Journal: The FEBS journal

Article Title: Trichostatin A epigenetically increases calpastatin expression and inhibits calpain activity and calcium-induced SH-SY5Y neuronal cell toxicity.

doi: 10.1111/febs.12572

Figure Lengend Snippet: Fig. 1. Trichostatin A decreases calpain 1 protein level and increases calpastatin protein and mRNA levels. After differentiation with all-trans retinoic acid for 5–6 days, SH-SY5Y cells were treated with various doses (150, 300, 600 nM) of TSA or DMSO only (0) for 24 h (A) or 300 nM TSA for the indicated times (0, 12, 24, 48 h) (B). The protein levels of calpain 1, calpain 2 and calpastatin were detected by western blot analysis. The protein level of b-actin was measured for a control. The blot shown is representative of at least three experiments. Quantifications were performed using densitometry and the results were normalized to b-actin. The mRNA levels of calpain 1 (C), calpain 2 (D) and calpastatin (E) in cells treated with 300 nM TSA for 24 h were detected by real-time PCR analysis. The results from each of three independent experiments were normalized to b-actin and expressed relative to the mRNA level of each gene in DMSO-treated control cells. Each bar shown is the mean fold increase above control SD, and the results are considered to be statistically significant at *P < 0.05 and **P < 0.01.

Article Snippet: SH-SY5Y cells grown in six-well plates were transiently transfected using FuGENE HD reagent (Roche) with 100 nM calpastatin siRNA (sc-29889; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology).

Techniques: Western Blot, Control, Real-time Polymerase Chain Reaction

Journal: The FEBS journal

Article Title: Trichostatin A epigenetically increases calpastatin expression and inhibits calpain activity and calcium-induced SH-SY5Y neuronal cell toxicity.

doi: 10.1111/febs.12572

Figure Lengend Snippet: Fig. 3. Trichostatin A increases calpastatin levels epigenetically via histone acetylations. Differentiated SH-SY5Y cells were treated as described in the legend to Fig. 1 and applied to ChIP analysis. ChIP analysis was performed with antibodies specific for acetylated H4K5 (H4K5-Ac), H3K9-Ac and H3K14-Ac. The DNA purified after ChIP was evaluated by semi- quantitative PCR. The input represents amplification of the total input DNA from whole cell lysates and mock ChIP samples without antibody. The DNA amount of the calpastatin promoter region after the ChIP assay was normalized to the input DNA level and expressed relative to that of DMSO-treated control cells. Each bar shown is the mean fold increase above control SD (n = 3), and the results are considered to be statistically significant at *P < 0.05 and **P < 0.01.

Article Snippet: SH-SY5Y cells grown in six-well plates were transiently transfected using FuGENE HD reagent (Roche) with 100 nM calpastatin siRNA (sc-29889; Santa Cruz Biotechnology) or control siRNA (sc-37007; Santa Cruz Biotechnology).

Techniques: ChIP-chip, Real-time Polymerase Chain Reaction, Control