For spontaneous apoptosis measurement by mitochondrial integrity analysis, HeLa cells were transfected with the indicated TNFR1-ECFP construct. Mitochondrial membrane potentials (ΔψM) were analyzed by staining cells with the mitochondrion-selective probe tetramethylrhodamine methyl ester (TMRM). The TMRM signal was measured by flow cytometry in ECFP-positive cells. For spontaneous apoptosis measurement by cell cycle analysis, HEK 293T cells were transduced with the indicated TNFR1 lentivirus at a multiplicity of infection of 50. Cell DNA content was measured by flow cytometry using propidium iodide (PI) staining. Following exclusion of debris and cell doublets, cells with a sub-diploid (G0/G1) DNA content were considered apoptotic.
