AXIIR on MM cells plays a role in adhesion of MM cells to BM stromal cells. (A) MM cells, MM1.S, and ANBL.6 were transfected with either siControl (siCon) or siAXIIR according to the manufacturer's protocol. RNA was prepared from untransfected MM cells (No siRNA) or MM cells transfected with either siControl (siCon) or siAXIIR. cDNA was reverse-transcribed and RT-PCR was performed. We observed between a 55% and 63% reduction in the level of AXIIR mRNA in MM^siAXIIR cells compared with MM^siControl cells in all the following experiments. (B) Untransfected MM1.S, MM1.S^siAXIIR and MM1.S^siControl cells were incubated with 1 μg/mL bio-AXII and after extensive washing were incubated with avidin-FITC. A secondary only control with avidin-FITC was included in the experiment (Control). Staining was visualized using confocal microscopy. (C-D) KM101 cells were seeded in 12-well plates and incubated at 37°C overnight. Untransfected MM cells, MM^siAXIIR, and MM^siControl cells (10⁶) were added to the KM101 cells for 3 hours at 37°C; (C) MM1.S and (D) ANBL.6. Nonadherent MM cells were removed after being washed 3 times with PBS, counted, and used to calculate the number of adherent cells. MM1.S^siAXIIR cells adhered significantly less compared with MM^siControl cells. (E) Stromal cells (5 × 10⁴) from either AXII^+/+ or AXII^−/− were plated in 24-well plates, and incubated overnight at 37°C. The next day, MM1.S, MM1.S^siAXIIR, and MM1.S^siControl cells (2.5 × 10⁵) were added to the stromal cells and incubated for 6 hours at 37°C. Nonadherent MM cells were removed after washing 3 times with PBS and counted. Adhesion between AXII^−/− and MM^siAXIIR cells compared with AXII^+/+ and MM1.S cells was reduced significantly. (F) MM1.S cells (1 × 10⁶) were incubated with AXII (1 μg/mL) for 48 hours at 37°C. At the end of 48 hours, total protein was collected and analyzed for RhoA expression by Western blotting. β-actin was used as a loading control. (*P < .05; **P < .01; ***P < .001)