FIGURE 1.

LPA₂-mediated protection from adriamycin-induced apoptosis is regulated by proteins interacting with its C-terminal tail. A, knockdown of TRIP6 expression reduces LPA₂-mediated ERK and AKT activation and protection from adriamycin-induced caspase-3/7 activation. LPA_½ DKO MEFs stably expressing FLAG-LPA₂ (DKO-LPA₂) were transduced with the lentivirus harboring a mouse TRIP6 siRNA (siTRIP6-1) or a scrambled control siRNA (siScramble). After starvation for 4 h, cells were treated with 2 μm LPA for 10 or 20 min, and immunoblotting was performed to determine the levels of activated phospho-ERK or phospho-AKT, respectively (right panel). The same blot was reprobed with an antibody specific to ERK, AKT, or TRIP6, respectively. The intensity of each protein was quantified by NIH IMAGE J software and the relative expression of phospho-ERK or phospho-AKT was normalized by the levels of total ERK or AKT. The same cells were starved in 0.1% fatty acid-free BSA-containing DMEM with or without 10 μm LPA for 1 h followed by the addition of 1.7 μm adriamcyin for 9 h, and caspase-3/7 activity was determined by cleavage of the luminogenic substrate containing the DEVD sequence and was normalized by protein concentrations (left panel). Data shown are the mean ± S.E. of five independent experiments. Statistic significance (p < 0.05) was determined by Student's t test. B, knockdown of NHERF2 expression attenuates LPA₂-mediated ERK activation and protection from adriamycin-induced apoptosis. DKO-LPA₂ MEFs were transduced with the lentivirus harboring a mouse NHERF2 siRNA (siNHERF2-3) or a luciferase control siRNA (siLuc). The starved cells were treated with LPA for 10 min and the levels of activated ERK, total ERK, NHERF2, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the whole cell lysates were determined by immunoblotting. Adriamycin-induced caspase-3/7 activation were assayed as described in A. Data shown are the mean ± S.E. of three independent experiments. C, inhibition of Siva-1 expression enhances LPA₂-mediated protection from adriamycin-induced apoptosis. DKO-LPA₂ MEFs were transduced with the lentivirus harboring a Siva-1 siRNA or a scrambled siRNA. The effect of LPA on adriamycin-induced caspase-3/7 activation was determined as described in A. Data shown are the mean ± S.E. of three independent experiments. Immunoblotting was performed to detect the expression of Siva-1 and control GAPDH in the whole cell lysates.