Supplemental Material

This article contains the following supporting material:

• Figure S1 - Expression of pluripotency marker genes in Mll2-deficient ES cells. Quantitative PCR analysis of Oct4 and Nanog gene expression in ES cell clones. Ct values were normalized against the internal standard gene ribosomal protein L19 (RPL19); error bars represent SD of triplicate analysis.
• Figure S2 - Histone 3 lysine 4 methylation in mll2 mutant ES cells. Western blot analysis for histone methyltransferase activity of Mll2. Protein preparations of wild-type (+/+) and mll2 mutant (-/-) ES cells, differentiated wild-type ES cells (neurogenic in vitro differentiation, day 9 (EBs +/+)) and wild-type somatic liver cells (liver +/+) were blotted and hybridized with H3K4me³ or H3K9me² specific antibodies, respectively. Ponceau stainings of the respective protein gels served as a loading control.
• Figure S3 - Apoptosis in mll2-/- ES cells is not repressed by caspase inhibition. (A) Exponentially growing wild-type, FLP-rescued and mll2-/- ES cells were counterstained for Annexin V and propidium iodide and analyzed by flow cytometry. As a positive control wild-type ES cells were induced with 12 μM camptothecin for 6 h to undergo apoptosis. Cells displaced to the right of the vertical line in dotblots are positive for Annexin V-APC and represent early apoptotic cells. Analysis was restricted to early apoptotic cells, which are Annexin V-positive but still PI-negative (quadrant Q4-2). Specificity of Annexin V binding was verified by blocking the cells with recombinant Annexin V protein prior to incubation with fluorescently labeled Annexin V-APC (indicated as blocked). (B) Functional test for Z-VAD-FMK activity. Wild-type ES cells were grown under indicated conditions, stained with a monoclonal fluorescein-conjugated antibody to activated-caspase-3 and subjected to flow cytometry. Events displayed to the right of the vertical line represent activated caspase-3 positive apoptotic cells. Cells analyzed were a) untreated, b) grown in the presence of 12 μM camptothecin, c) grown in the presence of 40 μM Z-VAD-FMK caspase inhibitor, d) grown in the presence of DMSO (mock control), and e) grown in the presence of 16 μM camptothecin and 40 μM Z-VAD-FMK caspase inhibitor. Data are obtained from analysis of 5000 events. (C) Effect of a broad-spectrum caspase inhibitor on apoptosis in mll2-/- ES cells. Proliferation rates of undifferentiated ES cells in the presence of the general caspase inhibitor Z-VAD-FMK. Mock control cells were grown under same conditions, but in the absence of Z-VAD-FMK (DMSO mock). ES cells were counted on four days of continued culture. Growth rates were obtained from triplicate counts; error bars indicate SD of wild-type (black lines), FLP-rescued (grey dashed lines) and mll2-/- (red dashed lines) counts. (D) Quantitative analysis of apoptotic marker gene transcripts (Bcl2, Caspase-3, -8, -9, AIF, Parp1, Apaf1) in viable and apoptotic ES cell fractions. Sorted fractions of Annexin V-negative and Annexin V-positive fractions were analyzed by quantitative RT PCR analysis. Ct values were normalized against the endogenous standard gene ribosomal protein L19 (RPL19). Data are means of triplicate analysis ± SD.
• Supplemental Material