Journal: Cardiovascular Research

Article Title: Control of sinus venous valve and sinoatrial node development by endocardial NOTCH1

doi: 10.1093/cvr/cvz249

Figure Lengend Snippet: Endocardial NOTCH1 regulates the SVV formation through myocardial NRG1. (A and B), RNA in situ hybridization shows the expression of Nrg1 in E10.5 control and Notch1eKO embryos. The high magnification of ventricle (A1 and B1) and SVV (A2 and B2) regions is showed on the right. The arrowhead and arrow indicate the endocardium and myocardium respectively (n = 3/group). (C) RT–qPCR analysis of the Nrg1 mRNA expression in E10.5 control and Notch1eKO hearts. The expression of Nrg1 was normalized to that of Gapdh (n = 3/group). (D) RNA in situ hybridization shows the expression of Hcn4 (arrow) in the cultured E9.5 control and Notch1eKO embryos with or without the NRG1 treatment for 24 h. a, atrium; sv, sinus venous. (E) RT–qPCR analysis of the Hcn4 mRNA expression. The expression of Hcn4 is normalized to that of Gapdh (n = 4/group). (F) Quantification of the ratio of the length/width of SVV in cultured embryos indicates the rescued SVV growth in the Notch1eKO embryos by NRG1 (n = 4/group). Statistical significance was calculated by one-way ANOVA and Tukey’s multiple comparisons test. *P < 0.05, ΔP < 0.01.

Article Snippet: 21 For rescue experiments, the culture media was supplemented with recombinant NRG1 proteins (1.25 × 10 −8 M) (R&D system, USA) or WNT2 conditioned media.

Techniques: RNA In Situ Hybridization, Expressing, Control, Quantitative RT-PCR, Cell Culture

Journal: Cardiovascular Research

Article Title: Control of sinus venous valve and sinoatrial node development by endocardial NOTCH1

doi: 10.1093/cvr/cvz249

Figure Lengend Snippet: Schematic summary of the roles of endocardial Notch1 in SVV and SAN formation. (A) A carton shows the normal development of SVV and SAN in the E10.5 control Notch1eKO or β-catmKO embryos. Red area marks the HCN4-expressing SAN (head and tail). The expression of HCN4 is reduced in the Notch1eKO embryos, but not affected in the β-catmKO embryos when compared to that in controls, whereas the elongation of SVV is affected in both mutant embryos. (B) A schematic working model shows the possible signalling mechanisms through which endocardial Notch1 regulates the SVV growth and SAN formation. Endocardial Notch1 promotes the SVV growth through regulating Nrg1 expression in the endocardium. Endocardial Notch1 also regulates myocardial Wnt2 to promote the SVV growth through β-catenin, as well as supports the SAN formation and maturation through regulating Tbx18.16 The downstream mediators of Notch1 in the endocardium that regulate Wnt2 expression in myocardium are unknown and future studies are needed to identify them.

Article Snippet: 21 For rescue experiments, the culture media was supplemented with recombinant NRG1 proteins (1.25 × 10 −8 M) (R&D system, USA) or WNT2 conditioned media.

Techniques: Control, Expressing, Mutagenesis

Journal: bioRxiv

Article Title: Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts

doi: 10.1101/2020.04.06.026971

Figure Lengend Snippet: A , expression of NRG1 in breast cancer subtypes (PAM50) extracted from METABRIC and TCGA datasets. Boxes indicate mean +/- quartiles and minimum and maximum values are represented by bars. Only statistically significant comparisons with luminal subtypes are depicted. ANOVA multiple comparison test (*** P < 0.001). B , expression of NRG1 in the epithelial and stromal compartment in 4 laser-capture microdissected (LCM) breast cancer datasets (GSE10797, n = 28; GSE14548, n = 14; GSE35019, n = 53; and GSE83591, n = 39). Boxes indicate mean +/- quartiles and minimum and maximum values in bars. Two-tailed paired Student’s t-test (* P < 0.05; ** P < 0.01; *** P < 0.001). C , viability of T47D (black) and MCF7 (grey) measured 72h after treatment with HER3 mAb lumretuzumab or pertuzumab at indicated doses. Values represent median of 3 independent experiments (n = 5). U-Mann Whitney two-tailed test was applied. No significant differences were observed. D , ectopic NRG-1β (50ng/mL) was added to T47D and MCF7 cell lines and viability quantified. The untreated control was set to 100% (not shown) and used to normalize the results from conditions where cells had been preincubated for 1 hour with lumretuzumab (Lum) or pertuzumab (Pert) at 10µg/mL. Two-tailed unpaired Student’s t-test (* P < 0.05; ** P <0.01, *** P <0.001) comparing each treatment with untreated condition. Bars represent average of two independent experiments +/- s.e.m. E , representative Western blot showing total levels and phosphorylation of HER3 and downstream effectors AKT and ERK1/2, 5min after addition of NRG-1β (50ng/ml). Some samples were either pre-incubated with mAbs lumretuzumab (Lum) or pertuzumab (Pert) at 10µg/ml for one hour.

Article Snippet: Prior to addition of CAF-CM or human recombinant NRG-1β (4711, BioCat), cells were pre-treated with either lumretuzumab or pertuzumab (10μg/ml) for 30min in low serum media (1% FCS).

Techniques: Expressing, Two Tailed Test, MANN-WHITNEY, Western Blot, Incubation

Journal: bioRxiv

Article Title: Stromal NRG1 in luminal breast cancer defines pro-fibrotic and migratory cancer-associated fibroblasts

doi: 10.1101/2020.04.06.026971

Figure Lengend Snippet: A , heatmap representing relative phosphorylation values of HER3, AKT and ERK1/2 in T47D and MCF7 cells after addition of CAF conditioned media (CM) for 5min with or without pre-incubation with lumretuzumab (Lum) (10µg/ml). Values represent median of three technical replicates and three biological replicates. Color intensities are ranked per each antibody (red = maximum, blue = minimum). B , relative proliferation of T47D and MCF7 cancer cells after 72h with different CAF-CM with lumretuzumab (red squares) or untreated (black circles). Dots represent mean +/- s.e.m of three independent replicates (n = 4). P values were determined by two-tailed U-Mann Whitney test for each CM (* P < 0.01; ** P < 0.001; *** P < 0.0005; **** P <0.0001). C , percentage of closure in a scratch assay of T47D or MCF7 cancer cells, after 21h of treatment with 10μg/ml lumretuzumab (red) or untreated (black), and with conditioned media (CM) of indicated CAFs. DMEM-F12 1% FCS was used as negative control and NRG-1β (50ng/ml) as positive control. Box plots correspond to the mean and s.e.m. of 2 independent experiments (n = 6 technical replicates). Two-tailed U-Mann Whitney test for each CM (* P < 0.01; **P < 0.001; *** P < 0.0005; **** P <0.0001).

Article Snippet: Prior to addition of CAF-CM or human recombinant NRG-1β (4711, BioCat), cells were pre-treated with either lumretuzumab or pertuzumab (10μg/ml) for 30min in low serum media (1% FCS).

Techniques: Incubation, Two Tailed Test, MANN-WHITNEY, Wound Healing Assay, Negative Control, Positive Control