Fig. 4.
PCI of 225.28-saporin is highly cytotoxic in drug-resistant melanoma cells that are BRAFV600E mutated and MITFlow/AXLhigh. (A) MITF (green) or (B) AXL (red) expression was examined by immunofluorescence (left panels). Right panels, merge of MITF or AXL and nuclear staining with DAPI (blue). Melmet q and Melmet 5 cells were treated with either (C) the chemotherapeutic agent dacarbazine or (D) the BRAF inhibitor vemurafenib for 72 h prior to examination of cell viability by the MTS assay. Figures show average of three independent experiments with ±SE bars. (E) Melmet 1 and (F) Melmet 5 cells incubated with 0.2 μg mL−1 TPCS2a (PCI no drug), or TPCS2a in combination with 100 pM streptavidin-saporin (PCI saporin) or the CSPG4-targeting immunotoxin 225.28-saporin (PCI 225.28-saporin) for 18 h, followed by a 4 h chase in drug-free medium prior to light exposure as indicated in the X-axis. Cell metabolic activity/viability was examined using the MTT assay 48 h post illumination. (G) Melmet 5 cells seeded at clonal density (1000 cells per 9.6 cm2) and treated as in F. Cells were assessed for clonogenicity 10–14 days post light exposure. Each figure shows a single representative experiment out of three independent experiments. Bars = SD of three technical replicates.