AR-42 down-regulates the expression of Kit protein and mRNA. (A) P815, C2, and BR cells and canine BMCMCs were treated with either AR-42 or 17-AAG (1μM AR-42 and 17-AAG for BMCMCs) at the indicated concentrations for 24 hours. Effects on the expression of phosphorylated Kit and total Kit were determined by Western blot analysis. The top band represents the mature form and the bottom band the immature form of Kit. (B) P815, C2, and BR cells (1.0 × 106) were treated with 1μM AR-42. Cells were collected at 6 hours after drug treatment and evaluated for cell-surface Kit expression by flow cytometry. PE-conjugated rat-IgG antibody was used as isotype control. Three independent experiments were performed, and 1 representative result is shown. (C) P815, C2, and BR cells were treated with AR-42 at the indicated concentrations and were collected at 4 and 8 hours after treatment, and real-time PCR for Kit was performed. Experiments were performed in triplicate and repeated 3 times. The difference between treatment groups and DMSO control group was analyzed using the Student t test. *P < .05.
