Figure 4

The proapoptotic activity of Livin is required for the generation of functional platelets. Control LAMA-84 cells (EV, empty vector) and LAMA-84 cells stably overexpressing WT Livin or Livin mutant lacking proapoptotic activity (Livin-RING) were treated with PMA for 4 days. Cells were harvested at 12, 24, 48 and 96 h. (a) Western blot analysis of Livin and XIAP proteins and their cleaved derivatives during LAMA-84 differentiation into MKs. Note the appearance of cleaved proapoptotic tLivin after 96 hours of differentiation induction with PMA. (b) Caspase-3 activation was assessed through the evaluation of caspase-3 enzyme activity (100 ng of protein) with Ac-DEVD-pNA (N-acetyl-Asp-Glu-Val-Asp-p-nitroaniline) in a colorimetric assay at day 4. Note that Livin transfection enhanced the caspase activity. (c) At day 4, culture-derived PLPs were collected from the medium for flow cytometric analysis of CD41 expression and the number of CD41+ platelet particles was quantitated. The PLP counts from cells transfected with either Livin or Livin-RING were significantly increased (n=3). (d) Functional assay for PLPs’ aggregation in response to arachidonic acid (AA) was performed to determine the potential biological activity of these PLPs produced by LAMA-84 cells. Both control and Livin transfectants produced functional PLPs, whereas those derived from the Livin-RING-transfected cells were not functional (n=4)