ACY-1215 in combination with bortezomib induces synergistic anti-MM activity. (A) MM.1S and RPMI8226 cells were cultured with bortezomib in the presence or absence of ACY-1215 (0-4μM) for 24 and 48 hours. Cytotoxicity was assayed by MTT. The isobologram analysis confirms the synergistic effect. (B) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 24 hours, followed by annexin/PI staining and flow cytometry analysis (left). MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 16 hours. Whole-cell lysates were immunoblotted with the indicated Abs. (C) MM.1S cells were treated with ACY-1215 (3μM), bortezomib (2.5nM), or combined therapy for 24 hours. Whole-cell lysates were immunoblotted with the indicated Abs. (D) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 12 hours. Cells were fixed and stained with 4′,6-diamidino-2-phenylindole (blue) and antiubiquitin Ab. The arrow indicates the ubiquitin-conjugated proteins. (E) MM.1S cells were treated with ACY-1215 (1μM), bortezomib (2.5nM), or combined therapy for 4 hours. Whole-cell lysates were immunoblotted with the indicated Abs. XBP-1 splicing was determined by WB and RT-PCR.