels were determined by qRT-PCR, as described previously [19], using specific primers to PIK3R3, and U6 as an endogenous control. PIK3R3 protein levels were determined by immunoblotting with PIK3R3 antibody, normalized to tubulin and quantified by densitometry. Mean and standard deviation are plotted at top, and a representative immunoblot is shown below. Mean values in the control groups are set to 1; *p<0.05, relative to corresponding control, using the student t-test. (B) Colony growth of control and PIK3R3-depleted SK-ES-1 cells as determined using a clonogenic assay (top panels and graph on left) and a soft agar assay of anchorage independent growth (bottom panels and graph on right). Representative images of individual wells from one experiment are shown. Quantifications represent the mean and standard error of the mean (SEM) of colony counts from independent experiments, each performed in triplicate (colony numbers in controls are set to 1; *p<0.05, relative to control, using the student t-test). (C) Levels of pAkt^Ser473 and total Akt were determined using immunoblotting as in Fig. 1, and quantified by densitometry. Plot shows relative pAkt^Ser473/Akt ratios from multiple experiments in control and PIK3R3-depleted SK-ES-1 cells; to facilitate comparison among different experiments, one value from control cells in each experiment is arbitrarily set to 1; bars show mean and standard deviation for each group. Statistical analyses did not reveal significant differences among the groups.