Figure 1.

Conditioned medium of megakaryocytes promoted T24 cell survival. K562 cells were treated with various concentrations of phorbol 12-myristate 13-acetate (PMA) (0–10 nM) for 4 days. (A) Proteins were extracted and subjected to Western blot analysis with indicated antibodies. Representative blots of four independent experiments are shown. The Western blots have been shown in Figure S1. (B) Cells were harvested and subjected to flowcytometric analysis for the measurement of surface presentation of CD41. (C) Cells were pelleted by centrifugation, spread onto glass slides, and stained with Wright–Giemsa dye. Scale bar: 300 µm. K562 cells were treated with PMA (0 and 10 nM) in the absence or presence of dipyridamole (10 µM), aspirin (100 µg/mL), or cytochalasin D (1 µg/mL) for 4 days. (D) Cells were harvested and subjected to flowcytometric analysis for the measurement of surface presentation of CD41. Bar graphs show relative genomic mean intensity of CD41-PE fluorescence. (E) The cultured media were harvested and centrifuged, and the resultant supernatants were named conditioned medium (CM). Equal amounts of CM and fresh medium were mixed and added to T24 cells (96-well) for 48 h. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) reduction assay. (F) Equal amounts of CM obtained from PMA treatments (0 and 10 nM) and fresh medium were mixed and added to T24 cells (96-well) in the absence or presence of cytochalasin D (1 µg/mL) for 48 h. Cell viability was measured by the MTS reduction assay. * p < 0.05 vs. untreated group and # p < 0.05 vs. PMA (10 nM) group or K562/PMA (10 nM) CM group, n = 4.