Fig. 1

Inhibition of class I HDACs induces apoptosis in JAK2^V617F-positive cells. a HEL cells were treated with 5 nM FK228 for 24 h (-, untreated control cells; +, treated cells). Immunoblotting shows full-length (fl.) and cleaved (cf.) PARP1 and cleaved forms of caspase-3; β-actin was used as a loading control; kDa, kilo Daltons. b HEL cells were treated with 5 nM FK228 for 24–48 h, stained with annexin-V-FITC/PI, and analyzed for early (annexin-V-positive) and late (annexin-V/PI-positive) apoptosis by flow cytometry. Left shows representative flow cytometry scans; right shows bar graphs with statistical evaluations. c HEL cells were incubated with 5 nM FK228 and/or 50 μM z-VAD-FMK for 24 h. PARP1 was detected by immunoblotting; α-tubulin served as a loading control. d HEL cells were treated as described in (c) and subsequently stained with annexin-V-FITC/PI and analyzed for apoptosis. e HEL cells were treated as described in (c), fixed, subsequently stained with PI, and analyzed for subG1 fraction accumulation via flow cytometry. f HEL cells were treated with 5 nM FK228 for 24–48 h. The indicated proteins were revealed by immunoblotting; β-actin was used as a loading control. g HEL cells were treated with 5 nM FK228 for 24 h and/or proteasome inhibitors (50 nM bortezomib; 10 μM lactacystin) for 6 h before being harvested. Immunoblotting shows the indicated proteins. h HEL cells were treated as described in (g) and analyzed for apoptosis via flow cytometry. i HEL cells were incubated with 5 nM FK228 or 5 µM MS-275 for 24 h, washed, plated into methylcellulose medium, and tested for colony formation. Left, representative images are shown. Scale bar, 200 μm. Right, graph depicts the numbers of colonies. The data are presented as mean ± SD of at least three independent experiments. Statistical analyses (unpaired t-test; one-way ANOVA; two-way ANOVA; Bonferroni correction; ns not significant; p values are as follows: *P  < 0.05; **P  < 0.01; ***P  < 0.001; ****P  < 0.0001)