Journal: Journal of Molecular Cell Biology
Article Title: Atad2 is a generalist facilitator of chromatin dynamics in embryonic stem cells
Figure Lengend Snippet: Atad2 is involved in ES cell differentiation. ( A ) Both control ES cells (WT, expressing an inactive shRNA) and cells with reduced amounts of Atad2 (KD, stably expressing an active anti- Atad2 shRNA) were seeded in LIF-depleted media for 6 days. Extracts were prepared and the indicated proteins were visualized. ( B ) WT and KD ES cells were seeded, and cell growth was monitored as a function of time. ( C ) WT and KD ES cells were cultured in the differentiation medium, and the diameter of EBs was measured after 6 days of differentiation and presented as box plots. Forty-eight EBs were measured for each condition, and the P -value (Mann–Whitney) corresponding to size differences between the two cell populations is indicated. The corresponding values are presented in Supplementary Table S3 . Six representative EBs for each condition are shown. ( D ) Control ES cells and cells with reduced amounts of Atad2 were placed in the differentiation medium for 3 days. RNA was prepared and the transcriptome was revealed following hybridization on Illumina chip. The heatmaps show genes differentially expressed between control cells (ct) and cells with Atad2 knockdown (Sh Atad2). Illustrations of GSEA categories of genes whose expression is regulated by Atad2 knockdown are shown. ( E ) WT and KD ES cells were placed in LIF-depleted media for 7 days. Cells were harvested every day and RNA were prepared. The expression of the indicated genes, specific representatives of differentiation into the three germ layers, was monitored by RT-qPCR and normalized with respect to the expression of actin . The data shown are representative of two independent experiments. Standard deviations are shown.
Article Snippet: The purified DNA was size-selected at 150 base pairs and processed for sequencing onto a single Illumina genome analyzer channel using the procedures recommended by the manufacturer.
Techniques: Cell Differentiation, Expressing, shRNA, Stable Transfection, Cell Culture, MANN-WHITNEY, Hybridization, Chromatin Immunoprecipitation, Quantitative RT-PCR