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Figure 8. Effect of FGFR inhibitor BGJ398 on neuroblastoma cell migration, cell death, and intracellular signaling. (A) SK-N-SH neuroblastoma cells were treated with increasing concentrations of BGJ398 and uniform scratch wounds were generated. Images from the IncuCyte Zoom^TM live-cell imager were obtained following the initial wound and 24h and 36h after treatment with BGJ398, with cells migrating into the scratch wound shown in purple; (B) Wound closure was calculated at regular intervals and plotted over time. Wound closure rates were also calculated and plotted versus concentrations of BGJ398; (C) SK-N-SH neuroblastoma cells were treated with 2.5, 5, or 10 µM BGJ398 for 72 h and were evaluated using a live cell imaging caspase activity assay. Images were obtained and analyzed using the IncuCyte Zoom^TM, with individual green dots representing cells with caspase activity. Images of untreated cells and of cells treated with 5 or 10 μM BGJ398 for 72 h are shown (left). Average green dot counts per field were normalized to control cells, and fold change was determined by comparing average green object counts in treated cells with average counts in untreated cells (right); (D) (top) SK-N-BE(2) cells were treated with or without BGJ398 (2.5 µM) for 24 h followed by stimulation of bFGF (20ng/ml) 15 min prior to protein extraction. Western blots were performed to determine effects on levels of total and phosphorylated FGFR1. (Bottom) SK-NBE(2), SK-N-SH, and NBL-S neuroblastoma cells were treated with or without BGJ398 (2.5 µM) for 24 h followed by stimulation of bFGF (20ng/ml) 15 min prior to protein extraction. Western blots were performed to determine effects on levels of total and phosphorylated MEK1/2 and ERK1/2 along with PARP and cleaved PARP; (E) SK-N-BE(2), SK-N-SH, and NGP neuroblastoma cells were treated with or without BGJ398 (2.5 µM) for 24 h followed by stimulation of bFGF (20ng/ml) 15min prior to protein extraction. Western blots were performed to determine effects on levels of total and phosphorylated FRS2 and ERK1/2. GAPDH was used as a loading control. FGFR: fibroblast growth factor receptor; PARP: poly-ADP ribose polymerase; FRS: fibroblast growth factor receptor substrate; BGJ398: infigratinib; ERK: extracellular signal-regulated kinase.