Validation of LIN9 target genes in ESCs.

<p>(A) & (C) Validation of microarray results by RT-qPCR. The expression of the indicated genes in control transfected cells and cells transfected with pSUPER-LIN9 was compared. (B) Expression of Cyclin B1 in control transfected ESCs and ESCs transfected with pSUPER-LIN9 was analyzed by immunoblotting. Tubulin was used as control for equal loading.</p

Cell cycle arrest in G2/M after depletion of LIN9.

<p>(A) Alkaline-phosphatase (AP) staining of control cells and LIN9 depleted cells. Scale bar: 200 µM (B) Expression of pluripotency markers Oct4 and Sox2 was analyzed in control-depleted cells and LIN9 depleted cells by RT-qPCR. (C) The cell cycle profile of LIN9 depleted ESCs and of control cells was analyzed by flow cytometry.</p

Gene expression changes after depletion of LIN9 in ESCs.

<p>(A) Number of up- and downregulated genes in LIN9 depleted cells identified by microarray analysis. For a list of regulated genes see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062882#pone.0062882.s002" target="_blank">Table S1</a> (B) & (C) GO analysis was applied to differentially expressed genes. Listed are the top fifteen overrepresented GO terms according to the p-value. For complete lists of GO terms with a p-value of less than 0.05 see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062882#pone.0062882.s003" target="_blank">Table S2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062882#pone.0062882.s004" target="_blank">S3</a>.</p

Identification of direct targets of LIN9 by ChIP-on-chip.

<p>(A) Functional categories of targets of LIN9 identified by ChIP-on-chip. LIN9 bound promoters were analyzed for enrichment of Gene Ontology terms. Shown are the top fifteen overrepresented GO terms according to the p-value. For a complete list bound promoters and GO terms with a p-value of less than 0.05 see Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062882#pone.0062882.s005" target="_blank">Tables S4</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0062882#pone.0062882.s006" target="_blank">S5</a>. (B) Mitotic genes are direct targets of LIN9 in ESCs. Comparison of gene expression data and ChIP-on-chip data. Shown are genes that are downregulated after depletion of LIN9 and that have a known function in mitosis. “√” indicates that binding of LIN9 to the promoter was detected by ChIP-on-chip. “−” indicates that no binding was detected. (C) Binding of LIN9 to the promoters of randomly selected mitotic targets genes was confirmed by conventional ChIP.</p

Direct target genes of LIN9 in ES cells as determined by microarray and ChIP-on-chip.

<p>Direct target genes of LIN9 in ES cells as determined by microarray and ChIP-on-chip.</p

Expression of Lin9 in pre-implantation embryos and ESCs.

<p>(A) Expression of <i>Lin9</i> mRNA in 3.5 and 4.5 dpc blastocysts was analyzed by <i>in situ</i> hybridization with a DIG-labeled Lin9 probe. As a control, Oct4 mRNA was analyzed. (B) Nuclear lysates from ESCs were immunoprecipitated with a LIN9 antibody or with nonspecific IgG as a control. Co-precipitated proteins were detected with the antibodies indicated. Input (5%) was also loaded on the gel for comparison.</p

Generation of a Biotag-LIN9 ESC line.

<p>(A) Scheme of N-terminal tagged LIN9 with the BirA recognition sequence. The biotin acceptor lysine is indicated in red. BirA: E. coli biotin ligase (B) LIN9 was immunoprecipitated from ESCs stably expressing BirA alone or BirA and Biotag-LIN9. LIN9 was detected by immunoblotting. The positions of endogenous and Biotag-LIN9 are indicated. (C) LIN9 was affinity purified with streptavidin-coupled magnetic beads and detected by immunoblotting. (D) LIN9 was affinity purified with streptavidin-coupled magnetic beads. Bound proteins were detected by immunoblotting.</p