Journal: Cancer cell

Article Title: MYC drives temporal evolution of small cell lung cancer subtypes by reprogramming neuroendocrine fate

doi: 10.1016/j.ccell.2020.05.001

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: RPM mice (n = 8) were given freshly prepared DAPT (GSI-IX) γ-secretase inhibitor (20 mg/kg, Apexbio cat no. A8200) diluted in corn oil from a 20 mg/mL stock in 100% EtOH to a 4 mg/mL working concentration.

Techniques: Western Blot, Plasmid Preparation, Recombinant, Staining, RNA Sequencing Assay, Software

Journal: bioRxiv

Article Title: Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor

doi: 10.1101/2022.04.07.487463

Figure Lengend Snippet: Notch1-Dll4 signaling regulates hMSCs osteogenic differentiation. (A) Representative bright field and fluorescence images of hMSCs in control, induction, DAPT and Jag1 treatment groups. Control group: cells were cultured and maintained in basal medium. Induction group: Cells were cultured in basal medium and induced by osteogenic induction medium after 2 days of cell seeding. DAPT group: cells were treated with DAPT (20 μM) daily. Jag1 group: cells were treated with Jag1 (40μM) daily. The bottom images are enlarged areas of a single hMSC. Green: Dll4 mRNA expression; red: ALP activity; blue: nucleus. Scale bar: 100 μm. (B) Mean fluorescence intensity of Dll4 mRNA expression under different treatments. (C) Comparison of hMSCs osteogenic differentiation efficiency under different treatments. Data represent over 100 cells in each group and are expressed as mean± s.e.m. (n=4, ***, p <0.001, **, p <0.01)

Article Snippet: For studying Notch1-Dll4 signaling, hMSCs were treated with 20 μM γ-secretase inhibitor DAPT or 20 μM Jag1 peptide (AnaSpec, 188-204) after osteogenic induction.

Techniques: Fluorescence, Control, Cell Culture, Expressing, Activity Assay, Comparison

Journal: bioRxiv

Article Title: Probing Notch1-Dll4 Signaling in Regulating Osteogenic Differentiation of Human Mesenchymal Stem Cells using Single Cell Nanobiosensor

doi: 10.1101/2022.04.07.487463

Figure Lengend Snippet: Notch1-Dll4 signaling regulates 3D hMSCs spheroids osteogenic differentiation. (A) Representative bright field and fluorescence images of hMSCs spheroids in control, induction, DAPT and Jag1 treatment groups. Green: Dll4 mRNA expression; red: ALP activity; blue: nucleus. Scale bar: 100 μm. (B) Quantification of ALP activity of hMSCs spheroids under different treatments. (C) Characterization of spheroids sizes under different treatments. Data represent over 50 spheroids in each group and are expressed as mean± s.e.m. (n=4, ***, p <0.001, **, p <0.01)

Article Snippet: For studying Notch1-Dll4 signaling, hMSCs were treated with 20 μM γ-secretase inhibitor DAPT or 20 μM Jag1 peptide (AnaSpec, 188-204) after osteogenic induction.

Techniques: Fluorescence, Control, Expressing, Activity Assay

Journal: Blood cancer discovery

Article Title: Tumor burden limits bispecific antibody efficacy through T cell exhaustion averted by concurrent cytotoxic therapy

