Figure 2.

CRT downregulation in WT motoneurons in vitro leads to death and implicates the Fas signaling pathway. Efficiency and specificity of CRT silencing with sh-CRTs in NSC34 cells (A, B) and in motoneurons (C, D). NSC34 cells were cultured for 24 h, then transfected with the EGFP vector or with shcrt1/2 or sh-control. Forty-eight hours later, cells were lysed and Western blot performed on the extracts (A). CRT levels were quantified relative to actin levels (B). Motoneurons were electroporated with the EGFP vector in combination with empty vector (e.v.), shcrt1/2, or sh-control and immunostained for CRT 48 h later (C). Immunostaining was quantified (D). Downregulation of CRT leads to motoneuron death (E, F). Motoneuron survival was assessed 1 and 2 d after electroporation with the EGFP vector in combination with e.v., shcrt1/2, or sh-control. All conditions were expressed relative to EGFP + e.v. condition (E). WT and CRT heterozygous (crt^+/−) Motoneurons were cultured and cell counting performed 1 and 2 d after seeding (F). The killing effect of CRT downregulation is dependent on Fas activation (G, H). WT motoneurons were electroporated as in (E). Motoneurons were treated 24 h later with Fas-Fc (1 μg/ml) and survival assessed 48 h later (G). WT motoneurons were electroporated as in (E), treated or not 12 h later with DETANONOate, and immunostained for Fas ligand 24 h later. FasL expression was quantified in EGFP-positive cells and expressed relative to EGFP alone condition (H). Data are means ± SD of three independent experiments: *p < 0.05, **p < 0.01, and ***p < 0.001.