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. Author manuscript; available in PMC: 2018 Apr 25.
Published in final edited form as: ACS Nano. 2018 Apr 2;12(4):3658–3670. doi: 10.1021/acsnano.8b00797

Figure 3.

Figure 3

DFMT overcame RTX resistance by avoiding Fc-mediated endocytosis. (A) Apoptosis induction in U-2932 and U-2932 4RH cells after consecutive treatments with RTX (1 μM, 1 h) /GAH (1 μM, 24 h) and Fab′-MORF1 (1 μM, 24 h)/P-(MORF2)10 (1 μM MORF2 equiv, 24 h). The apoptosis level was measured by Annexin V-FITC/PI assay. (B) The overall uptake (before surface CD20 shaving) and intracellular uptake (after surface CD20 shaving) of Cy5-labeled RTX with/without cross-linking by secondary antibody goat antihuman (GAH) IgG(Fc). (C) Intracellular internalization of Cy5-labeled Fab′-MORF1 with/without P-(MORF2)10 cross-linking after surface CD20 shaving. U-2932 and U-2932 4RH cells were consecutively treated with RTX (1 μM) or Fab′-MORF1-Cy5 (1 μM) for 1 h and subsequently cell culture medium (no cross-linking), GAH IgG(Fc) (1 μM), or P-(MORF2)11-Cy3 (1 μM MORF2) for 5 h, prior to CD20 shaving and flow cytometry quantification of Cy5 intensity. (D) Confocal images of internalization and accessible surface amount of RTX, F(ab′)2, and Fab′-MORF1 in U-2932 4RH cells. U-2932 4RH cells were treated with RTX-Cy5, F(ab′)2-Cy5, and Fab′-MORF1-Cy5 for 1 h and further incubated in cell culture medium for 3 h. The surface accessible RTX and F(ab′)2 were detected by AF488-labeled GAH IgG(H+L) staining at 4 °C for 20 min. P-(MORF2)11-Cy3 was also added at 4 °C to biorecognize the surface accessible Fab′-MORF1. Red: Cy5; Green: AF488/ Cy3; Yellow: overlay of red and green. (E) CD20 cycling in U-2932 4RH cells treated with RTX/GAH and Fab′-MORF1/P-(MORF2)10. U-2932 4RH cells were first treated with RTX or Fab′-MORF1 for 1 h, then GAH IgG(Fc) or P-(MORF2)x was added. At 0, 1, 3, 6 h time intervals, the amount of free available CD20 on U-2932 4RH surface were measured.