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. 2019 Oct 12;11(10):1544. doi: 10.3390/cancers11101544

Figure 2.

Figure 2

Effect of imatinib (IM), IPA-3, and their combination on cellular processes in K562 and KCL-22 cells. (A) Changes in the cell cycle distribution of K562 and KCL-22 cells after treatment with the indicated inhibitors. Each bar represents the mean ± SD. (n ≥ 4). A significant difference compared with the control group at p ≤ 0.003 is indicated by an asterisk (*). (B) Detection of apoptosis by using the Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. Data are expressed as the mean ± SD (n ≥ 5); * p ≤ 0.001 in comparison to control cells. (C) Changes in the mitochondrial membrane potential (MMP). Left panel: Depolarization of mitochondrial membrane measured by the green fluorescence of JC-1 stained cells. Data were expressed as mean  ±  SD (n ≥ 3); * p ≤ 0.03 in comparison to control cells. Right panel: Representative microphotographs present MitoStatus Red stained K562 cells treated with IM, IPA-3, and their combination for 24 h. A decrease in red fluorescence intensity reflects loss of MMP visualized under the fluorescent microscope (ZOE Fluorescent Cell Imager, Bio-Rad, Hercules, CA, USA), magnification 40×, scale bar 49 µm. (D) Western blots analysis of the indicated proteins. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a loading control. The changes in the protein levels were estimated by quantification of band intensities with the QuantiScan ver.3.0 (Biosoft, UK) densitometry analysis software. The protein levels were normalized to the signal of GAPDH (housekeeping protein). To calculate the normalized signal of each experimental target band, the signal intensities of each experimental target band were divided by the normalization factor. Normalization factor was determined by dividing the observed signal value for the GAPDH in each analyzed lane by the highest observed housekeeping protein signal on the blot. The results are presented as the arbitrary units (A.U.) calculated in comparison to control band. Abbreviations: PARP, Poly (ADP-ribose) polymerase; Bad, BCL2 associated agonist of cell death; cRaf, RAF proto-oncogene serine/threonine-protein kinase; STAT3, signal transducer and activator of transcription 3; Erk1/2, extracellular signal-regulated 1/2 kinase; γH2AX, phosphorylated form of H2A histone family member X.