Fig. 5
From: LPA1-mediated inhibition of CXCR4 attenuates CXCL12-induced signaling and cell migration
Enhanced CXCR4 responses in MDA-MB-231 cells targeted with sgLPAR1 using the CRISPR-Cas9 gene editing system. A, B MDA-MB-231 cells were transduced with sgScramble, sgLPAR1 #1, or sgLPAR1 #2, and transduced cells were selected with puromycin for two weeks. To evaluate LPA1 deficiency, LPA (1 μM, A) or alkyl-OMPT (1 μM, B)-induced calcium flux was measured in cells targeted with sgScramble or sgLPAR1. C LPA1-mediated migration was measured using a transwell migration assay with alkyl-OMPT (1 μM) in cells treated with sgScramble or sgLPAR1. D Intracellular calcium flux induced by CXCL12 (10 nM) was measured in cells targeted with sgScramble or sgLPAR1 (Left). The area under curve was measured for CXCL12-induced intracellular calcium levels (Right). E CXCL12-induced migration was evaluated in in cells targeted with sgScramble or sgLPAR1. F Cell surface expression of CXCR4 was measured by flow cytometry with anti-CXCR4 (4G10) primary antibody and anti-mouse antibody conjugated with APC. C, D Statistical significance was tested using unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01. C, E Migrated cells were counted from randomly selected images of 5 fields. Data represent the mean ± SD of n = 3 to 4 independent experiments performed in triplicate. E Statistical significance was tested using two-way ANOVA followed by Bonferroni’s multiple comparison test comparing “sgScramble” to “sgLPAR1 #1” (**P < 0.01; ***P < 0.001) and “sgScramble” to “sgLPAR1 #2” (#P < 0.05; ###P < 0.001; ####P < 0.0001)