Apoptosis induced by CA-NFAT1 in NIH3T3 fibroblasts is dependent on DNA binding.

<p>(A) Schematic representation of the primary structure of CA-NFAT1 MutDBD. The mutated residues are indicated in the Figure. (B, E, F, G) NIH3T3 cells were transduced with empty vector or retrovirus expressing CA-NFAT1 or CA-NFAT1 MutDBD. (B) The total lysate of transduced NIH3T3 cells was obtained for analysis of NFAT1 and GAPDH expression levels by Western Blot. The molecular weights are indicated in kilodaltons (kDa). (C) The ability of the NFAT1 DBD and NFAT1 MutDBD to bind to DNA was tested by EMSA. The NFAT1 DBD and NFAT1 MutDBD peptides were incubated with oligonucleotides corresponding to the NFAT responsive element in the IL-2 promoter. (D) Jurkat cells were transfected with expression vector (empty vector or vector containing the CA-NFAT1 or CA-NFAT1 MutDBD cDNAs), luciferase reporter vector pGL4.30 and the Renilla luciferase expression vector pRL-TK. After 24 hours, the luciferase activity was measured by the release of luminescence resulting from the oxidation of its substrate (luciferin), normalized with the Renilla vector and expressed as relative light units (RLU). (E) Proliferation was assessed by incorporation of crystal violet. The cells were plated in triplicate and analyzed for 120 hours. This graph is representative of three independent experiments. (F, G) NIH3T3 cells were stained with propidium iodide (PI) and analyzed by flow cytometry for cell death. (F) Analysis of cell death 48 hours after plating. The graph represents the average percentage of cells in sub-G0 in three independent experiments. (G) Representative graph of the cell death analysis shown in (F). The percentage of cells in sub-G0 is shown in the graph.