Fig 4. TQ inhibits migration and invasion of MM cell lines.

A, U266 cells (50x10⁴/well) were plated in 0.3 ml cell culture media with and without 15 μM TQ in the top chambers of 24-well transwell inserts with 8-mm pores. Cell culture medium (600 μl) containing the recombinant human B-cell chemoattractant, CXCL12 (SDF-1α) was added to the bottom chamber and incubated for 12 hours. After incubation, the insert was removed and calcein-AM (5 μM) was added to the wells and fluorescence was measured. Columns, mean; bars, SD. *, p < 0.05. B, U266 cells (50 x10⁴) in suspension were starved in serum-free RPMI-1640 for 3 h, and then loaded onto the Matrigel-coated inserts in the upper chambers of tissue culture inserts placed in 24 well plates. The wells of the plate were filled with 600-μl of 10% FBS-containing cell culture media with 100 ng/ml CXCL12 (SDF-1α). TQ (15 μM) was added with the cells to the upper chamber. Plates were then incubated at 37°C for 12 h. At the end of the incubation period, calcein-AM (5 μM) was added to the wells containing invasive cells, incubated at 37°C for 1 hour and fluorescence was measured. Columns, mean; bars, SD. *, p < 0.05. C, U266 cells were treated with TQ (15 μM) for 0, 12, 24, 36 and 48 h at 37°C. Whole-cell extracts were prepared, separated on SDS-PAGE, and subjected to Western blot analysis using antibody against CXCR4, COX-2 and MMP-9. The same blots were stripped and reprobed with β-actin antibody to show equal protein loading.