Figure 4

Effects of DMF on RANKL-induced EMT and EMT-related mRNA expression. (A) Analysis of 4T1 cell morphology after cell treatment of with 100 ng/mL RANKL or 100 μM DMF (× 40 magnification). (B) Total RNA was extracted, and the mRNA levels of vimentin, E-cadherin, N-cadherin, Snail, and Twist were determined by real-time PCR. The results are expressed as treated over control ratio after correction to GAPDH mRNA levels. The results are representative of 5 independent experiments. *p < 0.01, as compared to controls (ANOVA with Dunnett’s test). (C) 4T1 cells were pretreated with 100 ng/mL RANKL or 100 μM DMF for 24 h, after which 5 × 10³ cells were seeded into the upper compartments of chambers. Migration was analyzed by Boyden chamber assays using Falcon cell culture inserts. Invasive properties were analyzed using Falcon cell culture inserts covered with 50 μg of Matrigel per filter. For both assays, the lower chambers contained conditioned media (addition of RANKL in serum-free medium), which was used as a chemoattractant. After incubation for 24 h, the cells invading the lower surface were counted microscopically. The results are representative of 5 independent experiments. *p < 0.01 vs. the controls (ANOVA with Dunnet’s test). (D) 4T1 cells were incubated with 100 ng/mL RANKL or 100 μM DMF. After 60 min, the cytoplasmic fractions and nuclear fractions were extracted and then subjected to SDS-PAGE/immunoblotting with anti-NF-κB p65 antibody. Anti-β-actin and anti-lamin antibodies were used as the internal standard. (E) Quantification of the amount of NF-κB p65, normalized to the amounts of the corresponding proteins, respectively. The results are representative of 5 independent experiments. *p < 0.01, as compared to controls (ANOVA with Dunnett’s test).