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. 2016 Apr 13;7:11245. doi: 10.1038/ncomms11245

Figure 4. Reelin expression is regulated by RAS-PI3-Kinase pathway and is involved in migration.

Figure 4

(a) Graphical display of statistical analysis performed to identify genes undergoing significant changes of expression in Pik3caRBD cells as compared with wild-type counterparts. Statistically significant probes are shown in light blue (0.05 fdr). Reln reports are shown in red. (b) RNA from steady-state (st-st), serum-starved or EGF-treated (20 ng ml−1) fibroblasts was obtained and Reln mRNA levels measured by quantitative PCR (qPCR). Actin expression was used as an internal control for normalization. Independent triplicates were used for each time point. Error bars indicate mean±s.e.m. (c) Levels of Reln expression were measured by qPCR in Pik3caRBD cells containing an inducible active Akt construct (ER-MyrAKT) in the presence or absence of 4-hydroxytamoxifen (TX; 100 nM). Actin expression was used as an internal control for normalization. Independent triplicates were used for each time point. Error bars indicate mean±s.e.m. (d) Random migration after Reln silencing in Pik3caRBD cells. Migration was analysed by time-lapse video microscopy and cell tracing in the presence or absence of EGF (20 ng ml−1). Box and whisker plot was generated as indicated for Fig. 1a.ANOVA statistical analysis was performed with starved cells used as reference for each condition (NS, not significant; **P<0.01; ***P<0.001). (e) Recombinant Reln (1 μg ml−1) was added to the media of Pik3caWT and Pik3caRBD cells and random migration was then analysed in the same way as described for previous panel. (f) Reln expression levels in Pik3caWT, Pik3caRBD and Pik3caRBD RacV12 cells. Actin expression was used as an internal control for normalization. Independent triplicates were used for each time point. (g) Rac pull-down analysis in the presence of recombinant Reln. Recombinant Reln was added to the media of Pik3caWT MEFs and then Rac-GTP activity was determined in pull-down assays using GST-PBD of PAK1 (GST-PBD). Both total lysates and proteins bound to GST-PBD were analysed by western blot to detect Rac. (h) Representative graph showing Reln mRNA half-life. Pik3caWT, Pik3caRBD, Pik3caRBD p110a WT and Pik3caRBD ER-myrAkt (+TX) cells were treated with actinomycin D and levels of Reln mRNA were determined by qPCR at the displayed time points. Actin expression was used as an internal control for normalization. Independent triplicates were used for each time point.