Additional file 1 of FOXD1-dependent RalA-ANXA2-Src complex promotes CTC formation in breast cancer
Creators: name: Long, Yufei
name: Chong, Tuotuo
name: Lyu, Xiaoming
name: Chen, Lujia
name: Luo, Xiaomin
name: Faleti, Oluwasijibomi Damola
name: Deng, Simin
name: Wang, Fei
name: He, Mingliang
name: Qian, Zhipeng
name: Zhao, Hongli
name: Zhou, Wenyan
name: Guo, Xia
name: Chen, Ceshi
name: Li, Xin
Publisher: name: figshare
Year: 2022
Subjects: subject: Biochemistry
subject: Space Science
subject: Medicine
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Pharmacology
subject: Biotechnology
subject: Astronomical and Space Sciences not elsewhere classified
subject: Biological Sciences not elsewhere classified
subject: Information Systems not elsewhere classified
subject: Mathematical Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 1: Supplementary Fig. 1. (A) RT-qPCR analyses of FOXD1 mRNA in the indicated breast tumor cell lines and MCF-10A. Error bars, SEM. n = 3. (B) Western blotting analyses of FOXD1 expression in the indicated breast tumor cell lines and MCF-10A. (C)RT-qPCR analysis of FOXD1 expression in control and sh-FOXD1 cells (MDA231 and MDA468), or control and FOXD1 overexpression cells (MCF-7). Error bars, SEM. n = 3. **p &lt; 0.01, ***p&lt;0.001 by Student’s t test. (D) FOXD1 protein expression level in control and FOXD1 knockdown cells (MDA231 and MDA468), or control and FOXD1 overexpression cells (MCF-7) by immunoblotting. Supplementary Fig. 2. (A) Cell migration capacity of FOXD1 knockdown BC cells (MDA231 and MDA468), FOXD1 overexpression BC cell (MCF7), and their control cells was determined by the wound healing assays. Scale bar, 1000 μm. Error bars, SD. n = 3. **p&lt;0.01 by Student’s t-test. (B) Immunofluorescence images for E-cadherin and Vimentin expression in CtrL and FOXD1 knockdown BC cells (MDA231 and MDA468). Scale bar, 50 μm. (C) The morphology analysis of control and FOXD1 overexpression MCF-7 cells. Scale bar, 100 μm. (D) The cell proliferation in these transfected cells was determined using CCK-8 assays. Error bars, SD. n = 3. **p&lt;0.01, ***p&lt;0.001, ****p&lt;0.0001 by Student’s t-test. (E) Expression of FOXD1 across TCGA cancers (with tumor and normal samples). Supplementary Fig. 3. (A) Immunoblotting of FOXD1 after MDA231 cells transfected with pEXP-FOXD1 or with control. (B) Immunoblotting of RalA after MDA231 cells transfected with pEXP-RalA or with control. (C) Western blotting assay was used to examine the protein levels of RalA in MCF-7 cell transfected with the control or RalA-siRNAs (si-1, si-2, and si-3). (D) Western blotting assay was used to examine protein levels of FOXD1 in MDA231 cell stably overexpressed the vector or FOXD1. (E) Visualization of genotyping PCR product for each MDA231 single clonal cell line with indicated genotype. (F) Immunoblotting of RalA in MDA231 single clonal cell lines with indicated genotype. (G) WT and RalA KO MDA-MB-231 cells were subjected to transwell migration assays. Scale bar, 250 μm. Error bars, SD. n = 3. ***p&lt;0.001 by Student’s t-test. (H) The cell proliferation in MDA231 with WT, RalA-KO#6, or RalA-KO#26 was investigated using CCK-8 assays. Error bars, SD. n = 3. ****p&lt;0.0001 by Student’s t-test. ns. = not significant. (I) WT and RalA KO MDA-MB-231 cells were subjected to immunoblotting assays. (J) The cell proliferation in these transfected cells was determined using CCK-8 assays. Error bars, SD. n = 3. *p&lt;0.05, **p&lt;0.01, ****p&lt;0.0001 by Student’s t-test. Supplementary Fig. 4. (A) FOXD1 overexpression MDA231 in combination with SCH772984 (0, 1.25, 2.5 and 5 μM, 12 h) were subjected to western blotting assays. (B) FOXD1 overexpression MDA231 in combination with SCH772984 (0, 1.25, 2.5 and 5 μM, 12 h) were subjected to transwell migration assays. Scale bar, 250 μm. Error bars, SD. n = 3. ***p&lt;0.001 by Student’s t test. (C) Western blotting analyses of E-cadherin, N-cadherin, Vimentin, and GAPDH expression in MDA231 with FOXD1 overexpression alone or in combination with SCH772984 (5 μM, 12 h). (D) MDA231 cells with FOXD1 overexpression were treated with SCH772984 (0, 0.3125, 0.625, 1.25, 2.5 and 5 μM) for 12 hours, and the OD450 was measured by CCK-8 assay. Error bars, SD. n = 3. ****p&lt;0.0001 by one-way ANOVA. (E) The predicted model of the RalA-ANXA2-Src ternary interaction. (F) RalA-KO MDA231 cells transfected with constituently inactivated (G26A) or activated (G23V) RalA mutant respectively, were subjected to western blotting assays. For RalA-GTP assays, cells were harvested and subject to RalBP1-RBD pulldown assays to determine RalA-GTP level. Pulldowns were analyzed by immunoblotting with anti-RalA.