Fig. 4

Rolapitant promotes apoptosis of lung cancer cells by up-regulating DR5. A Flow cytometric analysis of Annexin V-FITC/PI staining in A549, H1299 and BEAS-2B cells treated with DMSO or Rolapitant (10, 15, 20, or 25 μM) for 48 h. Bar graph showed the percentage of apoptosis. B, C Proteomic analysis showed the up-regulated and down-regulated proteins in A549 treated with DMSO or Rolapitant (10 μM). Bubble plots showed the pathway of KEGG enrichment. Volcano plots illustrated the differentially expressed proteins in A549 treated with DMSO or Rolapitant (10 μM). D Flow cytometric analysis showed the different surface expression levels of DR5 receptor in A549 and H1299 treated with DMSO or the indicated concentrations (10, 15, 20, or 25 μM). Bar graph showed the mean fluorescence intensity of DR5 receptors on the membrane. E Western blot analysis of the three independent DR5 siRNA knockdown effects. F DR5 siRNA knocking down and control A549 and H1299 cells treated with DMSO or Rolapitant (10 μM) for 48 h were analyzed by Flow cytometric. Bar graph showed the percentage of apoptosis. G A549, H1299 and BEAS-2B cells were treated with DMSO or Rolapitant (10 μM) for 22 h and then exposed to TRAIL (100 ng/ml) for 2 h. Flow cytometric analysis of Annexin V-FITC/PI staining was used to detect the apoptosis cells. Bar graph showed the percentage of apoptosis. H Western blot analysis of DR5, Cleaved-Caspase-8, Cleaved-Caspase-3, PARP and Cleaved-PARPP levels in A549 and H1299 cells treated with DMSO or Rolapitant (10 μM) in the absence or presence of TRAIL (100 μg/ml). All the results were presented as the mean ± SD from 3 independent experiments. (n = 3, *p < 0.05, **p < 0.01, ***p < 0.001, ns = no significance, vs. DMSO)