Figure 4

Effects of histone deacetylase inhibitor on Igf2 knockout cells. Primary embryonic liver cells were derived from mice which carried a paternally-inherited deletion of Igf2 (Pat KO; lanes 1 and 2) or their wild-type littermates (WT; lanes 3 and 4). First passage cells were either treated with 500 nM TSA for 6 hr or left untreated as indicated. (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 primary cells (lanes 2 and 4). (B) Coomasie stained total protein loading control for the Western. (C) Northern hybridization of RNA derived from the same cells to a mouse probe for Tissue-type plasminogen activator (Tpa). Transcription levels can be seen to increase on TSA treatment (lanes 2 and 4) as in RD cells. (D) The membrane used in (C) was stripped and rehybridized with a probe for Igf2. Mice which carry a deletion of the gene on the paternally inherited allele (Pat KO) are missing the allele which is normally active but retain the silenced maternal allele and show no expression of the gene (lane 1). Reactivation of the silent maternal allele was not seen in the cells treated with TSA (lane 2). The two major transcripts ran as a single band here. (E) Rehybridization with an H19 probe shows no significant difference between the TSA treated and untreated samples, allowing for differences in loading. (F) 28S rRNA loading control for the Northern.