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. 2015 Sep 21;18(11):850–862. doi: 10.1080/15384047.2015.1078024

Figure 3.

Figure 3.

Mw overcomes the facilitative effects of PMA on B16F10 cell invasion. (A) B16F10 cells preincubated with Mw for 2hr and the cell suspension containing Mw and PMA (80nM) was seeded onto the upper well of 12-well microchemotaxis chambers. The randomly chosen fields were photographed (20X). The number of cells migrated to the lower surface was calculated. Data are mean ± SD of 3 independent experiments.πP < 0.05 versus untreated, *P< 0.05, **P< 0.001 vs. PMA alone. (B) 2×106 B16F10 cells were pretreated with Mw for 2hr and then stimulated with 80nM PMA for 24hr. The activity of MMP-2 and MMP-9 was assessed by gelatin zymography. (C) Western blot was performed in similarly treated cell to detect MMP-2 and MMP-9 using specific antibodies with anti-GAPDH as reference. (D) 2×106 B16F10 cells pretreated with Mw for 2hr and stimulated with 80nM PMA for 24hr. Gene expression of MMP-2, MMP-9, MMP-7, TIMP-1 and TIMP-2 was detected by RT- PCR analysis with GAPDH as control. (E) The protein expression of pIκBα, IκBα, pP65, P65, pc-Fos, c-Fos, pc-Jun, c-Jun and anti-GAPDH was assessed by western blot in B16F10 cells pretreated with Mw for 2hr and stimulated with 80nM PMA for 24hr using specific antibodies. (F) B16F10 cells treated similarly were immunoprecipitated using NF-κB (IP: NF-κB) and AP1 (IP: c-Jun) specific antibody. Semi quantitative RT-PCR amplified the putative NF-κB and AP1 binding sites at the MMP-9 promoter. Data are from one representative experiment performed at least thrice.