AR-42 induces Kit down-regulation and caspase-3/7 activation in malignant canine mast cells cultured ex vivo. Primary malignant canine mast cells were obtained from clinical patients by fine needle aspiration. Red blood cells were removed by hypotonic shock, and the purity of the cells was determinate by cytologic evaluation. (A) Fresh malignant canine mast cells from 17 individual tumor samples were treated with DMSO, 1μM AR-42, or 1μM 17-AAG in triplicate, and the WST-1 assay was used to assess cell viability after 48 hours of culture. Data from all 17 tumor samples was pooled, and the percentage of cell viability after AR-42 or 17-AAG treatment was calculated using the DMSO group as the 100% control (*P < .05). (B) Fresh malignant canine mast cells (from 4 different patients: MCT nos. 4, 6, 9, and 12 as indicated) were treated with or without 1μM AR-42 or 17-AAG in triplicate for 48 hours, and activation of caspases-3/7 was measured. The difference was analyzed between treatment groups (AR-42 and 17-AAG) and the DMSO control group for each individual tumor sample (*P < .05). (C) Fresh malignant canine mast cells were left untreated or treated with 1μM AR-42 or 17-AAG for 24 hours. Cells were collected and evaluated for Kit cell-surface expression by flow cytometry. The data presented here are from MCT patient no. 9; a total of 3 experiments from 3 different patients were performed once with similar results (data not shown). (D) Fresh malignant canine mast cells (from 4 different patients: MCT nos. 9, 14, 15, and 17 as indicated) were left untreated or treated with 1μM AR-42 or 17-AAG for 24 hours. Effects on the acetylation of histones H3 and H4 and p21 were determined by Western blotting.