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: Enrichment of cancer-related pathways from transcriptome-wide analysis. a. Pathway enrichment and b. Gene Ontology analysis of differentially expressed genes (DEGs) between the BT-474 and BT-474 NRP-1 cells from RNA sequencing based on a Log2 ratio cutoff ≥2 and concurrence in 2 replicates
Fig 3: Overexpression of NRP-1 caused differential cellular responses to cytotoxic treatment. a. Treatment with 0.5 uM Adriamycin + 300 nM Cyclophosphamide (AC) for 72 h significantly decreased the invasive capacity of the BT-474 NRP-1 cells but indicated an increasing trend in the invasion ability of BT-474 cells. Resistant BT-474 and BT-474 NRP-1 cells were generated by long-term treatment (eight months) with either four cycles of AC alone (4xAC) or a combination of four cycles of AC and four cycles of 20 nM Paclitaxel (4xAC + 4xPAC), and cellular responses were assessed based on their b, proliferation and c, clonogenic ability, whereby BT-474 4xAC cells indicated significantly increased proliferation and clonogenic capacity compared to BT-474 NRP-1 4xAC cells. d. Wound healing assay images (5× magnification, scale bar 500 μm) to determine the migratory capacity of chemoresistant cells 72 h post generation of the wound indicated decreased migration in BT-474 NRP-1 overexpressing 4xAC and 4xAC + 4xPAC resistant cells compared to BT-474 resistant cells. Fold change in colonies/well (panel c) is compared to the respective untreated BT-474 or BT-474 NRP-1 cells. Graphs represent the mean ± SEM of three independent experiments. Statistical analysis using ANOVA and Tukey post hoc test, p-value < 0.05 considered as statistically significant. ** p < 0.01, ***p < 0.001
Fig 4: 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 5: 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)
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