The effect of Pam3CSK4 on adhesion of HMCLs to BMSCs.

<p>HMCLs were stimulated with Pam3CSK4 for 24 hours and then exposed to HS5-coated wells for adhesion assay as explained in materials and methods. Pam3CSK4 decreased adhesion of OPM-1, OPM-2, and NCI-H929 cell lines to HS-5 in a dose-dependent manner. On the contrary, adhesion of L363, UM-6, UM-9 and U266 cell lines increased dose-dependently. The results are the statistical analyses of data in at least 3 separate experiments expressed as mean ± standard error of mean, *<i>P<0.05, **P<0.01, ***P<0.001.</i></p

HCML do not express of TLR2 and TLR5.

<p>Expression of TLR2 and 5 at protein level was determined by western blotting (panel A and B) and FACS (panel C). The immunoreactivity of the anti-TLR2 and -5 antibodies in western blotting and FACS analysis was confirmed with human intestinal lysate (panel A and B) and human PMBCs as positive controls (panel C), respectively. Data are representative for analysis of ≥2 independent experiments.</p

Expression of toll-like receptors in myeloma cell lines and primary bone marrow mononuclear cells from MM patients.

<p>+: positive; w+: weakly positive; −: negative; nd: not determined.</p><p>BMNC: CD138-positive cells gated from bone marrow mononuclear cells derived from MM patients (3 patients).</p

Expression analysis of TLR protein in HMCLs by Western blotting.

<p>All cell lines displayed a strong expression of TLR1, TLR3, TLR4, TLR7, TLR8, TLR9 proteins. Cell lysates were electrophoresed and blotted to PVDF membrane, which was probed with TLR-specific antibodies. To confirm the immunoreactivity of the antibodies, different positive controls were included. Beta-actin was served as loading control and was used to normalize expression levels between cells (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0060671#pone-0060671-g005" target="_blank">Figure 5</a>). Data are representative for analysis of ≥2 independent experiments.</p

TLR protein expression pattern in primary BMNCs from 3 MM patients analyzed by flow cytometry.

<p>CD138-positive cells were gated from the total cell population. Staining with specific antibodies for TLR1, TLR2, TLR3, TLR5, TLR7, TLR8, and TLR9 was compared with isotype-matched controls (NC).</p

Blocking experiments with anti-β7, anti-α4 and anti-αVβ3 antibodies for adhesion to HS-5 stromal cells.

<p>Panel A, Blocking with anti-β7. HMCLs were stimulated with Pam3CSK4 for 24 hours and then treated with anti-β7 antibody before adhesion to HS5-coated wells. Panel B, Blocking experiments with anti-α4 and anti-αVβ3 antibodies for adhesion to HS-5. HMCLs were stimulated with Pam3CSK4 for 24 hours and then treated with anti-α4 and anti-αVβ3 antibodies before adhesion to HS5-coated wells. The results are the statistical analyses of data in 3 separate experiments expressed as mean ± SEM, *<i>P<0.05, **P<0.01, ***P<0.001.</i></p