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cmi12263-sup-0001-si.tif12856K

Fig. S1. Bcl-2 expression in WT MEF and (lack of) effect on C12-induced activation of caspase 3/7. WT MEF grown on coverglasses (A) or in plastic dishes (B) were infected with retroviruses expressing GFP or Bcl-2 + GFP. The retroviral vector DNA plasmids (MSCV-PIG) expressing GFP or Bcl2-IRES-GFP were obtained from Dr A. Winoto (Univ. California, Berkeley). The Bcl2 vector has been described in Bassik et al. (2004). These plasmids were used to transfect Phoenix cells and produce retroviruses based on the system developed by Dr G. Nolan (Stanford Univ., http://www.stanford.edu/group/nolan/retroviral_systems/phx.html). Briefly, Phoenix cells were plated and grown overnight, then transfected with retroviral vector and Nolan helper vectors. The culture media containing retroviruses was collected 24–48 h post-transfection. The collected viral-containing media was filtered with 0.45 μm low protein-binding filter and kept frozen in small aliquots for future infection. WT MEF were plated and grown overnight to 50% confluence. Then the cells were infected by adding the retrovirus-containing media mixed with fresh media at 1:1 ratio. The expression of GFP and Bcl2-IRES-GFP was visualized by GFP fluorescence 24–48 h after the infection. This Bcl-2 construct elicits efficient reduction of apoptosis in Jurkat cells (Bassik et al., 2004).

A. Retroviruses expressing GFP and Bcl-2 + GFP expressed in nearly 100% of cells on the coverglasses.

B. Treatment of MEF with 50 μM C12 caused equivalent increases in caspase 3/7 activity in WT, GFP- and Bcl-2-infected MEF.

cmi12263-sup-0002-si.tif11443K

Fig. S2. C12-induced killing of WT and DKO NIH3T3 MEF. WT and DKO murine fibroblasts immortalized by the NIH3T3 spontaneous immortalization method (Zong et al., 2003) were used for measurements of PI uptake (cell death) or Western blots to test for Bak/Bax expression.

A. WT and DKO NIH3T3 cells were incubated with 10–50 μM C12 for 24 h then processed for PI uptake. There were no differences in cell killing between the two cell lines.

B. Western blots for Bak and Bax of WT and DKO NIH3T3 cells confirmed Bak/Bax expression in WT but not DKO cells. Data are averages ± SD, n = 3 experiments in each case.

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