Research Paper Volume 12, Issue 16 pp 16083—16098

Figure 5. Chidamide and Ibrutinib have synergistic effect on CLL cells. (A) Immunoblotting analysis of LC3 in MEC-1 cell line cells after ibrutinib (IB, indicated concentrations) treatment for 48 hours. Representative data shown are from three independent experiments. The bar graph represents the relative PARP cleavage/GAPDH ratio measured by immunoblotting. (B) CCK8 assay for detecting metabolically active cells. MEC-1 and JVM-3 cell lines were incubated with indicated regimens (10μmol/L Ibrutinib, 20μmol/L chidamide alone or in combination) for 24 hours. Viability of cells compared with the corresponding controls was shown from three independent experiments. (C) Flow cytometry using Annexin V–FITC/PI staining for cell death analysis as mentioned before. MEC-1 and JVM-3 cell lines were incubated with indicated regimens (10μmol/L Ibrutinib, 20μmol/L chidamide alone or in combination) for 24 hours. The bar graphs showed the percentage of apoptotic cells. (D) Combination analyses were performed following the median-effect method 37. MEC-1 and JVM-3 cell lines were exposed to chidamide and Ibrutinib whose concentration were in a constant ratio of 2:1 simultaneously for 24 hours. The concentrations of chidamide used for these experiments were (in μmol/l) 1/2/10/20/40/50 while those of ibrutinib were (in μmol/l) 0.5/1/5/10/20/25 (shown as blue dots, and increasing from left to right along the x axis). CIs for different levels of growth inhibition (fraction affected) were calculated using the CompuSyn software. Details of CI and DRI values shown in Table 1. (E) Immunoblotting analysis of LC3 in MEC-1 cell line cells after ibrutinib (IB, 10μmol/L) treatment for 24 hours in the presence or absence of chidamide (CHI, 20μmol/L). The bar graph represents the relative expression level of LC3-II with respect to the control group.