Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Representative images of EdU incorporation in MDA-MB-231 cells treated with EVs from DMSO or REV treated cells and quantification for three biological replicates. Statistical analysis was performed using two-sided T-tests (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001).) ( B ) Cellular component analysis of differentially expressed proteins using Funrich 3.1.4. software. ( C, D ) Migration and invasion of BT549 ( C ) and MDA-MB-231 ( D ) cells treated with DMSO (CIN LOW ) or REV (CIN HIGH ) using trans-well assays. Statistical significance was determined using two-sided T-tests (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Experiments were performed as biological triplicates. Scale bar: 100 µm. ( E ) Gene Ontology (GO) analysis on the proteins enriched in CIN+ EVs compared to CIN-EVs. ( F ) EFEMP1 levels in BT549 cell lysates and BT549 EVs treated with DMSO (CIN LOW ) or REV (CIN HIGH ) detected by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Software, Migration, Western Blot

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) qPCR quantification of EFEMP1 knockdown efficacy in BT549 cells: Statistical tests were done using a two-sided T-test (n=3; ***, p < 0.001). ( B ) qPCR quantification of EFEMP1 overexpression in BT549 cells. Statistical significance was assessed by a two-sided T-test (n=3; ****, p < 0.0001). ( C ) qPCR quantification of EFEMP1 knockdown efficiency in MDA-MB-231 cells. Statistical analysis using a two-sided T-test (n=3; ***, p < 0.001). ( D ) qPCR quantification of EFEMP1 overexpression. Statistical significance assessed by a two-sided T-test (n=3; ****, p < 0.0001). ( E-F ) Representative images ( E ) and quantification ( F ) of cell migration and invasion of BT549 cells with EFEMP1 overexpression assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( G, H ) Representative images ( G ) and quantification ( H ) of cell migration and invasion of MDA-MB-231 cells with EFEMP1 overexpression assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( I ) Representative images (left panel) and quantification (right panel) of scratch assays to quantify migration of BT549 cells with EFEMP1 knockdown. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ( J ) Representative images (left panel) and quantification (right panel) of scratch assays to quantify migration of BT549 cells with EFEMP1 overexpression. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. ( K-L ) Representative images ( K ) and quantification ( L ) of cell migration and invasion of BT549 cells with EFEMP1 knockdown assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( M, N ) Representative images ( M ) and quantification ( N ) of cell migration and invasion of MDA-MB-231 cells with EFEMP1 knockdown (two shRNAs) assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Knockdown, Over Expression, Migration

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 KD EVs assessed by trans-well assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( B ) Representative images (left panel) and quantification (right panel) of cell migration and invasion of MDA-MB-231 cells treated with EFEMP1 OEX EVs assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( C ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 scramble and EFEMP1 KD cells determined by Western blot. ( D ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 control and EFEMP1 OEX cells determined by Western blot. ( E ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from DMSO- or REV-treated BT549 scramble and EFEMP1 KD cells determined by Western blot. ( F ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 scramble and EFEMP1 KD cells determined by Western blot. ( G ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from MDA-MB-231 control and EFEMP1 OEX cells determined by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Migration, Western Blot, Control

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Schematic outline of the treatment of BT549 cells with EVs and experimental setup for downstream analyses. ( B, C ) Representative images ( B ) and quantification ( C ) of BT549 migration and invasion Transwell experiments following EFEMP1 knockdown (EFEMP1 KD ) compared to control (scramble) EVs. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( D, E ) Representative images ( D ) and quantification ( E ) of BT549 migration and invasion Transwell experiments following EFEMP1 overexpression (EFEMP1 OEX ) compared to control EVs. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. (F, G) Representative images ( F ) and quantification ( G ) of migration and invasion of control and CIN HIGH (REV-treated) BT549 cells using Transwell assays under Scramble control and EFEMP1 KD conditions. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Migration, Knockdown, Control, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Venn diagram of RNA sequencing analysis illustrating the downregulation of 29 genes associated with migration and invasion in BT549 cells treated with EFEMP1 KD EVs. The gene sets for migration (166 genes) and invasion (97 genes) were derived from the Cancer Single-cell State Atlas, underscoring the significant role of EFEMP1 in regulating cellular migration and invasion patterns. ( http://biocc.hrbmu.edu.cn/CancerSEA/goDownload ). ( B ) KEGG pathway analysis of the 290 deregulated genes in EFEMP KD cells. ( C ) Molecular Functions (MF) pathway analysis of the 290 deregulated genes in EFEMP KD cells. ( D) Schematic outline of the cell adhesion assay. ( E ) Quantification of cell adhesion of BT549 cells treated with EVs-derived from EFEMP1 KD cells. ( F ) Quantification of cell adhesion of BT549 cells treated with EVs-derived from EFEMP1 OEX cells.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: RNA Sequencing Assay, Migration, Derivative Assay, Cell Adhesion Assay

