Leal-Sanchez et al

Supplementary Table 1 Absolute cell numbers from thymus, spleen and lymph nodes of NLC and Daxx DN transgenic mice

Thymus, spleens and inguinal lymph nodes of mice of indicated age (in weeks) were removed. Single-cell suspensions were prepared and the total number of cells determined by counting in a Malassez cell. Values are the means ± standard deviation.

Supplementary Figure 1. Flow cytometric analysis of cell subsets in thymus and peripheral lymphoid organs from Daxx-DN Tg mice

Thymocytes or peripheral lymphocytes from spleen or lymph nodes from 6 to 10 week-old NLC and Daxx-DN Tg mice were stained with FITC-CD3, PE-CD4, biot-CD8 Mab, or FITC-Fas (Jo2ab). The percentages of double-negative, double-positive, and single-positive T cells of the total cell population are shown and are representative of at least six independent experiments.

Supplementary Figure 2. Localization of endogenous Daxx following Fas and CD3 activation.

(A) T cells were activated with anti-CD3 for 4 days and then stimulated or not with 100 ng/ml rhFasL plus 1 μg/ml anti-Flag antibody M2 for 6 minutes. Cells were fixed with 4% paraformaldehyde for 15 minutes and permeabilized with 0.2% Triton X-100 for 30 minutes. After blocking with BSA (3%) and FCS (5%), cells were incubated with anti-Daxx antibody (M112) and stained with anti-rabbit Alexa Fluor 488 (green) and DAPI (blue).

(B) 3A9 T cells were stimulated or not with 20 µg/ml anti-CD3ε for 15 minutes. Cells were stained following the same protocol as in (A) except anti-rabbit Alexa Fluor 546 (red) was used as secondary antibody.

Data shown is representative of >5 fields analyzed.

Supplementary Figure 3. Inhibition of Fas-mediated cell death in clones of Daxx shRNA expressing L12.10 Fas T cells expressing different amount of the Fas protein at the plasma membrane surface.

L12.10 cells (1x106/ml) stably expressing short hairpin Daxx siRNA (shRNA Daxx) or control siRNA (shRNA control) were incubated for 4 hours with titrated amounts of recombinant FasL plus 1 ug/ml anti-FLAG antibody. Daxx expression was analyzed by western blot.

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