Figure 1.
Expression of Lin9 in pre-implantation embryos and ESCs.
(A) Expression of Lin9 mRNA in 3.5 and 4.5 dpc blastocysts was analyzed by in situ 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.
Figure 2.
Impaired embryoid body formation after depletion of LIN9 in ESCs.
ESCs were transfected with a plasmid encoding a LIN9 specific shRNA. LIN9 mRNA (A) and protein levels (B) were compared with the levels in control-transfected cells by RT-qPCR and immunoblotting. (C) Embryoid body formation: Outline of the experiment. Equal numbers of LIN9 depleted ESCs or control cells were placed in hanging drops on lids of cell culture dishes. After two days, embryoid bodies were harvested and grown in suspension in the absence of LIF for up to 6 days. (D) Embryoid bodies formed in control cells and LIN9-depleted cells. Scale bar: 100 µM. See Figure S1 for additional examples of embryoid bodies formed in control cell and LIN9 depleted cells from an independent experiment.
Figure 3.
Cell cycle arrest in G2/M after depletion of LIN9.
(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.
Figure 4.
Gene expression changes after depletion of LIN9 in ESCs.
(A) Number of up- and downregulated genes in LIN9 depleted cells identified by microarray analysis. For a list of regulated genes see Supplemental Table S1 (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 Table S2 and S3.
Figure 5.
Validation of LIN9 target genes in ESCs.
(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.
Figure 6.
Generation of a Biotag-LIN9 ESC line.
(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.
Figure 7.
Identification of direct targets of LIN9 by ChIP-on-chip.
(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 Tables S4 and S5. (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.
Table 1.
Direct target genes of LIN9 in ES cells as determined by microarray and ChIP-on-chip.