PPARγ ligand inhibition of C/EBPβ mediated by PPARγ direct association with C/EBPβ. (A) HS-5 cells were pretreated with 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then incubated with KAS6/1 cells for 2 hours. After washing out KAS6/1 cells, adherent HS-5 cells were lysated. Nuclear extracts corresponding to 5 μg protein were incubated in the absence of antibody C/EBPβ or normal rabbit serum (NRS) in combination with a [³²P]-labeled C/EBP oligonucleotide probe. Arrows indicate migration location of each nonsupershifted C/EBPβ-DNA complex. (B) HS-5 cells were transfected with a 3 × C/EBPβ-binding element–pGL3 promoter luciferase construct. Cells were then pretreated with 1 μM 15-d-PGJ2, 10 μM troglitazone, or 50 μM GW9662 for 2 hours, and then incubated with or without KAS6/1 or ANBL-6 myeloma cells for 24 hours. Error bars represent the mean (± SE) from 3 independent experiments. (C) The PPARγ coimmunoprecipitates with C/EBPβ in KAS6/1 adhesion-induced HS-5 cells. HS-5 cells were treated with or without 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then added with KAS6/1 cells for 24 hours before lyses. Western blotting analysis with anti-C/EBPβ (top panel) or anti-PPARγ (bottom panel) was performed on anti-PPARγ immunoprecipitates. The densitometric relative units in each lane were indicated beneath each blot. (D) No significant changes in C/EBPβ expression in HS-5 cells. The HS-5 cells were treated with 1 μM 15-d-PGJ2 or 10 μM troglitazone for 2 hours and then simulated with KAS6/1 for 12 hours before lyses. Western blotting analysis was performed with anti-C/EBPβ. The densitometric relative units in each lane were indicated beneath each blot.