Vorinostat down-regulates the expression of JAK2V617F and affects its downstream signaling. (A) HEL cells bearing the JAK2V617F mutant were treated with the indicated concentrations of vorinostat for 24 hours. Acetylation of histone H4 and tubulin was detected by anti–acetyl-histone H4 and acetyl-tubulin immunoblotting. Activation of JAK2, Stat5, Stat3, Akt, and Erk1/2 was detected by immunoblotting using phosphospecific Abs. Membranes were reprobed for total proteins. (B) HEL cells were left untreated or treated with indicated concentrations of vorinostat, and c-Myc, Pim-1, PARP, and caspase 3 levels were analyzed by immunoblotting. Levels of Erk2 served as the loading control. (C) HEL cells were treated with the indicated concentrations of vorinostat for 24 hours. Total RNA was extracted and analyzed by the quantitative real-time PCR. The relative expression of JAK2 was normalized to 18S expression. Results are shown as means ± SEM from 3 independent experiments. (D) HEL cells were treated with the indicated concentrations of vorinostat for 24 hours, and lysates were immunoprecipitated with anti-HSP90 or anti-JAK2 Ab and immunoblotted with anti-JAK2 or anti-HSP90 Ab. (E-F) HEL cells were treated with vorinostat (1.0μM) or DMSO for 24 hours. Total RNA was extracted and reverse transcribed. Relative expression of SOCS1 and SOCS3 mRNA was determined by quantitative real-time PCR and normalized to 18S expression (E). Relative expression of GATA1, KLF1, FOG1, SCL, C/EBPα, PU.1, and NF-E2 mRNA was determined by quantitative real-time PCR and normalized to 18S expression (F). Results shown represent means ± SEM from 3 independent experiments. Asterisks indicate significant differences by Student t test. *P < .05; **P < .005.