HDACIs increase MLC phosphorylation in megakaryocytes. (A) Schema of the Rho/Rac/CDC42 pathway. (B) Western blot performed on fetal liver cell–derived megakaryocytes treated for 48 hours with increasing doses of panobinostat (Pan) and romidepsin (Romi) showing an increase in pMLC. Immunofluorescent staining performed on fetal liver cell–derived megakaryocytes treated for 48 hours with increasing doses of panobinostat and romidepsin demonstrating an increase in pMLC. Green indicates pMLC, red is tubulin, and blue is the nucleus stained with DAPI. (C) Western blot performed on Meg-01 cells treated for 24 hours with increasing doses of panobinostat and romidepsin showing an increase in pMLC levels, and on Meg-01 cells treated at 0, 12, 16, and 24 hours with 20nM panobinostat and 20nM romidepsin, showing increased pMLC from 12 hours on. (D) Immunofluorescent staining performed on Meg-01 cells treated for 24 hours with increasing doses of panobinostat and romidepsin, demonstrating an increase in pMLC. Green indicates pMLC, red is tubulin, and blue is the nucleus stained with DAPI. (E) Immunohistochemical staining of trephine sections of mice treated with panobinostat 10 mg/kg or vehicle for pMLC. The quantification of the fluorescent intensity of staining of mice treated with panobinostat 10 mg/kg or vehicle for pMLC was calculated using Metamorph v7.63 software. Megakaryocytes were isolated using a threshold for intensity of staining and manual elimination of other staining cells. At least 5 × 20 fields were examined per trephine.