Figure 2. BAP1 regulates the balance of HDAC2 and HDAC1 expression.

A. DUB siRNA library screen identifies BAP1 as a candidate regulator of class I HDACs. 72 hr after transfection with 40 nM siRNA pools against 92 human DUBs, their effect on HDAC1 and HDAC2 expression was screened by immunoblotting of nuclear extracts from A549 lung cancer cells. The main histogram shows mean log(2) values for HDAC2 relative to HDAC1 from two immunoblots; DUBs of interest and two non-targeting controls (siCON1 and siCON2) are indicated. Inset immunoblot panels show sections from the screen, highlighting DUB siRNAs that altered the HDAC expression ratio. The inset histogram shows an increase in HDAC1 and a decrease in HDAC2 expression for the siBAP1 samples from the replicate screens. B. BAP1 depletion decreases HDAC2 relative to HDAC1 in both cytosolic and nuclear protein extracts. A549 cells were transfected with 40 nM siRNAs for 72 hr. Quantification from immunoblotting shows the ratio of HDAC2 to HDAC1 expression from three independent experiments (error bars show SD, unpaired t-test, *P ≤ 0.05, **P ≤ 0.01). C. BAP1 depletion decreases HDAC2 and increases HDAC1 in other NSCLC cell lines. NCI-H460 or COR-L23 cells were transfected with 40 nM siRNAs for 72 hrs before immunoblotting of whole cell extracts. D–E. BAP1 siRNAs alter HDAC expression in MSTO-211H mesothelioma cells. Whole cell extracts were prepared 72 hr after transfection with siRNAs. A representative immunoblot D. and quantification from three independent experiments E. showing the HDAC2/HDAC1 ratio (left), and individual proteins normalized to actin (right); error bars show SD, one-way ANOVA with Tukey's post-hoc test: ****P ≤ 0.0001, ***P ≤ 0.001, **P ≤ 0.01, *P ≤ 0.05. F. Quantification of residual BAP1 for the siRNA experiments in B and D.