Fig. 7.

Inhibition of Type II mAb mechanisms of action. (A) Inhibition of dual-target DFMT apoptosis using an actin polymerization inhibitor (Latrunculin B). Raji cells were treated with combinations of dual-target DFMT with or without a pre-treatment of actin inhibitor (10 μM) for 24 h. Induced apoptosis was quantified using Annexin V/PI stain and flow cytometry. Geometric mean fluorescence of Annexin V+/PI+ cells are presented as a normalization to untreated control cells. (B) Inhibition of homotypic cell adhesion by co-treatment of Raji cells with dual-target DFMT with or without actin inhibitor (10 μM for 6 h at 37 °C). Bright field microscopy images depict Raji cells treated with dual-target DFMT (top) and cells treated with dual-target DFMT + Latrunculin B (bottom). Loss of homotypic cell adhesion (actin-dependent process) was observed. (C) Inhibition of lysosomal enlargement of dual-target DFMT pre-treated with or without actin inhibitor (10 μM for 24 h at 37 °C). Raji cells were subjected to 24 h dual-target DFMT therapy with or without Latrunculin B pretreatment and lysosomal enlargement was analyzed by LysoTracker Green geometric mean fluorescence quantified using flow cytometry. Bar graph represents mean fluorescence normalized to untreated control with accompanying fluorescence histograms. (D) Inhibition of lysosomal enlargement using the cysteine protease inhibitor, E-64. Raji cells were subjected to dual-target DFMT with or without pre-treatment of E-64 (5 μM for 24 h at 37 °C). After 24 h, cells were treated with LysoTracker Green and fluorescence was analyzed using flow cytometry. Fluorescence histograms of untreated control (black) are overlayed with dual-target DFMT (blue) and dual-target DFMT + inhibitor (red). All experiments were performed in duplicate. *** p < 0.001, ** p < 0.01 by One-Way ANOVA and Tukey test.