Additional file 2 of RNA-binding protein MSI2 isoforms expression and regulation in progression of triple-negative breast cancer
Creators: name: Li, Ming
name: Li, An-qi
name: Zhou, Shu-ling
name: Lv, Hong
name: Wei, Ping
name: Yang, Wen-tao
Publisher: name: figshare
Year: 2020
Subjects: subject: Biophysics
subject: Biochemistry
subject: Space Science
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Immunology
subject: FOS: Clinical medicine
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Additional file 2: Figure S1. MSI2 expression in breast cancer. a The CRN web portal (http://syslab4.nchu.edu.tw/) was used to interrogate GSE58135 datasets.MSI2–001(MSI2a) demonstrated downregulated in TNBC primary tumors compared to that in uninvolved breast tissue samples that were adjacent to TNBC primary tumors. b TCGA dataset. Levels of MSI2 mRNA across different breast cancer types in 737 breast tumors from the TCGA breast RNA-seq cohort(tcga-data.nci.nih.gov). c Transcripts abundance of MSI2 isoforms a-d between 25 TNBC tissues and 5 adjacent normal tissues (ANTs) of the RNAseq data. d qRT-PCR. MSI2a and MSI2b mRNA expression levels in 27 pairs of TNBC and normal tissues. e Kaplan–Meier survival curves comparing overall survival and disease-free survival of breast cancer patients with low vs. high MSI2a mRNA level. f qRT-PCR. MSI2b mRNA expression levels across different breast cancer types. g Receiver operating characteristic (ROC) curves of disease-free survival and overall survival showing the area under the ROC (AUROC) of MSI2b expression. h Kaplan–Meier survival curves comparing overall survival and disease-free survival of breast cancer patients with low vs. high MSI2a protein level. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S2. MSI2b knockdown showed no significant effect on TNBC cell growth and migration in vitro. a qRT-PCR assays. The efficiency of MSI2b knockdown in HS-578T cells. b CCK-8 assay for cell viability. Cell proliferation was assessed after MSI2b knockdown inHs-578T cells. c Wound healing and transwell migration assay. Images showing the migration capacity in MSI2b-knocked down Hs-578T cells. d Western blot assays of SLUG, ZO-1, vimentin, E-cadherin, N-cadherin, β-catenin, p-ERK1/2, and ERK1/2 expression in MDA-MB-468 and BT20 cells. e Tumorigenicity assay of MSI2a-overexpressing MDA-MB-231 cells (2 × 106) after subcutaneous injection in the flanks of nude mice (n = 5). The mice and tumors are shown. The quantification of tumor volume is shown below. Scale bar, 1 cm. Figure S3. Association of TP53INP1 expression with MSI2 expression. a Pathway analysis of enriched genes (log2 (fold change) below - 0.58 in RIP-seq) in HS-578T cells. The top three most significant pathways with enrichment scores are shown. b Expression of MSI2a and TP53INP1 mRNA was positively correlated in breast cancer. MSI2 and TP53INP1 mRNA expression levels were correlated using two published gene expression databases comprising 1215 breast tumors (c) from the TCGA breast RNA-seq cohort (tcga-data.nci.nih.gov) and 59 breast cell lines (d) from the Cancer Cell Line Encyclopedia breast cancer lines RNA-seq cohort. Statistical significance was determined using Pearson’s correlation. e Association of MSI2 and TP53INP1 mRNA expression in 166 TNBC tissues from the GSE76250 dataset. Statistical significance was determined using Pearson’s correlation.Figure S4. TP53INP1 inhibition of TNBC cell migration in vitro. a Transwell migration assay. Representative images and quantification showing the migration ability of MDA-MB-231 cells after TP53INP1 overexpression and of Hs-578 t cells after TP53INP1 knockdown. Scale bar, 100 μm. b Wound-healing assays. Representative images and quantification of the wound-healing assay results showing the wound-healing ability of MDA-MB-231 cells after TP53INP1 overexpression and of Hs-578T cells after TP53INP1 knockdown. Scale bar, 200 μm. c Western blot. TP53INP1-overexpressed MDA-MB-231 cells and TP53INP1-knocked down Hs-578T cells were analyzed by using western blot with the anti-TP53INP1, anti-P73, anti-DUSP10, anti-ERK, and anti-p-ERK antibodies, respectively. Vinculin was used as a control. *p &lt; 0.05, **p &lt; 0.01, ***p &lt; 0.001. Figure S5. U0126 impairment of TNBC cell growth and migration induced by MSI2 silencing. a Wound-healing assay. Cells were treated with 10 μM U0126 or DMSO for 24 h and subjected to the wound-healing assay. U0126 attenuated the effect of MSI2a silencing on the scratch-closure rate of Hs-578T and BT20 cells. b Transwell tumor cell migration assay. Cells were treated with 10 μM U0126 or DMSO for 24 h and subjected to a Transwell assay. U0126 attenuated the effect of MSI2a silencing on the migration abilities of Hs-578T and BT20 cells. *p &lt; 0.05. Figure S6. Association of TP53INP1 downregulation in human TNBC tissues with a poor TNBC prognosis. a Kaplan–Meier survival curves comparing overall survival and disease-free survival in TNBC patients with low vs. high TP53INP1 protein levels. b TCGA dataset. The level of TP53INP1 mRNA was analyzed using qRT-PCR in 27 pairs of TNBC tissues. c The levels of TP53INP1 mRNA across different breast cancer types were analyzed in 737 breast tumors from the TCGA breast RNA-seq cohort (tcga-data.nci.nih.gov) using qRT-PCR. d Kaplan–Meier survival curves comparing overall survival and disease-free survival in breast cancer patients with low vs. high TP53INP1 mRNA levels. *p &lt; 0.05, **p &lt; 0.01, and ***p &lt; 0.001.