Fig. 4. Involvement of nectin-3 in PDGF-induced phosphorylation of Akt and cell survival in NIH3T3 cells. (A) Inhibition of PDGF-induced phosphorylation of Akt by knockdown of nectin-3 in NIH3T3 cells. Confluent control and nectin-3-knockdown NIH3T3 cells were treated for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. ^*P<0.05 vs control NIH3T3 cells. (B) Increased number of apoptotic nectin-3-knockdown NIH3T3 cells. Confluent control and nectin-3-knockdown NIH3T3 cells were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand for 24 hours in the presence of 3 ng/ml PDGF. Apoptotic cells were detected using the TUNEL method. Nuclei were counterstained with DAPI and are seen in blue. The graph represents the mean percentage of TUNEL-positive cells assessed as described in Fig. 2C. ^*P<0.05 vs control NIH3T3 cells. Scale bars: 50 µm. (C) Requirement for the association of nectin-3 with afadin in order to obtain PDGF-induced phosphorylation of Akt. Four types of cells (control NIH3T3 cells, nectin-3-knockdown NIH3T3 cells, nectin-3-knockdown NIH3T3 cells rescued by FLAG-tagged full-length nectin-3 and nectin-3-knockdown NIH3T3 cells rescued by FLAG-tagged nectin-3 lacking the C-terminal four amino acids) were treated with PDGF for the indicated periods of time. Cell lysates were subjected to western blotting with the indicated antibodies. GAPDH was immunoblotted as a loading control. The relative band intensity was calculated as described in Fig. 2A. ^*P<0.05 vs control NIH3T3 cells. C, control; KD, nectin-3 knockdown.