Journal: International Journal of Molecular Sciences
Article Title: Functional Characterization of VS-186B, a Novel HDAC Inhibitor with Anticancer Activity
doi: 10.3390/ijms262311354
Figure Lengend Snippet: ( a ) Phosphatidylserine externalization was observed after 18 h of exposure to VS-186B, indicating apoptotic cell death. Cells were stained with Annexin V FITC and PI and analyzed through flow cytometry. The x-axis represents negative (untreated), vehicle (DMSO), and positive (H 2 O 2 ) controls, as well as VS-186B at its 24 h CC 50 (3.63 µM) and CC 50 × 2 (7.26 µM). The y-axis dictates the percentage of cells undergoing apoptosis. The summation of both early and late apoptotic subpopulations determines the total percentage of apoptotic cells. Representative flow cytometry plots demonstrate viable cells, early apoptotic cells, late apoptotic cells, and necrotic cells after CC 50 and DMSO treatment at 24 h. ( b ) After 18 h of exposure to VS-186B, there is significant ROS production. Flow cytometry plots indicate the percentage of cells induced by ROS after CC 50 and DMSO treatment. Gray represents the negative and vehicle controls and red represents the positive control and treatments. ( c ) Jurkat cells were treated with VS-186B for 10 h, and mitochondrial membrane depolarization was assessed by JC-1 staining and flow cytometry analysis. Flow plots indicate percentage of cells with depolarized mitochondria after CC 50 and DMSO treatment. ( d ) Treatment of VS-186B for 10 h resulted in caspase-3/7 activation, supporting the activation of intrinsic apoptotic cell death. Flow plots represent active caspase percentage comparing CC 50 and DMSO. Gray represents the negative and vehicle controls and red represents the positive control and treatments. Data represents the mean ± standard deviation (SD) of three independent measurements. A two-tailed Student’s paired t -test was performed by comparing DMSO to VS-186B samples. * p < 0.05, ** p < 0.01, *** p < 0.001. Black arrows point to the cell subpopulations shown in each flow cytometry plot.
Article Snippet: 400 μL of cell suspension was distributed into two flow cytometry tubes (unstained and PI-stained) and 5 μL of Propidium Iodide (PI; MP Biomedicals, Solon, OH, USA) at a final concentration of 5 μg/mL was added to each tube, and samples were analyzed by flow cytometry.
Techniques: Staining, Flow Cytometry, Positive Control, Membrane, Activation Assay, Standard Deviation, Two Tailed Test