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. 2014 Oct 23;289(50):34743–34767. doi: 10.1074/jbc.M114.576702

FIGURE 5.

FIGURE 5.

Regulation of PKCδ promoter activity by sodium butyrate treatment and ectopic expression of HDACs. A, PKCδ promoter activity is activated after treatment with NaBu. The PKCδ promoter reporter construct pGL3−1694/+289 or empty vector pGL3-Basic was transfected into MN9D (left panel) and NIE115 (right panel) cells. After 24 h of transfection, the cells were incubated with or without NaBu at concentrations ranging from 0.2 to 1 mm for 24 h. Cells were then harvested, and luciferase activities were determined and normalized by total cellular protein. Values are expressed as a percentage of the activity of the pGL3−1694/+289-transfected control and represented as means ± S.E. of three independent experiments performed in triplicate. **, p < 0.01; ***, p < 0.001; control versus NaBu-treated samples. B–E, other HDAC inhibitors stimulate PKCδ promoter activity in MN9D cells. The PKCδ promoter reporter construct pGL3−1694/+289 was transfected into MN9D cells. After 24 h of transfection, the cells were incubated with VPA (B), TSA (C), apicidin (D), or scriptaid (E) at the designated concentrations for 24 h. Cells were then harvested, and luciferase activities were determined and normalized by total cellular protein. Values are expressed as a percentage of the activity of untreated control (con) and represent the mean ± S.E. of three independent experiments performed in triplicate. ***, p < 0.001; control versus treated samples. F, PKCδ promoter activity is repressed by forced expression of HDAC proteins in NIE115 (black bar) and MN9D (blue bar) cells. Cells were cotransfected with pGL3−1694/+289 and 8 μg of HDAC1, HDAC4, HDAC5, or HDAC7 expression vector or the empty vector (EV) control. Luciferase activity was measured after 24 h of transfection and normalized by total cellular protein. Values are expressed as a percentage of the luciferase activity obtained from cells transfected with 8 μg of empty vector (EV) and represented as means ± S.E. of three independent experiments performed in triplicate. **, p < 0.01; ***, p < 0.001; EV- versus HDAC-transfected samples. G, effects of ectopic expression of HDAC proteins on butyrate-induced PKCδ promoter activation. MN9D (left panel) and NIE115 (right panel) cells were cotransfected with pGL3−1694/+289 and increasing concentrations of HDAC1, HDAC4, HDAC5, or HDAC7 expression vector (from 2 to 8 μg) or the empty vector (EV) control. After 24 h of transfection, the cells were incubated with or without NaBu (1 mm) for 24 h. Cells were then harvested, and luciferase activities were determined and normalized by total cellular protein. Values are expressed as a percentage of the luciferase activity obtained from NaBu-treated cells transfected with 8 μg of empty vector (EV) and are represented as means ± S.E. of three independent experiments performed in triplicate. Variations in the amount of total DNA were compensated with the corresponding empty vector. H, silencing of class I HDAC (HDAC1 and -2) up-regulated PKCδ protein expression in MN9D cells. MN9D cells were transiently transfected with siRNA-HDAC1, siRNA-HDAC2, and scrambled siRNA. Cells were collected 96 h after the initial transfection and then subjected to Western blot analysis. I, densitometric analysis. HDAC1, HDAC2, and PKCδ bands were quantified and normalized to that of β-actin. Values are shown as means ± S.E. of two independent experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001. J, multiple class I and IIa HDACs are expressed in MN9D and NIE115 cells. MN9D and NIE115 cell lysates were prepared and subjected to immunoblot for various HDACs and β-actin. Representative immunoblots are shown.