Fig. 7
From: LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration
LPA1 antagonists increase CXCR4 signaling and function. A HEK293A cells were transfected with CXCR4 and LPA1 and pretreated with AM966 at the indicated concentrations for 30 min. CXCL12 (10 nM)-induced Gαi1 activation was measured using BRET between Gαi1-Rluc8 and Gγ9-GFP2. B HEK293A cells were transfected with CXCR4 alone or together with LPA1 and pretreated with LPA1 antagonists AM966, AM095, Ro6842262, or BMS986020 at 10 μM for 30 min. Cells were treated with forskolin (3 μM) and CXCL12 (30 nM), and the effects of LPA1 antagonists on CXCR4-mediated cAMP responses were measured using the GloSensor cAMP assay. C MDA-MB-231 cells were pretreated with LPA1 antagonists at 10 μM for 30 min, and CXCL12 (30 nM)-induced inhibition of forskolin (3 μM)-induced cAMP accumulation was measured using the GloSensor cAMP assay. D, E The parental MDA-MB-231 (D) or LPAR1 knockout MDA-MB-231 cells (E) were pretreated with LPA1 antagonists at 10 μM for 30 min, and CXCL12 (10 nM)-induced migration was examined using a transwell migration assay. F MDA-MB-231 cells were pretreated with burixafor (1 μM) and/or AM966 (10 μM) and assessed for cell migration toward CXCL12 (10 nM) and/or alkyl-OMPT (1 μM) using a transwell migration assay. Migrated cells were counted from randomly selected images of 10 fields. A-C Data represent the mean ± SEM of n = 3 to 4 independent experiments performed in triplicate. D-F Data represent the mean ± SD of n = 3 independent experiments performed in triplicate. B Statistical significance was tested using two-way ANOVA followed by Bonferroni’s multiple comparison test (DMSO vs. LPA1 antagonists). *P < 0.05; **P < 0.01. C, D, and F Statistical significance was tested using unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001; ns, not significant