Fig. 2.

Knockdown of Pik3cb reduces self-renewal and decreases Nanog expression. mESCs were transfected with Pik3cb (p110β), Nanog or non-targeting (NT) Smartpool siRNAs and harvested 72 hours later. (A) Quantitative RT-PCR was conducted on quadruplicate samples to measure knockdown of Pik3cb RNA and levels of RNA encoding p110β were normalised relative to levels of β-actin RNA. Representative data from four independent experiments are shown with s.d.; ^*P<0.05 following a Student's t-test. (B) Quantitative RT-PCR was conducted to detect knockdown of Nanog RNA. Representative data are shown with s.d.; ^*P<0.05 and ^**P<0.01 following a Student's t-test. (C) Following transfection, cells were plated for 4 days and self-renewal assessed by alkaline-phosphatase staining. The average percentage of alkaline-phosphatase-positive, self-renewing colonies in each condition are shown with s.e.m. (n=4); ^*P<0.05 following a Student's t-test. The number of total colonies formed in each condition did not vary significantly. (D) Assessment of self-renewal by alkaline-phosphatase staining is depicted by the percentage of pure alkaline-phosphatase colonies (compact round red-stained colonies) with s.e.m. (n=4); ^* P<0.05 following a Student's t-test. (E) Immunoblotting was used to detect (i) Nanog and Oct4 protein levels or (ii) p85 protein levels, with SHP-2 reprobing used to confirm equal loading.