Fig. 1

Stable and transient PI3K-C2β downregulation inhibits 2D colonies formation. a Representative blot confirming downregulation of PI3K-C2β in all stable PC3 clones lacking PI3K-C2β (shPI3K-C2β) that were used in this study. Levels of PI3K-C2β in the corresponding control stable clones (sh scrambled) compared to parental PC3 cells are also shown. GAPDH was used as loading control. b, c PC3 cells and the indicated stable clones were plated as single cells in 6 well plates (200 cells/well) and incubated in complete media for 10 days. Cells were then fixed and stained with crystal violet, images were collected and 2D colonies were manually counted. Data in (b) indicate the number of colonies/well and are means ± s.d. of n = 2 independent experiments (PC3, n = 4). Representative images of colonies stained with crystal violet at the end of the experiment are shown in (c). d PC3 cells were transfected with siRNAs specifically targeting PI3K-C2β or PI3K-C2α. Control cells were transfected with a non-targeting siRNA (si control) or transfection reagent alone (oligo). Representative blots confirming efficient downregulation of the enzymes by all siRNAs used in this study. Tubulin and GAPDH were used as loading controls. e PC3 cells were transfected as in (d). Non transfected cells (NT) were also used as an additional control. After 24 h, cells were detached and plated as described in (b, c). Data indicate the number of colonies/well and are means ± s.e.m. of n ≥ 3 independent experiments. *p < 0.05 vs NT; #p < 0.05, ^##p < 0.01 vs oligo; ^$p < 0.05, ^$$p < 0.01 vs si control (two-tailed, unpaired t-Test with Welch’s correction)