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

Figure 4.

Figure 4.

Mw inhibits PMA-induced PKCα mediated B16F10 cell invasion. (A) B16F10 cells were treated with 80nM PMA, and PKC isotypes were analyzed in cytosol and membrane fractions by protein gel blotting. (B) B16F10 cells were pretreated for 1hr with Gö6976 (2uM), Rottlerin (0.5uM) or GF109203X (2uM), followed by 80nM PMA stimulation for 24hr. The MMP-9 mRNA (top), protein (middle) expression and activity (bottom) was analyzed by semi-quantitative RT-PCR, western blot and gelatin zymography respectively. Data are from one of 3 representative experiments. (C) Invasion assay was carried out in cells stimulated with 80nM PMA for 24hr after pretreatment with PKC inhibitors for 1hr. The randomly chosen fields were photographed (20X), and the number of cells migrated to the lower surface was calculated. Data are mean ± SD of 3 independent experiments. **P < 0.001 vs PMA alone treatment. (D) Confluent cells were scratched and treated with 80nM PMA for 24hr after pretreatment with PKC inhibitors for 1hr. The number of cells migrated into the scratched area was photographed (20X) and calculated. Data are shown as the mean ± SD of 3 independent experiments. **P< 0.001 vs PMA alone treatment. (E) B16F10 cells transfected with empty vector (EV), overexpressed full-length mouse PKCα (PKCαOV) or PKCδ (PKCδOV) isotypes as mentioned in materials and methods were subjected to semi-quantitative RT-PCR, protein gel blot and gelatin zymography for MMP-9 expression and activity respectively. Data are from one of 3 representative experiments. (F) B16F10 cells were pretreated with Mw for 2hr and then stimulated with 80nM PMA for 24hr. Western blot analysis was performed to detect the PKCα and PKCδ level in cytosol and membrane fractions. (G) B16F10 cells, transfected with overexpressed full-length mouse PKCα (PKCαOV) isotype were treated with Mw for 2hr followed by 80nM PMA stimulation for 24hr. The MMP-9 mRNA (top), protein (middle) expression and activity (bottom) were analyzed by semi-quantitative RT-PCR, western blot and gelatin zymography respectively. Data are from one of 3 representative experiments.