Additional file 1 of RGCC-mediated PLK1 activity drives breast cancer lung metastasis by phosphorylating AMPKα2 to activate oxidative phosphorylation and fatty acid oxidation
Creators: name: Cheng, Shaojie
name: Wan, Xueying
name: Yang, Liping
name: Qin, Yilu
name: Chen, Shanchun
name: Liu, Yongcan
name: Sun, Yan
name: Qiu, Yuxiang
name: Huang, Luyi
name: Qin, Qizhong
name: Cui, Xiaojiang
name: Wu, Mingjun
name: Liu, Manran
Publisher: name: figshare
Year: 2023
Subjects: subject: Biochemistry
subject: Medicine
subject: Microbiology
subject: FOS: Biological sciences
subject: Cell Biology
subject: Genetics
subject: Molecular Biology
subject: Physiology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer
subject: Computational Biology

description: Additional file 1: Supplemental Figure 1. A. Representative bio-luminescence images of lung-tropic metastasis in mice injected with 4T1 cells into fat pad for 30 days. B. Heatmap showing 20 most upregulated and downregulated genes between 4T1/LM3 and 4T1 parental cells. C. Western blot analyses of RGCC expression in the indicated parental and derivative cells (231: MDA-MB-231). Supplemental Figure 2. A-C. Western blotting to assess RGCC knockdown efficiency in 4T1/LM3 and MDA-MB-231/LM3 cells (A), and RGCC proteins in the indicated TNBC cells (B), or in ectopic RGCC overexpressing MDA-MB-468 and HCC1806 cells (C). D-G. Transwell assay was used to evaluate the invasion ability of 4T1/LM3 and MDA-MB-231/LM3 cells transfected with shNC or shRGCC (D, E), and ectopic RGCC overexpressing MDA-MB-468 and HCC1806 cells (F, G) (Columns are the average of three independent experiments;**P &lt; 0.01; 231: MDA-MB-231; MDA-468: MDA-MB-468). Supplemental Figure 3. A. A Venn diagram depicting the overlap transcriptional factors (TFs) between the altered TFs in MDA-MB-231/LM3 and the predicted TFs to potentially regulate RGCC expression by Promo Alggen database and JASPAR database. B. CEBPA mRNA levels in parental and derivative cells were determined by qRT-PCR. C. At the optimal cutoff prognostic index of 0.636, a total of 711 patient samples were divided into high risk (n=589) or low risk (n=192) group, according to the methylation level in CpG islands of CEBPA promoter. Supplemental Figure 4. A. Co-IP assays to confirm the direct interaction between RGCC and PLK1 in MDA-MB-231/LM3 cells using antibodies anti-RGCC or anti-PLK1, respectively. B. GST pulldown assay with purified HA-PLK1 and GST-RGCC to test the direct interaction between RGCC and PLK1. C-D. Molecular dynamics simulation assay to detect the stabilized binding between RGCC and PLK1 kinase domain (KD) (C), or between RGCC and PLK1 Polo-box domain (PBD) (D). The binding conformation of PLK1 was stabilized after a 22 ns simulation in (C) or a 20 ns simulation in (D). E. The binding mode of RGCC and PLK1 Polo-box domain (PBD). F-G. Western blot analysis of p-PLK1 (T210) protein levels in RGCC knocked down LM3 cells or RGCC overexpressing TNBC cells (F), and in various metastatic 4T1, MDA-MB-231 derived from brain, liver, lung and control parental cells (G). H. A proposed model explaining how RGCC promotes the intramolecular interaction between the PLK1 PBD and the kinase domain resulting in PLK1 kinase activity. I. WT or mutant (T210D, T210A) PLK1 was stably transfected into shNC or shRGCC with endogenous PLK1 knockout tumor cells, western blotting was used to determine p-AMPKα2 levels. J. Western blotting to check p-PLK1 and p-AMPKα2 levels in RGCC-overexpression cells by administration of Volasertib (a PLK1 kinase inhibitor). Supplemental Figure 5. A. Principal component analysis (PCA) of metabolites shows the percentage of variation for the first and second principal components. B. Heatmap analysis of significantly changed metabolites between RGCC-engineered MDA-MB-231/LM3 cells (sh RGCC vs sh NC) (n=6). C. The major signaling related with RGCC revealed by SangerBox analysis using microarray datasets GSE157684. D. Gene set enrichment analysis (GSEA) displays the relationship between RGCC expression and fatty acid oxidation and oxidative phosphorylation. E. Basal respiration of the indicated LM3 cells was detected using a Seahorse XF24 Extracellular Flux Analyzer. F. NADPH/NADP+ ratio of the indicated LM3 cells was detected using microplate reader. G. qRT-PCR was used to detect the levels of genes associated oxidative phosphorylation and fatty acid oxidation in RGCC knockdown MDA-MB-231/LM3 and RGCC overexpressing MDA-MB-468 cells. H. Cell viability was evaluated by CCK8 assay. (Data were presented as mean ± SD; **P &lt; 0.01,***P &lt; 0.001).