doi: 10.1158/2643-3230.bcd-21-0038

Figure Lengend Snippet: A. RMA summarized expression values of Tnfrsf17/BCMA mRNA of Vk*MYC derived lymphoma cell lines, normal plasma cells (PC), Balb/c plasmacytoma cell lines, Vk*MYC de novo MM and transplantable cell lines (tVK*MYC). Line at median is shown for each group. B. BCMA cell surface staining (white histogram) by FCM of cell lines or primary MM cells with (bottom panel) or without (top panel) GS inhibition (DAPT 1uM, 18 hours for in vitro and LY-411575-I at 5mg/kg for in vivo treatment). The gray histogram depicts negative control staining with secondary antibody only. C. Soluble BCMA levels quantified by ELISA in the serum of moribund tumor bearing or age matched WT control mice. Each symbol represents one untreated mouse, tested in duplicate. D. Surface BCMA quantified by FCM (geometric MFI) of ex vivo CD138+ tumor cells harvested from Vk12598 tumor bearing mice left untreated or treated for 48 hours with the GS inhibitor LY-411575-I at the indicated dose. Each dot represents an individual mouse. E. Tumor cell survival after incubation in vitro with splenocytes and BCMA/CD3-BsAb at two different concentrations, normalized to the untreated conditions. P values determined using multiple comparison T tests with Holm-Sidak adjustment. F. FCM analysis of T cells from killing assay in F, representative of triplicate tests. The proliferation index is in the upper left and the geometric MFI for the indicated markers is presented in the top right corner of both plots. G. M-spike levels (G/A relative to day 0) over time (days) in six de novo Vk*MYC mice treated with increasing doses of anti-BCMA/CD3 BsAb. H. M-spike levels (G/A) over time (weeks) in six de novo Vk*MYC mice treated with 1 mg/kg anti BCMA/CD3 BsAb on day 1,8. Each mouse is represented by a different colored histogram. # shows mice that succumbed to tumor burden.

Article Snippet: The gamma secretase inhibitors DAPT and {"type":"entrez-nucleotide","attrs":{"text":"LY411575","term_id":"1257853995","term_text":"LY411575"}} LY411575 -I were obtained from Cayman Chemical; DAPT was solubilized in DMSO and used at 1μM, {"type":"entrez-nucleotide","attrs":{"text":"LY411575","term_id":"1257853995","term_text":"LY411575"}} LY411575 -I was freshly suspended in 40% PEG300 + 5% Tween-80 + 45% saline before each in vivo administration.

Techniques: Expressing, Derivative Assay, Clinical Proteomics, Staining, Inhibition, In Vitro, In Vivo, Negative Control, Enzyme-linked Immunosorbent Assay, Control, Ex Vivo, Incubation, Comparison

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A to C ) Tumor weight of vehicle (VT)–, DAPT-, and Dll4 blockade–treated JE-3, BeWo, and MDA-MB-231 tumors ( n = 4 to 6 mice per group). ( D to F ) Representative images of CD31 + tumor vessels in vehicle-, DAPT-, and anti-Dll4 antibody–treated JE-3, BeWo, and MDA-MB-231 tumors. Red, CD31-positive signals; green, NG2-positive signals; yellow, overlapping signals. Arrowheads point to microvessel-associated pericytes. Scale bar, 50 μm. ( G to I ) Quantification of microvessel density and pericyte coverage in vehicle-, DAPT-, and anti-Dll4 antibody–treated JE-3, BeWo, and MDA-MB-231 tumors (5 to 10 random fields per group); data are presented as means ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques:

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A to C ) Left: Representative images of leakage of 70-kD LRD (lysinated rhodamine-labeled dextran) in vehicle (VT)–, DAPT-, and anti-Dll4 antibody–treated human JE-3, BeWo, and MDA-MB-231 tumors. Red, CD31-positive signals; green, extravasated 70-kD LRD; yellow, intravascular 70-kD LRD. Arrowheads indicate extravasated 70-kD LRD. Scale bars, 50 μm. Right: Quantification of extravasated 70-kD LRD ( n = 4 to 6 random fields per group); data are presented as means ± SEM. * P < 0.05; ** P < 0.01. ( D to F ) Left: Representative images of perfusion of 2000-kD LRD in vehicle-, DAPT-, and anti-Dll4 antibody–treated human JE-3, BeWo, and MDA-MB-231 tumors. Red, CD31-positive signals; yellow, perfused 2000-kD LRD. Arrowheads indicate positive signals of 2000-kD LRD. Scale bars, 50 μm. Right: Quantification of perfused 2000-kD LRD ( n = 4 to 6 random fields per group); data are presented as means ± SEM.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques: Labeling

