HDAC6 selective inhibitors or siRNA-induced HDAC6 knockdown fails to suppress oncogenic signaling or survival of MPN cells. IC₅₀ for this figure is provided in supplemental Table 3. (A-C) Cell Counting Kit-8 (CCK8) assays were performed similarly throughout in 96-well plates with cells seeded at a density of 0.1 million cells per mL with addition of indicated compounds and incubation at 37°C for 48 hours. CCK8 solution (Dojindo Molecular Technologies, Rockville, MD) was added, and absorbance at 450 nm was measured after 3 hours. Readings were normalized to dimethyl sulfoxide (DMSO)–treated wells. (A) MPN cell lines after treatment with increasing concentrations of nexturastat A. (B) Structure of FT108. (C) Survival after cell lines were treated with FT108 for 48 hours. (D-E) HEL92.1.7 MPN cells with a JAK2^V617F mutation were treated with indicated concentrations of (D) nexturastat A and (E) FT108 for 24 hours, and the indicated signaling proteins were assessed by western blot. (F) The IC₅₀ for panobinostat, entinostat, nexturastat A, and FT108 in suppressing each of the indicated HDACs was determined by an enzymatic assay. Color bar indicates concentrations, which ranged from 0 µM (green) to >10 µM (red). (G) Diagram showing experimental design and procedures relevant to panels H-J. (H-J) Signaling pathways, proliferation, and apoptosis were assessed in HEL92.1.7 cells 72 hours after transfection with HDAC6 siRNA or NTC siRNA. Mass spectrometry results are shown in supplemental Figure 2. (H) JAK-STAT pathway, ac-tubulin, and ac-H3 were assessed by western blot in HEL92.1.7 MPN cells treated with HDAC6 siRNA vs cells treated with non-targeting siRNA. (I) HEL92.1.7 MPN cell numbers were counted before and after HDAC6 knockdown. (J) Apoptosis of HEL92.1.7 MPN cells measured by flow cytometry after HDAC6 siRNA-directed knockdown. Statistical analysis was performed using Student t test (comparing NTC in each group). hs, hours; ns, not significant. **P < .01; *P = .01.