FIG. 6.

TDAG8 is dispensable for psychosine-induced inhibition of cytokinesis and formation of multinucleated cells. (A) Flow cytometry-based approach used to examine the effects of PSY on cell division and DNA content. Total splenocytes from TDAG8 WT and KO were stimulated with a soluble anti-CD3 antibody, as described in Materials and Methods. CD4⁺ and CD8⁺ subsets were identified by surface staining. The gate for cell cycle analysis (Hoechst 33342) included cells that had undergone no more than one division, as indicated by the CFSE profile. (B) Treatment with 15 μM PSY of splenocytes activated for 48 h with 100 ng/ml soluble anti-CD3 antibody resulted in the accumulation of cells with greater than 4 N DNA content. (C) Percentages of TDAG8 WT and KO CD8⁺ cells with greater than 4 N DNA content following treatment with 10 or 15 μM PSY. The error bars indicate SEM. (D) Morphological examination of psychosine-induced accumulation of multinucleated cells using DAPI-stained cytospins. These results are representative of three independent experiments.