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: (A) Representative brightfield images of primary RPM tumor cells cultured in DMSO or 10 μM DAPT treated every three days and visualized at indicated days. Red box on each row indicates the day that variant morphology was first observed. Scale bar, 100 μm. n = 4 biological experiments.

Article Snippet: DAPT treatments in vitro RPM time-series transition cell lines were treated with 10 μM DAPT (GSI-IX) γ-secretase inhibitor (Apexbio cat# A8200) from a 10 mM stock in DMSO starting on day 3 following initial digest.

Techniques: Cell Culture, Variant Assay

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: DAPT treatments in vitro RPM time-series transition cell lines were treated with 10 μM DAPT (GSI-IX) γ-secretase inhibitor (Apexbio cat# A8200) from a 10 mM stock in DMSO starting on day 3 following initial digest.

Techniques: Western Blot, Virus, Plasmid Preparation, Recombinant, Staining, Library Quantification, RNA Sequencing, Software

Journal: Scientific Reports

Article Title: Expression of B4GALNT1, an essential glycosyltransferase for the synthesis of complex gangliosides, suppresses BACE1 degradation and modulates APP processing

doi: 10.1038/srep34505

Figure Lengend Snippet: Whole cell lysate was prepared from cells treated with 1 μM DAPT for 12 h. Equal amounts of cellular proteins were subjected to immunoblot analysis with anti-APP antibody Y188 ( a ) or 82E1( c ). Longer (30 sec) and shorter (5 sec) exposure images are shown in panel a. β-actin served as an internal standard. Levels of α-CTF and/or β-CTF shown in panels a and c were quantified and presented in panels b and d, respectively. Data represent means ± s.d. (n = 6). Statistical analysis was performed by one-way ANOVA with Tukey-Kramer post hoc test (** P < 0.01).

Article Snippet: The γ secretase inhibitor DAPT was obtained from Peptide Institute, Inc. Cycloheximide, MG-132, chloroquine, Brij 98 were from Sigma-Aldrich.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Expression of B4GALNT1, an essential glycosyltransferase for the synthesis of complex gangliosides, suppresses BACE1 degradation and modulates APP processing

doi: 10.1038/srep34505

Figure Lengend Snippet: ( a,b ) BACE1 protein expression. GM3-expressing cells (M3-2) were incubated without or with GM3, GD3, GM2, GD2 or GM1 (30 μM each) for 21 h. Whole cell lysate was subjected to immunoblot analysis for BACE1, APP (22C11) and β-CTFs (Y188). β-actin served as an internal standard. BACE1, APP and β-CTF levels were quantified and plotted in panel b. Data are means ± s.d. (n = 3). Statistical analysis was performed by one-way ANOVA with Dunnett post hoc test, and statistical significance was not detected. IM, immature form; M, mature form. ( c,d ) Generation of β-CTF. GM3-expressing cells (M3-2) were treated with gangliosides as above. Twelve hours before harvesting cells, DAPT was added to culture medium at the final concentration of 1 μM. Whole cell lysate was subjected to immunoblot analysis with anti-Aβ (82E1) that detects only β-CTF. β-CTF level was quantified and plotted in panel d. Data represent means ± s.d. (n = 3). Statistical analysis was performed by one-way ANOVA with Dunnett post hoc test, and statistical significance was not detected.

Article Snippet: The γ secretase inhibitor DAPT was obtained from Peptide Institute, Inc. Cycloheximide, MG-132, chloroquine, Brij 98 were from Sigma-Aldrich.

Techniques: Expressing, Incubation, Western Blot, Concentration Assay

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