Anthracyclin treatment induces CRT expression on the cell surface and in the serum.CRT, GRP78, and CHOP mRNAs were determined by RT-PCR in HL-60 cells (A) stimulated for 20 hours with different concentrations of doxorubicin and (B) treated with 1μM of doxorubicin in a time course. (C) HL-60 cells were stimulated with 1μM of doxorubicin, and protein levels in whole cell lysates were determined by Western blot analysis; β-actin served as a loading control. (D) HL-60 cells were treated with 1μM of doxorubicin, and cs-CRT was determined by flow cytometry. (E) Again, HL-60 cells were treated with 1μM doxorubicin, and cs-CRT was determined by flow cytometry (black bars). White bars depict levels (ng/mL) of serum CRT detected by an N-terminus–specific CRT ELISA. Untreated control cells (0h) served as controls (1-fold). (F) HL-60 cells were treated with 1μM doxorubicin (dox; light gray bars) or with 0.5μM thapsigargin to induce ER stress (th; dark gray bars). Both stimulations were performed with (scattered bars) or without 1 hour before treatment of 10μM brefeldin A (BFA) to block anterograde traffic. (G) Cells from panel F were assessed for CRT protein levels and for XBP1 mRNA. u indicates unspliced; and s, spliced variant. β-actin and GAPDH served as loading controls, respectively. Because of limited slot number, Western blot and gel electrophoresis images were modified to arrange desired order. Vertical lines have been inserted to indicate a repositioned gel lane.