Figure 6.

Further mechanisms of cell death induction by T22-AUR in CXCR4⁺ DLBCL cells. (A and B) DAPI staining pictures and ratio quantification of MCs (yellow arrows) or apoptotic bodies (red arrows) in U-2932 cells treated with buffer or 125 nM-T22-AUR (24 and 48 h). Ratio quantification was measured dividing the number of cells undergoing MC or apoptosis in 10 fields of T22-AUR samples (24 and 48 h) by 10 fields of buffer-exposed samples. (C) Annexin/PI assay showing the total percentage of viable, early apoptotic and late apoptotic U-2932 cells treated with buffer or 125 nM T22-AUR (24 h and 48 h). These experiments were performed in biological triplicates. (D) IHC staining using γH2AX and cleaved PARP antibodies after buffer or 125 nM T22-AUR treatment (24 and 48 h) in U-2932 cells. (E) Ratio quantification of the IHC positive stained area marked by γH2AX and cleaved PARP in buffer or T22-AUR-treated samples. Ratio quantification was represented dividing the area of positive cells from 6 fields of T22-AUR-treated samples (24 and 48 h) by 6 fields of buffer-treated samples. (F) Anti-PARP, cleaved PARP, pro-caspase-3 and cleaved caspase-3 detection, by Western blotting, in U-2932 extracts treated with buffer or 125 nM T22-AUR for 24 h and 48 h. GAPDH and α/β tubulin were used as endogenous controls. All pictures were taken at 400x (scale bars= 50 μm). All data are shown as mean ± standard error. *p≤0.05; ***p≤0.005.

Abbreviation: MC, mitotic catastrophe.