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. 2009 Aug 24;29(21):5751–5762. doi: 10.1128/MCB.00415-09

FIG. 3.

FIG. 3.

PTHrP and forskolin induce the nuclear localization of HDAC4 and dephosphorylation of HDAC4 phospho-S246. (A) siRNAs targeting either Gapdh or Hdac4 knockdown expression of their respective proteins in chondrogenic MLB14 cells. (B) Knockdown of HDAC4 rescues Col10a1 expression in forskolin-treated MLB14 cells. MLB14 cells, induced to differentiate for 4 days in BMP2-containing medium, were treated as indicated; the ratio of Col10a1 to Tbp was determined by RT-qPCR. (C to H) USCs were transfected with an expression vehicle encoding GFP fused to HDAC4 (GFP-HDAC4) and cultured in either the absence or presence of forskolin. Localization of GFP-HDAC4- and DAPI-stained nuclei is displayed. (I) The percentage of chondrocytes displaying nuclearly localized GFP-HDAC4 when cultured in either the absence or presence of PTHrP or forskolin. (J) USCs were transfected with expression vehicles encoding either Flag-tagged wild-type HDAC4 (HDAC4-Flag) or a Flag-tagged mutant form of HDAC4 containing alanine in place of S246, S467, and S632 (HDAC4-3SA-Flag) and cultured in control medium or in medium containing either PTHrP or forskolin for an additional 1 or 4 h prior to cell lysis. Immunoprecipitated HDAC4-Flag was Western blotted and probed with antibodies to HDAC4-Flag, HDAC4 phospho-S246, HDAC4 phospho-S467, and HDAC4 phospho-S632.