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A to C ) Left: Representative images of tissue hypoxia in vehicle (VT)–, DAPT-, and anti-Dll4 antibody–treated human JE-3, BeWo, and MDA-MB-231 tumors. Green, pimonidazole-positive signals; blue, 4′,6-diamidino-2-phenylindole (DAPI). Scale bars, 100 μm. Right: Quantification of tumor hypoxia ( n = 4 to 6 random fields per group); data are presented as means ± SEM. FITC, fluorescein isothiocyanate. ( D to F ) Left: Representative images of tumor cell proliferation and apoptosis in vehicle-, DAPT-, and anti-Dll4 antibody–treated human JE-3, BeWo, and MDA-MB-231 tumors. Red, Ki67 + proliferating or cleaved caspase 3 + apoptotic cells; blue, DAPI. Scale bars, 100 μm. Right: Quantification of tumor hypoxia ( n = 4 to 6 random fields per group); data are presented as means ± SEM. PA index was calculated using the formula (% apoptotic cells/total cells)/(% Ki67-positive cells/total cells). Arrowheads indicate apoptotic cells. Scale bars, 50 μm. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques:

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A and B ) Tumor growth rates (A) and weights (B) of vehicle (VT)–, DAPT-, and anti-Dll4 antibody–treated vector-LLC and PlGF-LLC tumors (four to six mice per group). * P < 0.05; ** P < 0.01; *** P < 0.001. ( C ) Representative images of CD31 + tumor vessels in vehicle-, DAPT-, and anti-Dll4 antibody–treated vector-LLC and PlGF-LLC tumors. Red, CD31 + signals; green, NG2 + signals. Arrowheads point to pericyte coverage in tumor vessels. Scale bar, 50 μm. ( D ) Quantification of microvessel density and pericyte coverage 1 in vehicle-, DAPT-, and anti-Dll4 antibody–treated vector-LLC and PlGF-LLC tumors (5 to 10 random fields per group). Data are presented as means ± SEM.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques: Plasmid Preparation

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A ) Representative images of 70- and 2000-kD LRD, pimonidazole, Ki67, and cleaved caspase 3 in vehicle (VT)–, DAPT-, and anti-Dll4 antibody–treated vector-LLC and PlGF-LLC tumors. Arrowheads in the upper two rows of panels point to extravasated 70-kD (green) or perfused 2000-kD LRD (yellow). Tumor vessels were stained with CD31 (red). Tumor hypoxia in different groups was detected with pimonidazole probe (green in the middle rows of panels). Proliferating tumor cells were detected by Ki67 staining (red), and apoptotic tumor cells were detected by cleaved caspase 3 (red). Arrowheads in the bottom rows indicate apoptotic tumor cells. Scale bars, 50 μm. ( B to G ) Quantification of 70-kD LRD, 2000-kD LRD, and pimonidazole + , Ki67 + , and cleaved caspase 3 + signals in vehicle (VT)–, DAPT-, and anti-Dll4 antibody–treated vector-LLC and PlGF-LLC tumors ( n = 4 to 6 per group). Data are presented as means ± SEM. PA index was calculated using the formula (% apoptotic cells/total cells)/(% Ki67-positive cells/total cells). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques: Plasmid Preparation, Staining

Journal: Science Advances

Article Title: PlGF-induced VEGFR1-dependent vascular remodeling determines opposing antitumor effects and drug resistance to Dll4-Notch inhibitors

doi: 10.1126/sciadv.1400244

Figure Lengend Snippet: ( A to C ) qPCR quantification of Vegfr1 and Vegfr2 mRNA expression in vehicle (VT)– and DAPT-treated vector-LLC whole tumor tissues (A), isolated CD31 + endothelial cell fractions from vehicle- and DAPT-treated vector-LLC tumors (B), and vehicle- and DAPT-treated HUVECs (C). * P < 0.05; ** P < 0.01. ( D and E ) Tumor growth rates and weights of anti-Dll4 antibody– and vehicle-treated vector-LLC and PlGF-LLC tumors grown in wild-type or Vegfr1 tk–/– mice (six animals per group). * P < 0.05; ** P < 0.01. ( F to I ) Tumor microvessels and pericyte coverage of anti-Dll4 antibody– and vehicle-treated vector-LLC and PlGF-LLC tumors grown in wild-type or Vegfr1 tk–/– mice (four to six random fields per group). * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: The DAPT γ-secretase inhibitor was obtained from AbMole BioScience.

