Journal: Cell reports
Article Title: Endothelial Calcineurin Signaling Restrains Metastatic Outgrowth by Regulating Bmp2.
doi: 10.1016/j.celrep.2019.01.016
Figure Lengend Snippet: Figure 6. Calcineurin Regulates Bmp2 Expression in the Endothelium, a Potent Inhibitor of Tumor Cell Growth (A) caNFATc1 induces the expression of DSRC1, BMP2, and TISSUEF in HUVECs. The qRT-PCR analyses of the indicated genes; n = 3, **p = 0.0091 DSCR1, *p = 0.0257 BMP2, and *p = 0.0318 TISSUEF. (B) Loss of calcineurin reduces the expression of Cnb1, Dscr1, and Bmp2 in the BEC-2 population of lung ECs. *p = 0.0316, **p = 0.0029; n = 3 Cnb1WT-GFP; n = 4 Cnb1Dbec. (C) BMP2 induces the phosphorylation of Smad-1/5 in B16F10 cells. Cells were treated with 100 ng/mL BMP2 in the presence or absence of 1 or 3 mM DMH1. Western blot for the indicated proteins. (D) BMP2 does not affect B16F10 cell growth in 2D. Cells were treated with vehicle or 100 ng/mL BMP2 for 24 and 48 h and analyzed by CyQuant DNA-based assay. (E) BMP2 reduces B16F10 cell growth in 3D. Representative images of B16F10 spheroids treated with 100 ng/mL BMP2, BMP2 + 3 mM DMH1, or vehicle.
Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Rabbit anti-GAPDH Sigma RRID:AB_796208 Rabbit anti-P-Smad-1/5 Cell Signaling RRID:AB_491015 Rabbit anti-Smad1 Cell Signaling RRID:AB_10858882 Mouse anti-NFATc1 BD Bioscience RRID:AB_396478 Rat anti-CD45-PeCy7 eBioscience RRID:AB_2716950 Rat anti-CD31-Pe eBioscience RRID:AB_465631 Gp38-AlexaFluor647 In house Clone 8.1.1 Rat anti.EpCAM-eFluor450 Biolegend RRID:AB_10717090 Rat anti-CD31/PECAM-1 BD Bioscience RRID:AB_396660 Rabbit anti-collagen IV Millipore RRID:AB_2276457 Goat anti-CD13/Anpep R&D systems RRID:AB_2227288 Rat anti-Icam2 BD Bioscience RRID:AB_394784 Rabbit anti-cleaved caspase-3 Cell Signaling RRID:AB_2341188 Rabbit anti-Erg1/2/3 Santa Cruz RRID:AB_675518 Goat anti-Vegfr2 R&D systems RRID:AB_355500 Rat anti-GFP Biolegend RRID:AB_1279414 Rabbit anti-NG2 Millipore RRID:AB_91789 Rat anti-CD144 BD Bioscience RRID:AB_395707 Rat anti-CD45-FITC BD Bioscience RRID:AB_394610 Rat anti-CD3ε-e660 Biolegend RRID:AB_492861 Rat anti-CD8a-PerCP-Cy5.5 Biolegend RRID:AB_2075239 Rat anti-CD4-Alexa Fluor 700 Biolegend RRID:AB_493701 Rat anti-CD44-Pe-Cy7 Biolegend RRID:AB_830787 Rat anti-CD62L-Pe Biolegend RRID:AB_2270062 Rat anti-B220-Texas Red Thermo Fisher RRID:AB_10372805 Rat anti-CD11b-eFluor780 Thermo Fisher RRID:AB_1603193 Rat anti-Gr1-Alexa647 Biolegend RRID:AB_389331 Rat anti-Ly6C-PerCP-Cy5.5 Biolegend RRID:AB_1659241 Rat anti-NK1.1-PEe Biolegend RRID:AB_313395 Chemicals, Peptides, and Recombinant Proteins DMH1 Tocris Cat#4126 Recombinant human/mouse/rat-BMP2 R&D Cat#355-BM Tamoxifen Sigma Cat#T5648 EdU Santa Cruz Cat#sc-284628 Matrigel Corning Cat#356231 Luciferin Biosynth Cat#L-8220 Collagenase A Roche Cat#10103586001 Collagenase type IV Worthington Cat#LS004188 DNaseI Sigma Cat#11284932001 Fluoromount-G eBioscience Cat#00-4959-52 L-3,4-dihydroxyphenylalanine Sigma Cat#59-92-7 7AAD eBioscience Cat#00-6993-50 FITC-IB4 Invitrogen Cat#I-32450 (Continued on next page) e1 Cell Reports 26, 1227–1241.e1–e6, January 29, 2019
Techniques: Expressing, Quantitative RT-PCR, Phospho-proteomics, Western Blot, CyQUANT Assay
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