Figure 2: Class IIa HDACs are required for PTH-induced SOST suppression in vitro.

(a) Ocy454 cells were transfected with GFP-HDAC5 and then treated with PTH (50 nM) for the indicated times. Cytosolic (c) and nuclear (n) lysates were prepared and immunoblotted as indicated. (b) Ocy454 cells were treated with PTH (50 nM) for 30 min. Whole cell lysates were prepared and immunoblotted as indicated. Similar results were observed in four independent experiments. (c) Ocy454 cells with (WT, clone 17) and without (Null, clone 8) Gsα were treated with PTH (50 nM for 30 min) and analysed as in (a). (d) Ocy454 cells with (WT) and without (Null) Gsα were treated with either PTH (50 nM) or forskolin (5 μg ml⁻¹) for 30 min and analysed as in b. (e) Ocy454 cells were exposed to the indicated combinations of HDAC4-targeting sgRNAs (with Cas9) and HDAC5 shRNA-expressing lentiviruses, and whole cell lysates were analysed by immunoblotting as indicated. (f) WT, HDAC5 shRNA, HDAC4 KO and DKO Ocy454 cells were treated with PTH (1 nM) for 4 h, and SOST (left) and RANKL (right) mRNA transcript abundance was measured by RT-qPCR. For all cell culture experiments therein, values represent mean of n=3 biologic replicates. *indicates P<0.05 comparing the effects of PTH to vehicle for each cell line. Error bars represent SEM. (g,h) MEF2C chromatin immunoprecipitation was performed, and enrichment for the +45 kB enhancer determined (relative to control IgG ChIP). *indicates P<0.05 comparing fold enrichment of PTH versus vehicle by student’s unpaired two tailed t-test.