FIG. 3.

PTEN is required for CSIG to regulate p27^Kip1 expression. (A) Results of Western blot analysis of endogenous PTEN and p27^Kip1 expression in HEK 293, U87, and 2BS cells. (B) U87 cells were transfected with pIRES-CSIG (CSIG) versus empty vector (vector) (left panel) or with 20 nM siRNA targeting CSIG (siRNA CSIG) versus Ctrl siRNA (siRNA Ctrl) (right panel) for 48 h. Expressions of CSIG, PTEN, and p27^Kip1 were assessed by Western blot analysis; β-actin served as a loading Ctrl. Relative p27^Kip1abundances are expressed as increases relative to its level in control cells. (C) HEK 293 cells were transiently transfected with siRNA (20 nM) targeting PTEN (PTEN siRNA) (lanes 3 and 4) or siRNA Ctrl (lanes 1 and 2). Twenty-four hours later, cells were further transfected with pIRES-CSIG (CSIG) (left panel, lanes 2 and 4) versus an empty vector (vector; left panel, lanes 1 and 3) or with siRNA (20 nM) targeting CSIG (CSIG siRNA) (right panel, lanes 2 and 4) versus siRNA Ctrl (right panel, lanes 1 and 3). Cells were then cultured for an additional 48 h. Expression of CSIG, PTEN, and p27^Kip1 as well as the relative abundances of p27^Kip1 were evaluated as described for panel B. (D and E) FACS analysis of cells used in the right panel of panel C (D) and in the right panel of panel B (E).