Figure 4

Western blot of nuclear hormone receptors and proliferation in A375(DRO) cells with shPPARγ infection. A. – 60 μg of nuclear protein extract from A375(DRO), the SCR shRNA infected control cell and two clones of shPPARγ infections. Proteins were size-separated on a 10% SDS-PAGE gel and transferred to nitrocellulose. The blot was blocked with 10% nonfat milk and incubated with RXRγ, RXRα and RARβ primary antibodies and then secondary antibody with anti-rabbit IgG conjugated to horse-radish peroxidase as previously described. β-actin was measured as a loading control. B. – A375(DRO), the SCR infected and two shPPARγ infected sublines were grown in 2% fetal bovine serum RPMI in the presence of 1 umol/L of LGD1069, TZD or the combination for 9 days. Cell growth was analyzed using a nonradioactive cell proliferation assay. Proliferation was compared to that of cells grown in volume equivalent vehicle (DMSO – represented by the line). Proliferation of the SCR infected A375(DRO) was compared to the native cell line to confirm a similar response and then the shPPARγ cell lines were compared to the SCR condition for an assessment of decreased proliferation. Proliferation was statistically significantly attenuated compared to the A375(DRO) SCR subline in all treatment conditions (p < 0.001). Columns, mean; bars, SEM.