Expression of Hsp32 in neoplastic mast cells. Immunocytochemical detection of Hsp32 in primary neoplastic MCs in a patient with mast cell leukemia (A-B), in HMC-1.2 cells expressing KIT D816V (C-D), and in HMC-1.1 cells lacking KIT D816V (E-F), using an antibody against Hsp32. Before being applied, the antibody was preincubated with control-buffer (A,C,E) or a specific blocking-peptide (B,D,F). Acquisition of figures was performed by an Olympus DP11 camera connected to an Olympus BX50F4 microscope equipped with 100 ×/1.35 UPlan-Apo objective lenses (Olympus, Hamburg, Germany). Images were acquired with Adobe Photoshop CS2 software version 9.0 (Adobe Systems, San Jose, CA) and processed with PowerPoint software (Microsoft, Redmond, WA). (G) Western blot analysis of Hsp32 expression in HMC-1.1 cells and HMC-1.2 cells after exposure to control medium or hemin (10 μM; 37°C, 4 hours). Western blotting was performed using antibodies specific for Hsp32 or β-actin (loading control). (H) Detection of Hsp32 mRNA by RT-PCR in HMC-1 cells and highly enriched KIT-sorted neoplastic MCs obtained from 1 patient with mast cell leukemia (MCL) and 1 patient with mast cell sarcoma (MCS). RT-PCR was performed as described in “Materials and methods, Northern blot analysis and RT-PCR.” (I) Northern blot analysis of expression of Hsp32 mRNA in HMC-1.1 and HMC-1.2 cells after incubation in control medium or hemin (10 μM, 37°C) for 4 hours. Northern blotting was performed using DNA probes specific for Hsp32 or β-actin. As assessed by densitometry, hemin induced a 10-fold up-regulation of Hsp32 mRNA in HMC-1.1 cells, and a 20-fold up-regulation in HMC-1.2 cells.