Figure 2. Soluble PrP^C can mediate resistance to doxorubicin.

(A and B) Baseline doxorubicin IC₅₀ values (mean ± SEM from 3 independent experiments) comparing cell lines secreting different levels of PrP^C (****P = 0.001, comparing Hs578t/MDA436 versus MDA231/BT549; *P = 0.05, comparing HM.LNm5 and parental; see Supplemental Figure 2). P values were determined using 2-tailed Student’s t test. (C) Doxorubicin dose curves in various cell lines after depleting PrP^C with siRNA (confirmation of knockdown shown in Supplemental Figure 2, C and D). SCRsi, scrambled siRNA control sequence. (D) Doxorubicin dose curves for MDA231 and HM.LNm5 cells depleted of PrP^C and then supplemented with media from parallel cultures of PRNPsi or SCRsi transfectants. The experiment was also performed using equivalent media depleted of exosomes. (E) Western analysis of PrP^C fragments present in lysates from 5 cell lines treated with DMSO (control) or brefeldin A, compared with conditioned medium (CM) from untreated cells. Cleavage at the α site produces N1 and C1 fragments, while β-cleavage produces N2 and C2. Shedding of full-length or C1/2 fragments occurs by proteolytic cleavage at or near the glycosylphosphatidylinositol (GPI) membrane anchor. Blots were probed with the 3 different antibodies, the epitopes for which are indicated on the PrP^C schematic (drawn to scale). Protein preparations were first treated with the amidase peptide-N-glycosidase (PNGase-F) to resolve glycoprotein fragments at their respective molecular masses in denaturing conditions. CC, charged cluster domain; OR, octarepeat domain with dark-green dots as high-affinity copper-binding histidine residues; HD, hydrophobic domain; FL37, full-length glycosylated protein; FL27, full-length deglycosylated protein. N-linked glycosylation sites are also shown. (F) Summary plot of fold change across various units, demonstrating the positive association among cell lines in terms of intracellular accumulation of PrP^C protein levels after brefeldin A treatment (yellow; from Figure 1, G and H), levels of PrP^C protein secretion into conditioned media (light green; Figure 1I), dependence on PRNP expression for doxorubicin resistance expressed in units as doubling fold change (dark green; C), ratio of free to membrane-bound full-length PrP^C (light blue; E), and the baseline doxorubicin IC₅₀ (dark blue; Supplemental Figure 2, A and B). BT549 and HM.LNm5 were identified as the best models of soluble PrP^C-associated doxorubicin resistance.