Figure 1. PTEN affects β-catenin nuclear localization.

(a) Confocal microscopy revealed cells (labelled with arrows) with a heavily laden β-catenin (bcat) nuclear staining (b), in contrast to nearby PTEN-GFP-positive cells, where β-catenin staining could seldom be observed within the nucleus, arrowheads. Cells were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (c). Merged is shown (d). Scale bar, 10 μm. Immunofluorescence experiments for the Hs944T cells were performed three times with similar results. (e–h) Human melanoma Lyse cells mutated for NRAS (Q61K), which produce PTEN, were transfected with siScr (e,f) and siPTEN (g,h). Cells were labelled for β-catenin (e,g) and counter stained with DAPI (f,h). Scale bar, 10 μm. Immunofluorescence experiments for the Lyse cells were performed two times with similar results. (i,j) Confocal microscopy showing the localization of β-catenin in Tyr::Cre/°;PTEN^f/+=PTEN^f/+ (i) and Tyr::Cre/°;PTEN^f/f=PTEN^f/f (j) melanocytes. Note the increase of nuclear β-catenin staining in PTEN^f/f cells. Scale bar, 10 μm. Immunofluorescence experiments for the murine PTEN^f/+ and PTEN^f/f cells were performed four times with similar results. (k) Immunoblot analysis of PTEN, AKT (total and phosphorylated form Ser473), GSK3β (total and phosphorylated form Ser9), β-catenin (total, phosphorylated form Thr41/Ser45 and Ser675) and β-actin proteins in Hs944T transfected with either expression vector encoding GFP (CMV::GFP) or PTEN (CMV::PTEN-GFP). Cells expressing either exogenous GFP or PTEN treated with LY294002 or LiCl for 1 h. It is noteworthy that a higher concentration of GSK3β antibody reveals a second upper band. Western blot analyses were performed two times for all antibodies with similar outputs. (l) Immunoblot blot analysis for GFP, PTEN, AKT (total and pSer473), β-catenin pSer675 and β-actin of Hs944T lysates co-transfected with either empty vector GFP or PTEN and WT or constitutively active p110 E545K mutant. Cells were starved for 2 h with 0.1% serum before lysis. Western blot analyses were performed two times for all antibodies with similar outputs. (m) Immunoblot blot analysis for PTEN, AKT (total and pSer473), β-catenin pSer675 and β-actin of Hs944T lysates co-transfected with a constitutive active form of p110 (p110 CAAX) or a kinase-dead form (p110 KD) in the presence of GFP or PTEN. Western blot analyses were performed three to six times, depending on the antibody with similar outputs.