Fig 1: NUPR1 increases TEV release and packages NUPR1 in TEVs to promote cancer progression. (A) Western blot analysis for NUPR1 using 10 μg of EO771 TEVs isolated from 2 separate collections 24 hrs after cells were treated with vehicle, 10 μM reserpine, or 100 μM paclitaxel; (B) RNAseq analysis of EV markers using RNA from normal human lung bronchial epithelial cells BEAS-2B and BEAS-2B engineered to overexpress NUPR1; (C) Nanoparticle tracking analysis of TEVs from nickel transformed BEAS-2B cells and nickel transformed BEAS-2B cells expressing shRNA for NUPR1; (D) Western blot analysis for NUPR1 in EVs from nickel transformed BEAS-2B cells and nickel transformed BEAS-2B cells expressing shRNA for NUPR1. The EVs were further characterized for EV markers CD63 and GAPDH and the non EV biomarker GM130; (E) NUPR1 RNA expression from mouse Ch25h−/− endothelial cells treated with vehicle, TEV, or TEV with 10 μM reserpine. TEVs: Tumor-derived extracellular vesicles; NUPR1: Nuclear protein 1; EV: Extracellular vesicle.
Fig 2: Suppressing TEV release delays tumor growth and prevents loss of IFNAR1. (A) Tumor growth curve of EO771 tumor-bearing WT mice treated intravenously (iv) with vehicle, 1 mg/kg of reserpine (every other day), 2 mg/kg paclitaxel (twice a week), or both reserpine and paclitaxel (n = 5, each group); (B) ELISA for CD63 to quantify TEVs from serum of WT mice treated intravenously (iv) with vehicle, 1 mg/kg of reserpine (every other day), 2 mg/kg paclitaxel (twice a week), or both reserpine and paclitaxel with EO771 with tumors of similar size; (C) Flow cytometry analysis comparing change in mean fluorescent intensity (ΔMFI) of IFNAR1 in CD45+ cells from peripheral blood of WT mice with EO771 with tumors of similar size treated intravenously (iv) with vehicle, 1 mg/kg of reserpine (every other day), 2 mg/kg paclitaxel (twice a week), or both reserpine and paclitaxel (n = 5); (D) Tumor growth curve of 4T1-GFP tumor-bearing WT mice treated intravenously (iv) with vehicle and given vehicle chow (n = 5), iv vehicle and reserpine chow (n = 9), iv injection with 2 mg/kg paclitaxel (twice a week) and vehicle chow (n = 5), or iv injection with paclitaxel and reserpine chow (n = 5); (E) ELISA for CD63 to quantify TEVs from serum of WT mice with similar size tumor from (D); (F) Flow cytometry analysis comparing change in mean fluorescent intensity (ΔMFI) of IFNAR1 in CD45+ cells from peripheral blood of WT mice with EO771 with tumors of similar size from (D); Quantitative data are represented as mean ± SEM; P values: * P < 0.05; ** P < 0.01; *** P < 0.001, and ns for not significant from Anova test (panels A and B) or Student’s t test (panels B, C, E, and F).
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