TSP-1 is transcribed, translated, and secreted upon monocyte apoptosis. (A) TSP-1 secretion by apoptotic monocytes correlates to early apoptosis. TSP-1 concentrations in apoptotic monocyte culture media were measured by TSP-1 EIA at several time points after apoptosis induction. TSP-1 levels peaked 8 hours following induction of apoptosis, corresponding to early apoptosis, as shown in Figure 1. Data are mean ± SD of 3 experiments. (B-C) The N-terminal heparin-binding domain of TSP-1 appears exclusively in the extracellular milieu of apoptotic monocytes. (B) Smear at average 130 kDa represents differentially degraded TSP-1 monomers (arrows) as verified by mass spectrometry. A degraded 26-kDa fragment (thick arrow) is also seen. TSP-1 monomers appear as 3 bands at 130 kDa or more, as verified by mass spectrometry (arrows). TSP-1 is seen only in apoptotic monocytes. (C) TSP-1 differentially degraded/glycosylated monomers appear as 3 bands at 130 kDa or more, and all perform full peptide coverage at mass-spectral analysis (arrows). TSP-1 is evidently seen only in apoptotic monocytes. Protein from 3 × 10⁷ cells, from culture media of viable (0 hours, B) or apoptotic monocytes (8 and 16 hours, B), and from whole cell lysed viable (0 hours, C) or apoptotic monocytes (8 and 16 hours, C) was loaded and separated by SDS-PAGE, as described under “Proteomic-based identification of secreted proteins from cells undergoing apoptosis.” Proteins were transferred to the PVDF membrane and exposed to mouse anti-human triclonal TSP-1 antibody and then to goat anti-mouse HRP. ECL results are presented. (D-E) TSP-1 mRNA is transcribed upon monocyte apoptosis. Total mRNA was extracted from 10⁷ monocytes at various time intervals following serum withdrawal apoptosis induction, and then reverse-transcribed and enhanced by polymerase chain reaction. (D) TSP-1 mRNA and β-actin mRNA were enhanced by PCR using specific primers, and their relative abundance was measured by ethidium bromide photospectrometry. Viable monocytes (0 hours) had very low levels of TSP-1. The level increased as apoptosis progressed. TSP-1 mRNA was not detected in viable iDCs or mDCs. (E) Relative abundance of TSP-1 mRNA adjusted to β-actin mRNA level, as measured by densitometry. TSP-1 mRNA levels peaked at 9.5 hours, in agreement with a state of early monocyte apoptosis.