β-catenin directly affects proliferation and cell cycle in MM. (A) Wnt3A stimulation of MM1.S (top) and OPM1 (bottom) cells increases proliferation. Cells were treated with control (Con CM) or Wnt3A-conditioned medium (Wnt3A CM) for 24 or 48 hours and assayed for 3H-thymidine incorporation. (B) β-catenin (β-Cat shRNA) knockdown decreased proliferation of OPM1 and MM1.S cells. (C) Partial rescue in proliferation of OPM1 or MM1.S β-catenin shRNA (β-Cat shRNA) cells after 24 hours treatment with Wnt3A CM. In panels B and C, cells had been stably transduced and sorted for GFP-positive cells within 4 days after infection, followed by a growth curve (B) or treatment with control or Wnt3A CM for 24 hours, followed by 3H-Thy proliferation assay (C). The difference in proliferation starts out modestly but is more apparent over a period of 6 days, as is shown in panel B, whereas in panel C the experiment was performed with the use of cells technically at the day 2 time point of panel B. (D) A representative immunoblot shows a slight-but-consistent increase in β-catenin after 24-hour Wnt3A CM treatment in β-catenin shRNA OPM1 cells. (E) β-catenin (β-Cat shRNA) knockdown inhibits proliferation in MM1.S, RPM18226, and MMS1 cells. (F) Immunoblot analysis confirms the reduction of β-catenin in the different MM cells stably transduced with β-catenin shRNA (β-cat). Proliferation assays were performed in triplicate and repeated at least twice. The average and SEM of 2 to 3 experiments are shown. (G) Primary MM patient samples with β-catenin knockdown (β-Cat shRNA) also show decreased proliferation. Proliferation assays were performed in triplicate. The error bars and statistical significance were calculated from the triplicate dataset. (H) Increase in G1 and G2/M cell-cycle phases and decrease in S phase in β-catenin shRNA MM1.S cells compared with control cells. The FACS data represent the average of triplicate analyses repeated twice.
