Fig. 4.

Lurbinectedin downregulates the expression of CCR7 on B-CLL cells and inhibits the migration towards CCL19 and CCL21. CLL PBMC were cultured for 24 h at 37 °C. Then, Lurbinectedin 1 nM, 3 nM or vehicle were added for 24 h. After that, cells were labeled with anti-CD19-PECy5 and anti-CCR7-PE (a) or anti-CXCR4-PE (b) antibodies and analyzed by flow cytometry. Figure shows the individual values and the mean ± SEM, n = 14. c, d The effect of Lurbinectedin over CLL CD19⁺ cell migration was measured by a migration assay on transwell plates (for more details on the procedure see the methods section). The results show individual values and mean ± SEM of the migration index to CCL19 (n = 7) and CCL21 (n = 6). The statistical analysis was performed using Friedman test followed by Dunn’s post-test. e CLL B lymphocytes were treated with Lurbinectedin 3 nM or vehicle for 24 h. Cells were stimulated with the chemokines and whole protein extracts were obtained. Cell extracts were then analyzed by Western blot using antibodies directed toward P-Erk 1/2. Figure shows a representative example of the bands obtained. f Quantification of the experiment described on (e) as mean ± SEM of the intensity ratio of the P-Erk vs actin, n = 8. Statistical analysis was performed by two-ways ANOVA followed by Tukey multiple comparison post-test. g CLL B lymphocytes were treated with Lurbinectedin 3 nM or vehicle for 24 h. Cells were stimulated with the chemokines and stained for P-Akt as was described in materials and methods. Results showed representative histograms and the corresponding MFI. Isotype control is depicted in grey. h Mean ± SEM of the MFI of p-Akt staining are shown (n = 9). The statistical analysis was performed by a two-ways ANOVA followed by Tukey’s post-test of multiple comparisons. Lur Lurbinectedin, MFI mean fluorescence intensity, N.S no statistically significant difference; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001