Techniques: Expressing, Plasmid Preparation, Isolation

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet: Vegfaa drives cardiomyocyte proliferation by endocardial notch signaling (A) LSFM images of Tg(myl7:h2b-GFP) larvae at 24 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection. (B) Ventricular cardiomyocyte number in Tg(myl7:h2b-GFP) larvae at 24 and 48 hpi treated with PBS 0.1% BSA or zfVegfaa 0.1% BSA injection, n = 20. Unpaired t test. (C) Images of injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, imaged at 48 hpi. Non-myocardial EdU signal is excluded post-acquisitionally. (D) Percentage of EdU+ cardiomyocyte nuclei from uninjured and injured ventricles from Tg(myl7:h2b-GFP) larvae, EdU stained and bathed in vehicle or AV951, n = 13–36. Unpaired t test. (E) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or DAPT, acquired at 48 hpi by LSFM. (F) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or DAPT, n = 24–40. Unpaired t test. (G) Images of injured Tg(myl7:nlsDsRed) larvae treated with vehicle or AG1478, acquired at 48 hpi by LSFM. (H) Ventricular cardiomyocyte number in uninjured and injured Tg(myl7:h2b-GFP) larvae at 48 hpi treated with vehicle or AG1478, n = 24. Unpaired t test. (I) Treatment strategy for the injection of uninjured larvae with zfVegfaa and continuous bathing in AG1478 solution. (J) Hypothesized signaling pathway active in uninjured and injured larval hearts driving cardiomyocyte proliferation. (K) LSFM-acquired z plane showing notch expression colocalizing with endocardium in Tg(Tp1:venus-PEST;kdrl:hsa.HRAS-mCherry) , abbreviated in the figure to Tg(Tp1:venus-PEST;kdrl:mCherry) . AG1478 abbreviated to AG; white box, zoom panel. (L) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following zfVegfaa injection and bathing in AG1478, n = 28. Fisher’s exact test. (M) Treatment strategy for the lasering of larvae and continuous bathing in AG1478 solution. (N) Representative z plane images of uninjured, injured, and injured AG-treated ventricles from Tg(tp1:venus-PEST) larvae at 48 hpi. BA, bulbous arteriosus; AVV, atrioventricular valve; white arrowheads, laterally inhibited cardiomyocytes; cyan arrowheads, notch+ endocardium; cyan box, zoom panel. Fisher’s exact test. (O) Proportion of larvae with notch+ endocardium at 6, 24, and 48 hpt following laser injury and bathing in AG1478, n = 18. (P) Cardiomyocyte number at 48 hpi following injection with recombinant Vegfaa and continuous bathing in DAPT or AG1478, n = 22–25. One-way ANOVA followed by Holms-Sidak’s multiple comparison post-hoc tests. All images are maximum intensity projections of 3D LSFM stacks unless otherwise stated. Scale bars, 50 μm. Data are represented as mean ± SEM, ∗ p ≤ 0.05, ∗∗ p ≤ 0.01, ∗∗∗ p ≤ 0.001, and ∗∗∗∗ p ≤ 0.0001.

Article Snippet: To inhibit notch signalling, larvae were bathed in gamma secretase inhibitor DAPT (Cambridge Bioscience) 0-48 hpi.

Techniques: Injection, Staining, Expressing, Recombinant, Comparison

Journal: Developmental Cell

Article Title: Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

doi: 10.1016/j.devcel.2022.05.014

Figure Lengend Snippet:

Article Snippet: To inhibit notch signalling, larvae were bathed in gamma secretase inhibitor DAPT (Cambridge Bioscience) 0-48 hpi.

Techniques: Recombinant, In Situ, Imaging, Software