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Figure 1 | Molecular Cancer

Figure 1

From: Reactivation of a silenced H19 gene in human rhabdomyosarcoma by demethylation of DNA but not by histone hyperacetylation

Figure 1

Effects of histone deacetylase inhibitor on rhabdomyosarcoma cells. Cells were treated for 6 hr (lanes 1, 2) or 15 hr (lane 3–5) with 500 nM of either Trichostatin A (TSA) or 5'-aza-2'deoxycytidine (AzaC) as indicated at top. (A) Western analysis of protein derived from the cells using the anti-acetylated histone 4 (AcH4) antibody shows that treatment with TSA markedly increases the amount of acetylated histone in the cells at 6 hr (lanes 1 and 2). While treatment for 15 hr also increases acetylation levels, the effect is less marked, presumably due to a compensatory mechanism in the cell (lanes 3 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a probe for Tissue-type Plasminogen Activator (TPA). Transcription levels can be seen to increase in parallel with the marked increase in histone acetylation (lanes 1 and 2), indicating that acetylation plays an important role in determining levels of TPA transcript in the cell. The less marked increase in acetylation seen at 15 hr has only a slight effect on transcript levels (lanes 3 and 4). (D) The membrane used in (C) was stripped and rehybridized with a probe for H19, which is normally silent in these cells. No signal was detected in the normal or TSA-treated samples (lanes 1–4), but some reactivation could be seen after even a brief treatment with a low level of AzaC (lane 5). (E) Rehybridization with an IGF2 probe shows no difference between the TSA treated and untreated samples at 6 hr (lanes 1 and 2) or 15 hr (lanes 3 and 4). IGF2 transcripts accumulate in the cells during the log phase of growth in culture and at 15 hr, basal levels of IGF2 have increased from those seen at 6 hr (compare lanes 3 and 1). The two major IGF2 transcripts of 6 kb and 4.9 kb are visible. (F) 28S rRNA loading control for the Northern.

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