DKK1 induces drug resistance via receptor CKAP4. (A) Representative protein levels of LRP6 and CKAP4 in WT and BR MM cells. (B) LRP6 and CKAP4 expression in 6 patients with disease progression before and after BTZ-based treatment. (C) Western blotting shows the efficacy of 2 shRNAs targeting CKAP4 in HEK293T cells, and validates the knockdown efficacy of shRNA#1 in MM cells (D). (E) Representative of CD138 protein level in MM cells stably expressing shRNA targeting CKAP4 or non-target control (NT Ctrl), and subsequently treated with rhDKK1 (100 ng/mL) for 48 hours. (F) Western blotting shows the cleavage of PARP as a marker of apoptosis in MM cells stably expressing NT Ctrl or CKAP4-shRNA (CKAP4 KD) treated with BTZ and rhDKK1, or anti-DKK1 (G) for 24 hours. (H) Schematic illustration of CRD1 domain deletion in human DKK1 gene, and positions of 3 primers to validate the expression of truncations. CRD1, cysteine-rich domain; FL, full length; P1, position for primer 1; P2, position for primer 2; P3, position for primer 3 for the real-time PCR. (I) qPCR data shows DKK1 FL or ΔCRD1 truncation expressions using 3 different primers. (J) Cleavage of PARP in MM.1S and OPM-2 cells treated with FL or ΔCRD1 DKK1 in presence of BTZ for 24 hours. (K) Quantification of apoptotic MM.1S and OPM-2 cells treated with BTZ in presence of FL DKK1 or ΔCRD1 DKK1.