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. 2013 Aug 8;288(37):26753–26763. doi: 10.1074/jbc.M113.470260

FIGURE 6.

FIGURE 6.

Knockdown of endogenous RanBPM by shRNA promotes BLT2-mediated chemotaxis and ROS generation. A, HaCaT cells were transfected with either control shRNA (−) or RanBPM shRNA (+). At 48 h after transfection, the transfected cells were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 3 h. The migrating cells were then fixed and stained with hematoxylin/eosin. The migratory activity was expressed as a percentage of the control. B, PHK cells expressing either control shRNA or RanBPM shRNA were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 3 h and then assayed for chemotaxis as described above. C, HaCaT cells expressing either control shRNA or RanBPM shRNA were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 5 min and then assayed for ROS generation. D, PHK cells expressing either control shRNA or RanBPM shRNA were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 5 min and then assayed for ROS generation. E, 253J-BV bladder cancer cells were stained with primary antibodies against human BLT2 (Cayman Chemical) and RanBPM (Santa Cruz Biotechnology), and analyzed by FACS analysis. The results shown are representative of three independent experiments with similar results. F, dose dependence of LTB4-induced chemotactic motility was determined in 253J-BV bladder cancer cells, as described above. G, 253J-BV bladder cancer cells were transfected with either control shRNA (−) or RanBPM shRNA (+), and the amount of RanBPM mRNA was analyzed by semiquantitative RT-PCR. H, 253J-BV cells were transiently transfected with control shRNA or RanBPM shRNA. At 48 h after transfection, the transfected cells were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 3 h and then assayed for chemotaxis as described above. The migratory activity was expressed as a percentage of the control. I, 253J-BV cells expressing either control shRNA or RanBPM shRNA were exposed to 300 nm LTB4 (left panel) or 100 nm 12HHT (right panel) for 5 min and then assayed for ROS generation. The data are presented as the means ± S.D. of three independent experiments.