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Correlation analysis of EFEMP1 and STAT1 mRNA expression using DepMap data. ( B ) Correlation analysis of EFEMP1 and STAT1 mRNA expression in DepMap-included breast cancer cell lines. ( C ) qPCR quantification of IL6, IL8, CXCL1, CXCL10, EFEMP1, STAT1, STAT3, CD63 and CYLD in BT549 WT and STAT1 KO cells. Statistical analysis was done using two-sided T-test (N = 3; *p< 0.05, **, p< 0.01, ***p < 0.001, ****p < 0.0001). ( D, E ) Representative images ( D ) and quantification ( E ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from WT of STAT1 KO BT549 cells, treated with DMSO or REV, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm. ( F, G ) Representative images ( F ) and quantification ( G ) of cell migration and invasion of BT549 cells co-cultured with EVs isolated from STAT1 KO BT549 cells with or without EFEMP1 overexpression, assessed by transwell assays. Statistical significance was determined by a two-sided T-test (n= 3 biological replicates), *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar: 100 µm.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Expressing, Migration, Cell Culture, Isolation, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from BT549 and STAT KO BT549 cells treated with DMSO or REV determined by Western blot. ( B ) CD63, CD81, EFEMP1, Calnexin and beta-Actin protein levels in whole cell lysates and EVs from STAT KO BT549 cells treated with or without EFEMP1 overexpression determined by Western blot.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Western Blot, Over Expression

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A ) Experimental setup for investigating the invasive behaviour of EV-treated MDA-MB-231 cells in zebrafish embryos. Xenografted embryos were monitored until 1-day post-transplantation (dpt). ( B ) Representative images of embryos xenografted with MDA-MB-231 cells treated with control (left column), EFEMP1 KD EVs (right column), or EFEMP1 OEX EVs (bottom rows) at 1 dpt. Arrowheads point to migrated MDA-MB-231 cells. Scale bar: 200 µm. ( C ) Quantification of MDA-MB-231 cell migration treated with EVs as indicated in large number of zebrafish embryos. Significance was tested using a chi-square test (****, p=3.4084×10 -30 ). ( D ) Quantification of EV-treated MDA-MB-231 cell migration toward the zebrafish embryo’s tail region. Cells were treated with control or EFEMP1 KD EVs as indicated. Significance between groups (control or EFEMP KD EVs) was tested using a chi-square test and migration patterns (categorized as 0, 1-4, and >4 cells migrated cells). p=2.0401×10 -25 . ( E ) Quantification of EV-treated MDA-MB-231 cell migration toward the zebrafish embryo’s tail region. Cells were treated with control or EFEMP1 OEX EVs as indicated. Significance between groups (control or EFEMP KD EVs) was tested using a chi-square test and migration patterns (categorized as 0, 1-4, and >4 cells migrated cells). p=0.0382.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Transplantation Assay, Control, Migration

Journal: bioRxiv

Article Title: Chromosomal instability promotes cell migration and invasion via EFEMP1 secretion into extracellular vesicles

doi: 10.1101/2024.10.21.619397

Figure Lengend Snippet: ( A-B ) Scatter plots showing the correlation between EFEMP1 expression and aneuploidy score in DepMap included cancer cell lines ( A ) or DepMap included breast cancer cell lines ( B ). ( C ) Kaplan-Meier survival curves for TGCA-included breast cancer patients, showing overall survival for grade 3 (blue) ( n = 952), grade 2 (yellow) ( n=771 ) and grade 1 (tangerine) ( n=169 ) breast cancer. ( D-F ) Kaplan-Meier survival curves for TGCA-included breast cancer patients stratified for low or high EFEMP1 expression per grade (grade I, D ; grade II, E , grade III, F ). Significant differences between EFEMP1 expression groups were tested using a Log-rank Test.

