Fig 1: NRP-1/TNC/Integrin β3/FAK/Akt/NF-kB schematic signaling pathway. Schema to illustrate the identified mechanism by which NRP-1 activates TNC-dependent integrin β3 signaling via phosphorylation of FAK/Akt-473/NF-κB p65. In addition, NRP-1 upregulates TNFR2 expression and downregulates BCRP/ABCG2, TNFR1, and PI3K/Akt-308. Solid black lines indicate identified direct interactions in the pathway whereas dashed lines indicate indirect effects of NRP-1 overexpression via unidentified mechanisms. The key denotes the arrows and colors used in the illustration
Fig 2: Confirmation of DEGs from transcriptome analysis. a. KEGG pathway analysis of DEGs between BT-474 and BT-474 NRP-1 cells identified from RNA sequencing. Real-time qPCR was implemented on shortlisted DEGs obtained from RNA sequencing to confirm their differential expression. BT-474 NRP-1 cells significantly upregulated b. TNC expression and downregulated c, ACE, d, APOD, e, ATF3, f, DDIT3 and g, P2RX6 expression. Cells transfected with the empty plasmid indicated similar expression to the BT-474 cells. Graphs represent the mean ± SEM of 3 independent experiments. Statistical analysis using independent samples t-test, p value < 0.05 considered as statistically significant ** p < 0.01, ***p < 0.001
Fig 3: Differential spheroid formation capacity and molecular profiles in NRP-1 overexpressing chemoresistant cells. a. Spheroid formation (20× magnification, scale bar 100 μm) assessed after 24 and 72 h in BT-474 and BT-474 NRP-1 resistant cells indicated multiple smaller spheroids in 4xAC resistant cells but similar phenotypes between the 4xAC + 4xPAC and their respective untreated controls. b. Representative western blots to indicate NRP-1 overexpression inversely correlates with a downregulation of breast cancer resistant protein (BCRP/ABCG2) in untreated cells and on short term AC treatment for 72 h. GAPDH protein expression is indicated as a loading control. c. Representative images from western blot analysis of BT-474 and BT-474 NRP-1 untreated, 4xAC and 4xPAC cell lysates blotted with the indicated antibodies. NRP-1 and integrin β3 pathway molecules indicate a similar expression profile of significantly upregulated expression in BT-474 4xAC cells but a downregulation in BT-474 NRP-1 4xAC cells compared to their respective untreated controls. Images are representative of independent experiments with comparable outcomes. GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)
Fig 4: NRP-1 overexpression activates integrin β3 and TNFR2 pathways. Representative western blot images of protein lysates from untransfected BT-474, BT-474 NRP-1 and empty vector control cells blotted with indicated antibodies involved in a. Integrin signaling, and downstream signaling targets FAK, Akt, GSK3-β and NF-kB b. siRNA-mediated TNC downregulation decreased phosphorylation of FAK and Akt-473. c. Blots show levels of tumor necrosis factor receptors (TNFRs). GAPDH protein expression is indicated as a loading control. (The prefix P beside the antibody names indicates the phosphorylated form)
Fig 5: NRP-1 overexpression in BT-474 cell line triggers tumorigenic features. a. Comparison of NRP-1 expression in three breast cancer cell lines MDA-MB-231, MCF-7 and BT-474. NRP-1 was stably overexpressed in the BT-474 cell line and confirmed at the level of protein expression by western blotting and immunofluorescence staining (40× magnification, scale bar 10 μm). b. Wound healing assay images (5× magnification, scale bar 500 μm) taken on day 0 and day 3 indicate increased migration in BT-474 NRP-1 cells compared to control cells 3 days post wound generation. c. NRP-1 overexpression reduced spheroid formation (20× magnification, scale bar 100 μm). d. Western blotting indicated decreased expression of mature form of E-cadherin (lower band) and β-catenin along with e, significantly increased vimentin and f, NRP-1 gene expression. Gene expression is relative to the control BT-474 and normalized to β-Actin and GUSB reference genes expression. The graph represents the mean ± SEM of three independent experiments. Statistical analysis using independent samples t-test, p-value < 0.05 considered as statistically significant ** p < 0.01
Supplier Page from DNASU for NRP1 (Homo sapiens) in pDONR221 (Gateway donor/master vector)