Fig. 3

FOXD1 activates the ERK1/2 signal by regulating RalA in BC cells. A KEGG analysis of the FOXD1 binding sites detected by ChIP-seq (up panel) and CUT&Tag-seq (down panel). B Heat map showing RNA differential expression genes (by RNA-seq) between control and FOXD1 knockdown cells. The genes were identified with significant expression change and fold change > 1.5. C Venn diagram of FOXD1 target selection. For both ChIP-seq and CUT&Tag-seq analysis, the genes with FOXD1 promotor binding in − 1000 ~ 0 bp to TSS were selected. D Representative images of ChIP-seq and CUT&Tag-seq results with FOXD1 antibody on RalA genomic region. RalA-peak represents the DNA region that was cloned and used in (E). E Dual luciferase reporter assay with FOXD1 overexpression. Schematic view of FOXD1 binding region on RalA TSS. Truncations were ligated to pGL4.10[Luc2] vector. Dual luciferase assay was performed with co-transfection of pEXP-FOXD1 and full-length or truncated reporter vectors. Error bars, SD. n = 3. *p < 0.05, **p < 0.01 by Student’s t-test. ns. = not significant. F Dual luciferase assays of MDA231 cells or MCF-7 co-transfected with a RalA-fragment 3 (F3) promoter-reporter plasmid and overexpression vectors for FOXD1 (gradient dilution). Error bars, SD. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001 by Student’s t-test. G Dual luciferase assays demonstrated the expression of RalA-F3 (WT or mutant form) by MDA231 or MCF-7 cells transfected with pEXP-FOXD1 or with control. Error bars, SD. n = 3. *p < 0.05, **p < 0.01 by Student’s t-test. ns. =not significant. H ChIP-qPCR assay using FOXD1 antibody or control IgG in MDA231 shows the binding of FOXD1 on the RalA promoter. Error bars, SD. n = 3. ****p < 0.0001 by Student’s t-test. I Association of RalA expression with FOXD1 expression at clinical BC primary tumors. n = 80. J Western blot analyses of RalA and MAPK pathway relative protein expression in CtrL and FOXD1 knockdown cells (MDA231 and MDA468), Ctrl and FOXD1 overexpression cells (MCF-7). GAPDH served as a loading control. K IHC analyzed the expression of FOXD1, RalA, p-ERK1/2, and Vimentin in xenograft tumors from mouse models. Scale bar, 50 μm. Error bars, SD. n = 3. *p<0.5, **p<0.01 by Student’s t-test. L RalA activation assays in the indicated cells. Bar graphs showing quantification of GTP-RalA/Total-RalA ratio. Error bars, SD. n = 3. *p< 0.5, **p< 0.01 and ***p< 0.001 by Student’s t-test