FIG. 3.

Knockdown of the associated HDACs reveals their crucial roles in the repression. (A) Transfection with an siRNA construct (2 to 4 μg) targeting the HDACs found associated with the respective genes was employed to knock down the HDAC expression levels in αT3-1 cells, and the efficiency was verified by Western analysis. The left lane (−) shows the protein in nontransfected cells, the middle lane (+) is following transfection with the specific siRNA, and in the right lane (c) the sample is from cells transfected with a control siRNA sequence. Also shown in the lower box in each row is the loading control, GAPDH or polymerase II. (B) Total RNA was isolated 48 h after transfection with the various siRNA HDAC constructs, and RT-PCR analysis of LHβ and FSHβ mRNA levels was carried out. The primers designed to amplify the LHβ transcript targeted two different exons, while primers targeting FSHβ and 60sRP were as described in the legend of in Fig. 1. Also shown are untreated (first lane) or GnRH-treated (100 nM for 24 h; next three lanes) controls. (C) ChIP was carried out to test the association of HDACs 1 and 5 (for LHβ) or 2 and 3 (for FSHβ) following siRNA-mediated knockdown of HDAC4. All transfections were carried out in duplicate 48 h before harvest. siHDAC4, HDAC4-targeting siRNA construct; α, antisera against.