Figure 3

Cytotoxicity of the peptide in vitro and ex vivo. (A) Determination of the cytotoxicity of Pep and TAT-Pep peptides on the MEC-1 cell line. Increased concentrations (0 to 100 µM) of Pep and TAT-Pep were incubated with the MEC-1 cell line, and the number of dead cells was measured using the Incucyte live-cell imaging device with Cytotox Dye (green). Fluorescence was monitored for 24 h. (B) Measurement of the cytotoxicity of the TAT-Pep peptide on CLL-B cells from patients. Increased concentrations (0 to 30 µM) of TAT-Pep were incubated with CLL-B cells, and the number of dead cells was measured using the Incucyte live-cell imaging device with Cytotox Dye (green). Fluorescence was monitored for 48 h. Experiments were conducted on 30 different patients, each in technical triplicates. (C) Evaluation of TAT-Pep’s cytotoxicity on CLL-B cells from 6 patients bearing TP53 mutations (Patients ID numbers: 2, 6, 7, 9, 11 and 12). The same protocol as in (B) was applied. (D) Cytotoxicity of the TAT-Pep peptide on PBMCs from healthy donors as well as isolated healthy B cells. The same protocol as in (B) was applied. Increased concentrations (0 to 30 µM) of TAT-Pep were incubated with PBMCs or B cells from 6 different individuals, each in technical triplicates. In all analyses, the ‘Green Object Count/Phase Object Count’ ratio was determined using the Incucyte analysis software to obtain the percentage of dead cells. The area under the curve for each condition was then calculated, and they were compared using a t-test.