Journal: Oncogene

Article Title: Epigenetic targeting of neuropilin-1 prevents bypass signaling in drug-resistant breast cancer

doi: 10.1038/s41388-020-01530-6

Figure Lengend Snippet: a Endogenous FGFR1 was immunoprecipitated from D2.A1 whole-cell lysates. These precipitates were probed by immunoblot with antibodies for NRP1. Total levels of NRP1 (input) and use of a non-specific antibody (IgG) are shown as controls. b Immunoblot analyses showing differential phosphorylation of ERK1/2 (pERK1/2) upon FGF2 (20 ng/ml) stimulation in control (scram) and NRP1-depleted (shNRP1-01 and -02) D2.A1 cells. Expression of total ERK1/2 (tERK1/2) served as a loading control. c Immunofluorescent staining for phosphorylated-ERK1/2 (pERK1/2) in control (scram) and NRP1-depleted (shNRP1-02) D2.A1 cells grown on 3D culture scaffolds. Cells were not stimulated (NS) or stimulated with FGF2 (20 ng/ml) for 10 min. Data in a – c are representative of at least three independent analyses. d Bioluminescent quantification of control (scram) and NRP1-depleted (shNRP1) D2.A1 cells growing under 3D culture conditions in the presence or absence (NS) of FGF2 (20 ng/ml). e Bioluminescent quantification of NMuMG cells stably expressing control vectors (eGFP and empty (MT)) or FGFR1-GFP and NRP1 in 3D culture in the presence or absence (NS) of FGF2 (20 ng/ml). For d , e , readings were taken 6 days after plating and are the mean ± SE of two independent experiments completed in triplicate resulting in the indicated P values.

Article Snippet: FGF2, PDGFa, and HGF were purchased from GoldBio (St. Louis, MO).

Techniques: Immunoprecipitation, Western Blot, Expressing, Staining, Stable Transfection

Journal: Oncogene

Article Title: Epigenetic targeting of neuropilin-1 prevents bypass signaling in drug-resistant breast cancer

doi: 10.1038/s41388-020-01530-6

Figure Lengend Snippet: a Correlation between EMT transcription factors and NRP1 gene expression in the METABRIC dataset. b Phase-contrast microscopy showing the epithelial and mesenchymal phenotypes of control (YFP) and Twist-overexpressing HMLE, MCF-10A, and NMuMG cells. c RT-PCR showing NRP1 upregulation following Twist overexpression. Data are normalized to NRP1 levels in the control (YFP) cells for each cell type and are the mean ± SE of duplicate experiments completed in triplicate. d Immunoblot analyses showing expression of NRP1 and other EMT proteins in response to Twist overexpression. e RT-PCR showing NRP1 in control (YFP) and Twist-overexpressing cells following 7 days of treatment with the indicated concentrations of JQ1. Data are the mean ± SE of triplicate experiments resulting in the indicated P values. f Immunoblot analyses of NRP1 expression in control (YFP) and Twist-overexpressing HMLE and MCF-10A cells following 7 days of treatment with the indicated concentrations of JQ1. g RT-PCR analyses of NRP1 expression in afatinib-resistant (BMAR) cells following 7 days of treatment with JQ1 (50 nM). Data are the mean ± SE of triplicate experiments. h Immunoblot analyses of FGFR1 and NRP1 expression in BMAR cells following 7 days of treatment with JQ1 (50 nM). Expression of tubulin served as a loading control. i Phase-contrast images showing 3D spheroid growth of BMAR cells in response to FGF2 (20 ng/ml) in the presence or absence of JQ1 (50 nM). j Measurement of BMAR spheroid radius at the indicated time points, under the conditions described in i . Data are represented as a boxplot including the measurement of at least three images per condition and are representative of two independent experiments.

Article Snippet: FGF2, PDGFa, and HGF were purchased from GoldBio (St. Louis, MO).

Techniques: Expressing, Microscopy, Reverse Transcription Polymerase Chain Reaction, Over Expression, Western Blot