FIGURE 9.

Inactivating ACAT1 by gene KO or by pharmacological inhibition decreases the integrin β 1 (CD29) level in manganese ion-activated macrophages. A and B, the effect of ACAT1 inhibition on CD29 expression level in RAW 264.7 mouse macrophage cells depends on manganese treatment. RAW 264.7 macrophages were plated in 6-well dishes in DMEM plus 10% fetal bovine serum and grown in the same medium. Cells in DMEM plus 10% fetal bovine serum were treated with 1 μm of an ACAT1-specific inhibitor (K604) and 0.02% ethanol or with 0.02% ethanol only for 24 h until ∼90–100% confluency. A, cells were harvested without manganese ion treatment. B, cells were rinsed twice with Hanks' buffer without calcium or magnesium ions and then incubated with the same buffer and 5 mm manganese ions for 30 min at 37 °C. Afterward, cells were harvested in radioimmune precipitation assay buffer with protease inhibitor mixture. The extracts were used for immunoblotting analysis using a mouse monoclonal antibody against CD29 (Santa Cruz Biotechnology, catalog no. sc-374429) to monitor CD29 expression. C–E, Acat1 KO or ACAT1 enzyme activity inhibition decreases CD29 expression in manganese-activated peritoneal macrophages. Peritoneal macrophages isolated from WT and Acat1^−M/−M mice (C), WT and global Acat1^−/− mice (D), or Apoe KO mice treated without or with 1 μm ACAT1-specific inhibitor K604 for 24 h (E) were subjected to manganese activation and then harvested for immunoblot analysis in the same manner as described in A and B. Quantitation of immunoblots was performed by ImageJ analysis. *, p < 0.05; ***, p < 0.001. E, p = 0.09.