Fig. 2. Involvement of afadin in PDGF-induced Akt activation and cell survival in NIH3T3 cells. (A) Inhibition of the PDGF-induced phosphorylation of Akt by knockdown of afadin in NIH3T3 cells. After 16 hours of serum starvation, control (denoted by C) and afadin-knockdown (KD) NIH3T3 cells were treated with 3 ng/ml 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. Bars in the graph under each blot represent the relative band intensities of phosphorylated Akt (top), Src (middle) and PDGF receptor (bottom) normalized to the total amount of each protein as compared with the values from control cells treated with PDGF for 5 minutes, which are expressed as 1. Grey bars, control cells; white bars, KD cells. Error bars indicate s.e.m. ^*P<0.05 vs control NIH3T3 cells. (B) Increase in serum-starvation-induced apoptosis by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were serum-starved for 16 hours and then treated with or without 3 ng/ml PDGF or 3 ng/ml EGF for 24 hours. Apoptotic cells were detected using the TUNEL method. Nuclei were counterstained with DAPI and are seen in blue. Bars in the graph to the right represent the mean percentages of TUNEL-positive cells in three independent experiments. In each experiment, the number of TUNEL-positive cells among a total of 100 cells observed in four different fields of view was counted. Error bars indicate s.e.m. ^*P<0.05 vs paired control cells; ^#P<0.05 vs control cells without growth-factor treatment; P<0.05 vs afadin-knockdown cells without growth-factor treatment. (C) Increase in Fas-ligand-induced apoptosis by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were serum-starved for 16 hours and then stimulated with 100 ng/ml Fas ligand (Jo2 mAb) concomitant with or without 3 ng/ml PDGF or 3 ng/ml EGF for 24 hours. Apoptotic cells were counted as described in B. Error bars indicate s.e.m. and symbols indicate the same as shown in B. (D) Increased production of cleaved caspase-3 by knockdown of afadin in NIH3T3 cells. Confluent control and afadin-knockdown NIH3T3 cells were cultured in the same conditions described in B and C. Cell lysates were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control. (E) Inhibitory effect of Akt-CA on apoptosis in afadin-knockdown NIH3T3 cells. Confluent afadin-knockdown NIH3T3 cells transfected with GFP or Akt-CA-GFP 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. Cells were immunostained with the anti-cleaved-caspase-3 mAb and nuclei were counterstained with DAPI. The graph represents the mean percentage of cleaved-caspase-3-positive cells assessed as described in B. Error bars indicate s.e.m. ^*P<0.05 vs control NIH3T3 cells. (F) Reduced production of cleaved caspase-3 by the expression of Akt-CA in afadin-knockdown NIH3T3 cells. Cell lysates from control and afadin-knockdown NIH3T3 cells cultured in the same conditions as described in E were subjected to western blotting with the anti-cleaved-caspase-3 pAb. GAPDH was immunoblotted as a loading control. Scale bars: 50 µm.