Fig. 4

FOXD1-RalA-ERK1/2 signaling cascade mediates CTC formation and cell migration. A BC cells (MDA231 and MDA468) with FOXD1 knockdown alone or in combination with RalA were subjected to transwell migration assays. MCF-7 with FOXD1 overexpression alone or in combination with si-RalA were subjected to transwell migration assays. Scale bar, 250 μm. Error bars, SEM. n = 3. ***p<0.001 by Student’s t-test. B, C EMT relative markers (E-cadherin, N-cadherin, Vimentin, Snail, β-catenin) and MAPK pathway relative proteins expression levels were measured via western blotting. GAPDH detection was shown as a control. D The mRNA levels of RalA in the clinical BC primary tissues with CTC ≥ 5 or CTC<5 were analyzed by qRT-PCR. Error bars, SEM. CTC ≥ 5, n = 40; CTC<5, n = 40. *p < 0.05 by Student’s t-test. E RalA mRNA levels in clinical BC primary tissues of early-stage BC patients were analyzed by qRT-PCR. Error bars, SEM. CTC ≥ 5, n = 27; CTC<5, n = 31. *p<0.05 by Mann-Whitney test. F Western blotting analyses of RalA expression in clinical BC primary tumors with CTC ≥ 5 or CTC<5. GAPDH detection is shown as a control. G IHC analyzed RalA, p-ERK1/2 and p-MEK1/2 expression in clinical BC primary tumors with CTC ≥ 5 or CTC<5. Scale bar, 50 μm. Error bars, SD. n = 5. *p < 0.05, **p < 0.01 by Student’s t-test. H Overall survival analysis of BC patient with high RalA expression, by TCGA database