Effects of extracellular FGF2 on cell survival and transcriptional activation.

<p>(a) ELISA. PC-9 cells were treated with various concentrations of gefitinib (indicated) for indicated days, and conditioned media were collected. The FGF2 level in the media was examined by an ELISA analysis. Data are shown as mean ± SD (<i>n</i> = 3). (b) Effects of PD173074 (PD), an FGFR inhibitor, on cell viability under the presence of gefitinib. PC-9 cells were treated with 0.5 and 1.0μM of PD173074 or DMSO (a vehicle) for 1h prior to exposure to 0, 0.1, 1.0 and 10μM of gefitinib. After 3day-incubation, cell viability was examined. Data are shown as mean ± SD (<i>n</i> = 3). The data of DMSO represent the results treated only by gefitinib. Significant difference between PD173074 and DMSO, <i>i</i>.<i>e</i>., between the presence and absence of PD173074, was examined by one-way analysis of variance (ANOVA) (Dunnett’s test; ***p<0.001). (c) Effects of extracellular FGF2 on cell viability under the presence of gefitinib. PC-9 cells were treated with FGF2 or DMSO together with gefitinib, and cell viability was examined and analyzed as in b (<i>n</i> = 3). (d) Inhibition of extracellular FGF2 by neutralizing antibodies. PC-9 cells were treated with 0.1 or 1μM of gefitinib under the presence of various levels of anti-FGF2 antibodies (α-FGF2). After 3day-incubation, cell viability was examined as in b. Significant difference between α-FGF2 and a control IgG was examined by Student’s t-test (two-tailed; *<0.05, **p<0.01, ***p<0.001). (e, f) Effects of extracellular FGF2 on transcription factors. The pGL4-NF-κB-RE and pGL4-SRE plasmids were introduced together with phRL-Tk into PC-9 cells. After 24h-incubation, the cells were treated with various levels of FGF2, and further incubation was carried out. After 6h, dual-luciferase assay and data analyses were carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201796#pone.0201796.g001" target="_blank">Fig 1b</a> (<i>n</i> = 3). (g) Influence of PD173074 on NF-κB activation. The pGL4-NF-κB-RE and phRL-Tk plasmids were transfected into PC9 cells as in e, and the cells were treated with PD173074 for 1h prior to gefitinib treatment. After 6h gefitinib treatment, dual-luciferase assay was carried out as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0201796#pone.0201796.g001" target="_blank">Fig 1b</a> (<i>n</i> = 3).