Figure 1. ART induces specific, iron-depended PCD in pancreatic cancer cell lines.

A. BxPC-3, and Panc-1 pancreatic cancer and non-neoplastic HPDE epithelial cells were treated with ART (50 μM) alone or in combination with iron-saturated holo-transferrin (HTF, 20 μg/ml) or the iron chelator deferoxamine (DFO, 0.1 mM) for 24 or 48 hours. Following, cell death was assessed using the exclusion dye PI (1 μg/ml). Data is presented as fold-change in PI intensity relative to drug-free control conditions. Statistical significance was tested vs. cells treated under control conditions (*) or ART alone (^#)(n = 3; ^#,*, p ≤ 0.05; **,^##p ≤ 0.005). B. Panc-1 cells were subjected to ART, HTF, or ART and HTF. At 24 hours, 300 surviving cells were re-seeded for a colony formation assay. Colony count following 11 days of re-seeding is presented as fold change compared to control conditions. Statistical significance was tested vs. control (*) or ART alone (^#) (n = 3-4; *,^#, p ≤ 0.05; **,^##, p ≤ 0.005). C. Panc-1 cells were subjected to ART, DFO, or ART and DFO for 24 hours. Following colony formation assays were performed and analyzed as in (B). D. Panc-1 cells were stained with Alexa Fluor Human Transferrin (HTF⁵⁴⁶, 5 μg/ml). Following, endolysosomal HTF⁵⁴⁶ was detected by fluorescence microscopy at 30 minutes, 6 hours and 24 hours fluorescence of exposure to ART or control conditions. Representative images of three independent experiments are shown. E, Panc-1 cells were pre-treated with ART or control conditions for 24 hours. Following, cells were stained with HTF⁵⁴⁶ and endolysosomal HTF⁵⁴⁶ fluorescence detected at 30 minutes, 6 and 24 hours. Representative images of three independent experiments are shown. Scale bars, 10 μm.