PKT16 treatment provokes cell cycle arrest and a mitochondrial type of caspase-independent PCD in CLL cells. (A) Cell viability was determined by Annexin-V/PI colabeling in CLL cells that were preincubated with the vehicle (−) or QVD (+) (1 µM, 20 minutes) and were treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours). The percentages refer to the Annexin-V-positive staining (n = 4). (B) PLCγ1-Y783 phosphorylation was detected by immunoblot analysis in untreated (Co.) or PKT16-treated (100 µM, 6 hours) B lymphocytes. Equal loading was confirmed by whole PLCγ1 detection. This blot was repeated 3 times with similar results. (C) Cell death was measured by Annexin-V/PI co-labeling in B cells untreated (−) or treated with PKT16 (100 µM, 6 hours) or venetoclax (4 nM, 24 hours) and preincubated with the vehicle (Control) or the external Ca²⁺ chelator BAPTA (10 mM, 30 minutes). Annexin-V positive cells were quantified and expressed as a percentage (n = 5). (D) Representative Ca²⁺ mobilization recorded with the Fura-2 dye in a fluorimeter in untreated (Control) and PKT16 (100 µM) or venetoclax-treated (4 nM) B cells. Ionomycin (Iono; 1 μM) was used as a control to show the maximum response. (E) The loss of ΔΨm was measured in the CLL B cells untreated (Control) or treated with PKT16 (100 µM, 6 hours). A representative flow cytometry plot is shown. The percentage in the cytofluorometric plot refers to cells with low ΔΨm after PKT16 treatment. The data from 5 patients are presented in a histogram. (F) Mitochondrial and cellular ROS levels were recorded by flow cytometry with the help of the mitosox and hydroethidine dyes in CLL B cells untreated (Control) or treated with PKT16 (100 µM, 6 hours). Percentages in the cytofluorometric plots refer to cells with high ROS levels. The data from 7 patients are presented in a histogram. (G) Flow cytometry cell cycle analysis performed in control and PKT16-treated (100 µM, 6 hours) OSU-CLL cells by BrdU and 7-AAD (DNA content) colabeling. (Left) Representative cytometric panels of control and PKT16-treated B cells. Percentages refer to cells in the indicated cell cycle phase. (Right) Percentage of cells in phases S, G2/M, and G0/G1 was quantified and expressed as a plot (n = 5). (H) Assessment of oligonucleosomal DNA fragmentation in OSU-CLL cells untreated (Control) or treated with PKT16 (100 µM, 6 hours) or etoposide (100 µM, 24 hours). This gel was repeated 3 times with similar results. Statistical significance in panels A, C, E, and F was calculated by the Student t test and by a Mann-Whitney U test in panel G. Symbols and bars represent mean ± SD.