S4 Jurkat cells do not secrete fortilin without anti-Fas IgM challenge. the 7-AAD staining as described in the Materials and Methods section of the manuscript. 7-AAD cannot traverse through the intact plasma membrane. The cells with red fluorescence 7-AAD signal have disrupted plasma membrane. At least 200 cells were counted and the 7-Put index was calculated as (the PE859 number of 7-AAD positive cells)?/?(the number of total cells)???100. mmc3.zip (15M) GUID:?F5280CBA-375A-4D50-874A-5ECC75DD0EDE Fig. S4 Jurkat cells do not secrete PE859 fortilin without anti-Fas IgM challenge. Abbreviations: NS, not statistically significant. 5??105 Jurkat cells were seeded at each cell of 6-well plates using PRMI media supplemented by 5% FBS. Next day, cells were washed once with PBS and re-suspended in 1?mL of fresh RPMI media with 5% FBS. At times 0, 6, and 12?h, 500?L of cell suspension was harvested from each well in triplicates and subjected to centrifugation at 100?for 5?min. The supernatant was transferred to a fresh microfuge tube and stored at ??80?C for fortilin ELISA. mmc4.zip (336K) GUID:?138670BB-7EE0-4AB3-A8E4-276DC88EEDB7 Fig. S5 Comparison of fortilin with other apoptosis biomarkers and LDH. Abbreviations: Cyt C, cytochrome c; n-DNA, nucleosomal DNA; fCK-18, fragmented cytokeratin-18; LDH, lactate dehydrogenase. LDH is usually a cell death marker that is passively released through the damaged plasma membrane without apoptosis-specific modification. Although it is usually passively released from the cells unmodified, Cyt C can still be an apoptosis marker as Cyt C is usually released from the mitochondrial intermembrane space into the cytosol in the apoptosis-specific process. While it is usually passively released from the cells, fCK-18 is usually a caspase-cleaved product of the original cytoskeleton protein CK-18. Based on the data described in Fig.?6F and H, both Cyt C and fCK-18 rely on the compromise in plasma membrane integrity for their release into extracellular space. n-DNA is an apoptosis-specific degradation PE859 product of nuclear DNA by the caspaseactivated DNAse (CAD) and released before plasma membrane changes occur detectable by 7-AAD and LDH-release (Fig.?6G). Fortilin is unique because it does not undergo apoptosis-specific modification and is released in the very early phase of apoptosis, most likely via exosomes (Fig.?6E). mmc5.zip (1.0M) GUID:?29B75B9B-7262-4850-86E2-21155602AAC0 Abstract Background Billions of cells undergo apoptosis each day in the average PE859 normal adult. The ability to readily assess the degree of apoptosis in human diseases is usually hampered by the lack of sensitive and specific serum biomarkers of apoptosis. Fortilin is usually a novel prosurvival molecule that protects cells against various noxious stimuli. While fortilin is usually secreted into the extracellular space under certain conditions, the relationship between the serum concentration of fortilin and the presence and extent of apoptosis in vivo remains unknown. Methods & results Using a newly developed fortilin ELISA system, we show here that fortilin exists in the normal human and mouse circulation. We further demonstrate that fortilin serum levels are significantly elevated SELE in patients with solid cancer, in response to anti-cancer chemo- or radiation therapy. The elevation of fortilin serum levels is usually more robust and sensitive than that of such previously-reported serum biomarkers of apoptosis as fragmented cytokeratin-18, cytochrome c, and nucleosomal DNA. In addition, targeted apoptotic liver damage induced by Jo2 anti-Fas (CD95) antibody consistently and significantly increased serum fortilin levels in C57BL/6J mice. Finally, when challenged by anti-human-Fas IgM antibody, Jurkat leukemic T cells apoptosed and released fortilin into the medium before plasma membrane integrity was compromised. Conclusions Taken together, these data suggest that serum fortilin levels reflect the degree and extent of apoptosis occurring in vivo. General significance Fortilin is a viable serum biomarker of in vivo apoptosis and can be utilized to noninvasively assess the status of in vivo apoptosis in humans. for 5?min, transferred the medium to fresh microfuge.