iJC-TCRβ induces apoptotic cell death in both non-T and T cells that is regulated by its Ig domain. (A) S49.1 T cells (i) or thymocytes from TCRβ-deficient mice (ii) were transfected with EGFP-iJC (green) and 5 hours or 7 hours later, respectively, were labeled by the TUNEL method (rhodamine staining) and counterstained with Hoechst for nuclear staining. The merge panels show colocalization of the Hoechst and the TUNEL signals. All pictures were taken by fluorescence microscopy (100× oil-immersion objectives). (B,C) 293 cells (Bi-vi,Ci-xvi) or Jurkat cells (Bvii,viii) were transfected with the indicated constructs. At 48 hours (293) or 11 hours (Jurkat) after transfection, the cells were fixed, and those transfected with Flag-iJC were stained with anti-Flag antibody, a biotin-conjugated anti–mouse antibody, and a streptavidin Oregon Green 488 conjugate. All cells were stained with propidium iodide and analyzed for their cell-cycle distribution. EGFP-negative or Flag-negative cells are the cell populations that remained untransfected in each experiment; EGFP-positive or Flag-positive cells are the transfected cell populations; cells transfected with empty vectors are pCDNA3 (Bv,Cxi) and pEGFP-F (Bvi,Cxii). Bar, 5 μM.