Figure 7. EZH2^GOF DLBCL cells also over express BBAP E3 ligase, HDAC1,2 target H4K91ac and inhibition of HDAC1,2 activity decreases H4 monoubiquitination following doxorubicin treatment.

A: Whole cell lysate was prepared from Karpas-422, SUDHL4, SUDHL8, NALM6, HeLa, NIH3T3, and mouse fibrosarcoma cell lines. Western analysis was performed with anti-BBAP antibody and histone H3 was used as a loading control. Whole cell lysate of HeLa cells was prepared following transfection of cells with either non-targeting or two different BBAP siRNAs. These extracts served as negative controls for the signal from BBAP antibody. B: SUDHL4 and Karpas-422 cells were treated with DMSO or 2μM ACY-957 for 48h prior to chromatin extraction. Western analysis was done using anti-H4K91ac or anti-H4K16ac and histone H4 served as a loading control. C: HeLa cells were transfected with either wild type H4 or H4K91R mutant constructs for 65 hours prior to extraction of histones by trichloroacetic acid. Western blot analysis was performed with anti-FLAG antibody. H4ub, ubiquitinated form of H4. * : cross-reacting bands. D: SUDHL4 cells and Karpas-422 were treated with DMSO or 2μM ACY-957 for 24h and histones were extracted by TCA precipitation. Western blotting with anti-H4 was performed to detect H4 and monoubiquitinated H4. SE, short exposure. E: SUDHL4 cells and Karpas-422 were treated with DMSO or 2μM ACY-957 for 48h and treated with 50nM doxorubicin for 4h or 8h prior to histone extraction. Histones were extracted by TCA precipitation and western blotting with anti-H4 was performed to detect H4 and monoubiquitinated H4. SE, short exposure and LE, long exposure.