FIG. 2.

Ectopic Rai expression increases Ret-induced survival and differentiation of PC12coRα cells. (A) Western blot (WB) analysis of Rai expression with 50 μg of whole-cell lysates from the indicated cell lines and polyclonal antibodies against the Rai CH1 region (upper panel) or vinculin for normalization (lower panel). α-Rai, anti-Rai antibodies. (B) The percentage of apoptosis was measured in PC12coRα cells infected with either the Pinco retrovirus expressing Rai (Rai) or the empty vector (Ctrl) and maintained for 48 h in complete medium (serum), SF medium (0), or SF medium supplemented with GDNF at concentrations of 1, 10, and 100 ng/ml. The percentage of apoptotic cells was measured by FACS analysis of phosphatidylinositol-stained nuclei. Values shown are the means (± standard errors) of three independent experiments, each performed in triplicate. (C) Morphology of control (Ctrl) and Rai-expressing (Rai) PC12coRα cells grown in medium containing 1% serum and supplemented with different concentrations of GDNF (0, 1, 10, 100 ng/ml). Photographs were taken 7 days after the addition of GDNF. These results are representative of three experiments which gave comparable results. (D) Neurite outgrowth was quantitated by counting the cells with neurites longer than the size of two cell bodies. Results were expressed as the increase in percentage (n-fold) with respect to that of unstimulated control cells.