The transcription factor PLZF controls the proapoptotic phenotype of iNKT cells. (A) Expression of PLZF in expanded GFP⁺iNKT targeted by a control shRNA (sh-control) or specific for PLZF (sh-PLZF) by flow cytometry. Gray histograms, isotype control; black lines, anti-PLZF. One representative experiment of 4. (B) Staurosporine-induced apoptosis of GFP⁺iNKT cells targeted with a control shRNA (sh-control) or 2 shRNA specific for PLZF (sh-PLZF-1-, n = 6; sh-PLZF-2-, n = 3). (C) Same as in panel B except that GFP⁺iNKT cells were analyzed for FAS-induced apoptosis (left panel, n = 5). In the right panel, cells were repeatedly treated with anti-FAS antibodies every 24 hours and the proportions of GFP⁺ cells in the cultures were determined. Data represent the ratio of the percentage of GFP⁺ cells in the culture to the percentage of GFP⁺ cells at time 0. One representative experiment of 2 with 2 donors (Nos. 4 and 9). (D) Expression of PLZF in expanded Vβ2 T cells infected by a lentiviral empty vector (empty vector) or containing a PLZF transgene (Tg-PLZF). Gray histograms, isotype control; black lines, anti-PLZF. One representative experiment of 4. (E) Staurosporine-induced apoptosis in GFP⁺ Vβ2 T cells expressing the empty vector (empty vector) or a PLZF transgene (Tg-PLZF; n = 3, donors Nos. 4, 6, and 9). (F) Same as panel C. FAS-induced apoptosis in GFP⁺ Vβ2 T cells expressing the empty vector (empty vector) or a PLZF transgene (Tg-PLZF; n = 4, donor Nos. 4, 6, and 9). (G) Expression of PLZF in Jurkat cells infected by a lentiviral empty vector (empty vector) or containing a PLZF transgene (Tg-PLZF). Gray histograms, isotype control; black lines, anti-PLZF. (H) FAS-induced apoptosis in Jurkat cells expressing PLZF (Tg-PLZF) or not (empty vector; n = 4). (I) Staurosporine-induced apoptosis in Jurkat TAg cells infected by a lentiviral empty vector (empty vector) or containing a PLZF transgene (Tg-PLZF) PLZF (Tg-PLZF; n = 3). (J) Activity of the promoter P1 of caspase 3 measured by a reporter luciferase assay in Jurkat TAg cells expressing PLZF (Tg-PLZF) or the empty vector. Data of 2 independent experiments with quintuplicates, mean ± SD. (K) Expression of actin, PLZF, caspase 3 (CASP3), cleaved caspase 3 (CASP3 cleaved), and Ku70 (as loading control) in wild-type Jurkat TAg cells (WT) or expressing PLZF (Tg-PLZF) or the empty vector by Western blotting. On the right, quantification of caspase 3 and cleaved caspase 3 expressions in Jurkat TAg cells expressing PLZF compared with the empty vector and normalized on actin expression. One representative experiment of 2. Paired, 2-tailed t tests in panels B, C, E, F, H, and I with data mean ± SD and n = number of independent experiments with duplicate (*P < .05, **P < .01, ***P < .001).