JCS022822 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. p53 status does not affect long-term survival of ES cells. (A) Undifferentiated ES cells were plated onto six-well plates, γ-irradiated at various doses and cultured for 8 days. Number of surviving cells was counted thereafter. Three independent experiments were performed in duplicates and data represents mean ± s.e.m. (B) Long-term survival of ES cells was assayed by the colony formation assay. 1000 undifferentiated ES cells were irradiated (1 or 5 Gy), and surviving colonies were stained with crystal violet solution after 10 days and photographed. Data from two independent experiments are shown for 1 Gy irradiation.

• Supplemental Figure S2 -

  Fig. S2. Oncogenic Ras does not co-operate with loss of p53 function in regulating ES cell growth. (A) Undifferentiated ES cells were stably transfected with linearized pBabe Ras^V12 plasmid and selected with 1 µg/ml puromycin for 8-10 days. Expression of Ras was determined by semi-quantitative RT-PCR. (B) 1×10⁵ cells were plated onto six-well plates and counted daily. Three independent experiments were performed in duplicates and data represents mean ± s.e.m.

• Supplemental Figure S3 -

  Fig. S3. p63 and p73 are not involved in ES cell death. (A) The expression of p63 and p73 genes in mouse ES cells was analyzed by semi-quantitative RT-PCR using specific primers. p53^+/+ and p63^−/−p73^−/− MEFs were included as positive and negative controls, respectively. (B) Silencing of p73 expression using p73-siRNA. RT-PCR analysis was performed to determine efficiency of gene silencing. (C) 1×10⁵ undifferentiated ES cells were transfected with scrambled or p73-siRNA 36 hours prior to treatment with 1 µg/ml doxorubicin for 12 hours. Cell viability was determined by flow cytometric analysis after annexin-V/propidium iodide staining. Data represents mean ± s.e.m. of two independent experiments.