ACY-1215 selectively inhibits HDAC6. (A) Chemical structure of ACY-1215. (B) MM.1S cells were cultured with control medium or ACY-1215 (0-5μM) or SAHA (0-5μM) for 6 hours. MM.1S, MM.1R, and RPMI8226 cells were cultured with control medium or ACY-1215 (0.25 and 1μM) for 18 hours. (C) CD138⁺ patient cells were treated with control medium or ACY-1215 (2μM) for 4 hours. Whole-cell lysates were subjected to Western blotting using the indicated Abs. Increased acetylated α-tubulin was observed. CD138⁺ patient cells were fixed and double-stained for anti–human CD138⁺ and acetylated α-tubulin (left panel) and for anti–human CD138⁺ and acetyl-histone H3 (right panel). A significant increase in acetylation of α-tubulin was observed, whereas no significant difference was observed in the acetyl-histone H3 in the treated sample compared with control. (D) PBMCs from 4 healthy donors were stimulated with PHA and cultured with increasing doses (0-4μM) of ACY-1215 and SAHA for 48 hours. Cell growth was assessed by MTT assay (left panel). CD4⁺ T cells purified from human blood were stimulated by CD3/CD28 Dynabeads for 7 days in the presence of compounds. Cell viability was assessed using alamarBlue (right panel)