Figure 4

Protein levels and collagenolytic activity more strongly inhibited by dual treatment with LG268 and rosiglitazone. (a) SW-1353 cells were pretreated for 24 hours in serum-free media with 50 nM LG268, 50 nM rosiglitazone, or both, followed by treatment with interleukin-1-beta (IL-1β) for 24 hours. Protein was trichloracetic acid-precipitated from 1 mL of the conditioned media and resuspended in 40 μL of Laemmli buffer, and the entire sample was resolved using Tris-HEPES-SDS-PAGE and then transferred to a polyvinylidene difluoride membrane that was probed with anti-MMP-1 or anti-MMP-13 antibodies. (b) SW-1353 cells were embedded in a type I collagen matrix diluted to 1 mg/mL with serum-free media containing 50 nM LG268, 50 nM rosiglitazone, or both compounds. After gelation of the collagen, an additional 1 mL of serum-free media containing 50 nM LG268, 50 nM rosiglitazone, or both compounds was added on top of the gelled collagen and allowed to incubate for 24 hours. IL-1β was then added to the media to stimulate MMP production, and after 24 hours the media was recovered and quantified. Collagen breakdown is indicated by media quantities over 1 g, with the additional media being released from the collagen gel during destruction. Y values are the amount of media recovered over 1 mL. P values were calculated for the difference from the IL-1β-treated sample using the Student t test (*P < 0.05, **P < 0.005, ***P < 0.0005). 268, LG100268; MMP, matrix metalloproteinase; NoTx, no treatment; Rosi, rosiglitazone.