Article Snippet: The primary antibodies for Western blots used in this study were EFEMP1 (1:300, Novus Biologicals, NBP1-77040), β-Actin (1:2000, Cell Signaling Technology, 4970 or 3700S), STAT1 (1:1000, Cell Signaling Technology, 9172S), CD63 (1:1000, Novus Biologicals, NBP2-42225), CD81 (1:1000, Novus Biologicals, NB100-65805SS), and Calnexin (1:1000, Novus Biologicals, NB100-1965SS).

Techniques: Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

doi: 10.1016/j.mcpro.2022.100223

Figure Lengend Snippet: ADAMTS7 candidate substrates identified in all TAILS experiments

Article Snippet: To validate EFEMP1 cleavage in a binary assay, purified recombinant human HA-tagged EFEMP1/Fibulin-3 (R&D, 8416-FB) provided in PBS was dialyzed into TBS pH 8.0 to prevent precipitation with the CaCl 2 in the assay buffer.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: TAILS Identifies Candidate Substrates and Biomarkers of ADAMTS7, a Therapeutic Protease Target in Coronary Artery Disease

doi: 10.1016/j.mcpro.2022.100223

Figure Lengend Snippet: Validation of TAILS substrate EFEMP1 and cleavage site preference. A , EFEMP1/Fibulin-3 protein domains, amino acid sequence of the atypical EGF repeat linker, and location of the ADAMTS7 cleavage sites. Abbreviated EFEMP1 domains: signal peptide (SP), N-terminal region (N), EGF repeats (E). B , concentrated medium from HUVEC expressing Ad-Luc, Ad-mWT, or Ad-mEQ assessed by Western blot under nonreducing conditions. Anti-EFEMP1 antibody recognizes an epitope C-terminal to the ADAMTS7 cleavage sites. C , quantitation of semi-tryptic or semi-chymotryptic peptides from HUVEC medium matching novel cleavage sites from the endogenous EFEMP1 protein. The total area was greater for the 123.124 cleavage site compared to the adjacent 124.125 cleavage site. Additional cleavage events observed were also found in the Luc and EQ controls. D , in vitro cleavage of HA-EFEMP1 by purified full-length mouse ADAMTS7 S3A assessed by Western blot. The antibodies to the N-terminal HA epitope and C-terminal EFEMP1 epitope recognized the EFEMP1 more strongly under nonreducing conditions. A band at 100 kDa under nonreducing conditions is consistent with a purified HA-EFEMP1 dimer, which was also sensitive to ADAMTS7 cleavage. E , overnight digest of HA-EFEMP1 by mouse ADAMTS7 S3A assessed by Coomassie staining. F , quantitation of semi-tryptic or semi-chymotryptic peptides from the atypical EGF1 repeat region from HA-EFEMP1, showing a consistent preference for the 123.124 cleavage site. ADAMTS7, A disintegrin and metalloproteinase with thrombospondin motifs 7; E, EGF repeats; HUVEC, Human umbilical vein endothelial cells; N, N-terminal region; SP, signal peptides; TAILS, terminal amine isotopic labeling of substrates.

Article Snippet: To validate EFEMP1 cleavage in a binary assay, purified recombinant human HA-tagged EFEMP1/Fibulin-3 (R&D, 8416-FB) provided in PBS was dialyzed into TBS pH 8.0 to prevent precipitation with the CaCl 2 in the assay buffer.

Techniques: Sequencing, Expressing, Western Blot, Quantitation Assay, In Vitro, Purification, Staining, Isotopic Labeling