Figure 8.

Histone deacetylase (HDAC)-4 degradation occurs in cytoplasmic and nuclear compartments. A: Schematic diagram of the HDAC4 variants: N-terminally (ter) FLAG-tagged full-length HDAC4 construction, nuclear localization signal (NLS), nuclear export signal (NES), and L1062A (the Leu 1062 in HDAC4 was replaced by Ala). B: NIH/3T3 cells were transfected with wild-type (WT) FLAG-HDAC4 or FLAG-HDAC4 variants, and the expression of the HDAC4 variants and their localization were measured through immunofluorescence staining (anti-FLAG staining in green). C: The stability of the HADC4 variants in the NIH/3T3 cells cultured on plastic or in three-dimensional (3D) collagen was measured by Western blot analysis against FLAG. D and E: Stability of wild-type FLAG-HDAC4 and L1062A mutant in NIH/3T3 cells cultured on plastic or embedded in 3D collagen together with overexpression of cathepsin (Cts)-H was measured by Western blot analysis. F: Overexpression of CtsH tagged with hemagglutinin-epitope in NIH/3T3 cells to induce degradation of endogenous HDAC4 was determined by Western blot analysis. G: The NIH/3T3 cells stably expressing CtsH and HDAC4 were embedded in 3D collagen and challenged with an increased concentration of tumor necrosis factor (TNF)-α. The expression of MMP13 was determined by real-time quantitative RT-PCR analysis. All experiments were performed at least twice. Data are expressed as means ± SEM. ^∗P < 0.05, ^∗∗P < 0.01. Scale bars = 20 μm. coll, collagen; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFP, green fluorescent protein; vec, vector.