Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: (A) Schematic showing the experimental design to harvest cleared 24 h culture supernatants from control (NSC) and AnxA6 downregulated (A6sh5) MDA 468 cells. (B) Detection of secreted proteins by antibody arrays. Quantification of secreted proteins by ImageJ particle analysis. Bars represent the spot density relative to controls (CTLs). (C) Validation of AnxA6-dependent secretion of MCP-1. Cell type-specific expression of MCP-1 in TNBC cells. (D) Secreted MCP-1 in control (NSC) and AnxA6 downregulated (A6sh5) BT-549 and MDA-468 cells.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Control, Particle Size Analysis, Expressing

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: The altered expression of AnxA6 affects the secretion and enrichment of cholesterol in EVs from TNBC cells. Extracellular vesicles (EVs) were purified from culture supernatants from control (NSC) and AnxA6 downregulated (sh5) BT-549 or MDA-468 TNBC cells and from empty vector control and AnxA6 upregulated MDA-468 cells. (A-C) EV particle analyses. (D) Verification of isolated EVs by transmission electron microscopy. Bars denote 500nm. (E-F) Determination of cholesterol in EVs from control and AnxA6 downregulated TNBC cells (E) and empty vector control and AnxA6 upregulated MDA-468 cells (F) . Bars represent EV-associated cholesterol/particle. ** denotes p<0.01

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Expressing, Purification, Control, Plasmid Preparation, Isolation, Transmission Assay, Electron Microscopy

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: Chronic lapatinib (Lap-R) induced expression of AnxA6 promotes the loading of cholesterol in EVs from MDA-468 cells. (A) Western blot showing inhibition of EGFR activation in MDA-468 cells following chronic treatment with lapatinib. (B) Enrichment of AnxA6 and RAB7 in EVs from lapatinib resistant MDA-468 cells. (C) AnxA6 dependent enrichment of cholesterol in lapatinib resistant MDA-468 cells. Bars represent cholesterol levels/10^6 cells. (D) Lapatinib withdrawal leads to increased secretion of MCP-1 in AnxA6 expressing NSC cells (more apparent in NSC-LapRW than the NSC-LapR), and decreased secretion in AnxA6 downregulated A6sh5-LapRW and A6sh5-LapR.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Expressing, Western Blot, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: Empty vector and Flag-AnxA6 transfected MDA-468 cells were grown to 70% confluency and either treated with or without DSP. Immunoprecipitation of AnxA6 from cell lysates was carried out by using anti-flag M2 antibody or anti-AnxA6 antibody (A) . Detection of AnxA6 and SNAP23 in the immune complexes was carried out by western blotting. (B) Proximity ligation assay (PLA) confirmed the interaction of AnxA6 with SNAP23. All experiments were repeated three times, and representative results are presented. NXL: Non-cross linked, XL: Cross-linked. (C) Validation of the interaction of AnxA6 with SNAP23 by GST Pulldown Assay. GST and GST-AnxA6 were expressed in E. coli BL21 and purified to homogeneity. Equal amounts of purified GST and GST-AnxA6 were used to pull down assays using cell lysates from MDA-468. Ponceau stained membrane. (D) Membranes were probed with antibodies against the indicated proteins.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Plasmid Preparation, Transfection, Immunoprecipitation, Western Blot, Proximity Ligation Assay, GST Pulldown Assay, Purification, Staining, Membrane

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: Control AnxA6 expressing and AnxA6 depleted BT-549 cells were treated with ( A and B ) 5 mM Ca 2+ for 5 min, fixed, and processed for immunogold TEM. Images are representative transmission electron micrographs at the same magnification. Arrows indicate the location of AnxA6 within each cell.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Control, Expressing, Transmission Assay

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: Cell count in various samples (A) . Cell viability is reduced in MDA-468 compared to HCC70 and BT-549 (B) . Cell viability is lower in AnxA6 neutralizing antibody treated BT-549 compared to untreated and IgG BT-549 cells (C) . In MDA-468, AnxA6 neutralizing antibody strongly inhibited cell viability when compared to the untreated and IgG (D) . * denotes p<0.05; ** denotes p<0.01; *** denotes p<0.001.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Cell Counting

Journal: bioRxiv

Article Title: Annexin A6 modulates the secretion of pro-inflammatory cytokines and exosomes via interaction with SNAP23 in triple negative breast cancer cells

doi: 10.1101/2024.10.22.619710

Figure Lengend Snippet: Chronic lapatinib (Lap-R) induced expression of AnxA6 promotes the loading of cholesterol in EVs from MDA-468 cells. (A) Western blot showing inhibition of EGFR activation in MDA-468 cells following chronic treatment with lapatinib. (B) Enrichment of AnxA6 and RAB7 in EVs from lapatinib resistant MDA-468 cells. (C) AnxA6 dependent enrichment of cholesterol in lapatinib resistant MDA-468 cells. Bars represent cholesterol levels/10^6 cells. (D) Lapatinib withdrawal leads to increased secretion of MCP-1 in AnxA6 expressing NSC cells (more apparent in NSC-LapRW than the NSC-LapR), and decreased secretion in AnxA6 downregulated A6sh5-LapRW and A6sh5-LapR.

Article Snippet: Immunoprecipitation was performed as previously described ( ) and the bound proteins were analyzed by western blotting using the following antibodies: anti-AnxA6 (Santa Cruz Biotechnology sc-271859), anti-β-actin (Sigma, A1978), EGFR (Cell Signaling Technology, C74B9), pEGFR (Cell Signaling Technology, D95F2), GAPDH (Santa Cruz Biotechnology sc-25778), CD63 (Proteintech, 25682-1-AP, USA), Rab7 (Cell Signaling Technology, D7A5), Flotillin-1 (Santa Cruz sc-25506), SNAP23 (Abcam, ab-131242 ER 8538), anti-hCCL2/MCP-1 (R & D Systems, MAB679), anti-hCXCL8/IL-8 (R & D Systems, MAB208).

Techniques: Expressing, Western Blot, Inhibition, Activation Assay