Figure 1.
PtdSer-dependent cell proliferation. A, PtdSer-dependent growth stimulation of TIM4- and MERTK-expressing Ba/F3 cells. Ba/F3 cells (2.5 × 105) expressing TIM4, MERTK, or both TIM4 and MERTK were cultured in 0.5 ml of RPMI 1640 medium containing 10% FBS. After incubation for the indicated periods, viable cells were counted after staining with trypan blue. Right panel, Ba/F3 cells expressing TIM4 and MERTK were cultured for 72 h in medium containing the indicated concentration of D89E, and the number of viable cells was expressed as the percentage of that in the absence of D89E. The experiments were carried out three times, and average values were plotted with S.D. (error bars). The values were statistically analyzed by Student's t test. **, p < 0.01; ***, p < 0.001. B, apoptotic cell–stimulated, PtdSer-dependent cell growth. Ba/F3 cells (2.5 × 105) expressing TIM4, MERTK, or both TIM4 and MERTK were cultured in 0.5 ml of medium for the indicated periods in the presence of 2.5 × 106 apoptotic thymocytes (left panel). Center and right panels, Ba/F3 cells (2.5 × 105) expressing both TIM4 and MERTK were cultured in 0.5 ml of medium for 72 h in the presence of the indicated concentration of apoptotic thymocytes (center panel) or in the presence of 6.25 × 105 apoptotic thymocytes and the indicated concentration of D89E. After incubation, the trypan blue–negative viable cells were counted. Right panel, the number of viable cells was expressed as the percentage of that in the absence of D89E. The experiments were carried in triplicate, and average values were plotted with S.D. (error bars). *, p < 0.01; **, p < 0.001; Student's t test. C, DNA synthesis of Ba/F3 cells expressing TIM4 and MERTK without IL-3. 2.5 × 105 Ba/F3 cells (a, d, and e) and Ba/F3 cells expressing MERTK and TIM4 (b and c) were cultured at 37 °C for 72 h without (a, b, and c) or with (d and e) IL-3 in the absence (a, b, d, and e) or presence (c) of 2.5 × 106 apoptotic thymocytes. The culture was supplemented with (a–d) or without (e) 10 μm BrdU and incubated further for 4 h. The incorporated BrdU was then detected with FITC-labeled anti-BrdU Ab.