Expression of cell-cycle regulatory proteins in BM myeloma cells. (A) Expression of cyclin D and other cell-cycle proteins by immunohistochemistry on bone marrow trephine biopsies (all 60 × 0.75). Representative sections from 2 cases with t(14;16) (cases 1 and 6, both with circulating plasma cells) and 2 cases with t(11;14) (cases 20 and 30 [with circulating plasma cells] showing the expression of the appropriate D-type cyclin, phosphorylated pRb, CDK4 and CDK6). (B) Bone marrow–derived CD138⁺ cells from a case with t(14;16) (patient 13, with circulating plasma cells) were cultured in CM (control) or CM with APRIL for up to 72 hours, and analyzed by Western blotting for expression of cell-cycle proteins. (C) Expression of cell-cycle proteins in BM CD138⁺ cells from cases 1 (with circulating plasma cells) and 7 harboring t(14;16) and t(4;14), respectively, at time of purification, and after 72 hours of culture with or without APRIL. (D) Effect of APRIL on the expression of cyclin D1 and other cell-cycle proteins in bone marrow–derived CD138⁺ MM cells with t(11;14) (patients 30 [with circulating plasma cells] and 31). Culture conditions as for panel B. (B-D) Protein expression was quantified using ImageJ 1.43, and results are expressed as a ratio to control.