To determine the NF-κB contribution to motility of cells, MMP-2/MMP-9 production was analyzed. (a) Original MCF-7 cells were incubated with the indicated concentrations of IMD-0354 in serum-free DMEM media for 48 h, and conditioned media were collected as samples for gelatin zymography. PMA (100 ng/mL) was used as a PKC activator. The 70-kDa and 90-kDa bands represent gelatinolytic activity of MMP-2 and MMP-9 in the conditioned media, respectively, and human fibrosarcoma HT-1080 cells were used as a positive control. These cropped gels are used in the main figures and full-length gels are included in the
supplementary information ( Supplementary Fig. S9). Photos are representative of 3 individual experiments. (b) PMA-induced MMP-9 activities were suppressed by IMD-0354. mRNA obtained from the MCF-7 cells was extracted at 16 h after the incubation begun, and then downregulation of MMP-9 gene expression was detected by real-time RT-PCR. Each column represents the mean ± SE of 3 different experiments in duplicates; *, P < 0.01 as compared with PMA alone. (c) Furthermore, original MCF-7 cells were treated with various concentrations of IMD-0354 in the Matrigel-coated invasion chamber. After 24-h incubations, the cells invading into the Matrigel, which mimicked the basement membrane, were fixed, stained, and counted. Photographs show typical features of invading cells in each group. Arrows indicates migrated cells. Bars in photos indicate 200 μm. (d) Treatment of IMD-0354 for 24 h suppressed tumor cell invasion in a dose-dependent manner. Each value represents the mean ± SE of 3 different experiments in duplicates; *, P < 0.01 as compared with PMA alone.