LPA mediates cell proliferation through STAT-3 activation.

<p>Subconfluent monolayers of HCT-116 cells were FBS depleted overnight and treated with LPA at the indicated times. a) Total lysates were obtained and prepared for western blotting using a specific antibody against the phosphorylated form of STAT-3 at tyrosine 705 (p-STAT3). The band images were quantified by optical density, and the score was calculated using the ratio between p-STAT3, STAT3 and α-tubulin. LPA increased STAT-3 phosphorylation after 5–15 min of treatment. b) Immunofluorescence of HCT-116 cells corroborated the increase in pSTAT-3 after LPA treatment (green, arrows) and displayed its nuclear location (nucleus, blue). Scale bar, 20 μm. c) The relative cell number of LPA-treated HCT-116 cells was evaluated using crystal violet staining. STAT-3 inhibition using STA 21 prevented the increase in cell numbers after 48 h of LPA treatment. Statistical analyses were performed using <i>two-way</i> ANOVA with <i>post-hoc</i> Bonferroni test (*** p<0.001, vs control; ### p<0.001, vs. LPA). d) Cell cycle analyses through PI staining indicated that STAT-3 inhibition reduced the number of cells at the S-G2/M phase after LPA treatment. Statistical analyses were performed using <i>one-way</i> ANOVA (*** p<0.001, vs. control; ### p<0.001, vs. LPA). Data are presented as mean ± SEM.