primary antibodies against notch1 (d1e11)  (Cell Signaling Technology Inc)

Journal: Frontiers in Immunology

Article Title: Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer

doi: 10.3389/fimmu.2023.1244159

Figure Lengend Snippet: SS has gamma secretase modulator (GSM) activity and inhibits Notch1 cleavage. APP C99 and NOTCH1 were transiently transfected into HEK 293T wild type, PS1, and PS2 KO cells. After 16 hours, cells were treated with 5, 12.5, 25, 50, and 75 μM SS or SF in fresh media. Conditioned media were collected after 24 hours and assayed by Aβ ELISA as described in the method section (A, B) . Similarly, H4 cells stably overexpress Notch1 or APP C99 were treated with 25, 50, 75, and 100 μM SS; conditioned media were collected after 24 hours and assayed by Aβ ELISA (C) . Human MDA-MB-231 cells were treated with 10, 25, 50, and 100 μM SS for 48 hours, after which the expression of total Notch1 and cleaved Notch1 (CN1) was measured by Western Blot analysis (D) .

Article Snippet: Membranes were incubated with primary antibodies against Notch1 (D1E11), Notch1-IC (Val1744:D3B8) (Cell Signaling), GAPDH, and β-tubulin (Santa Cruz Biotechnology).

Techniques: Activity Assay, Transfection, Enzyme-linked Immunosorbent Assay, Stable Transfection, Expressing, Western Blot

Journal: Frontiers in Immunology

Article Title: Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer

doi: 10.3389/fimmu.2023.1244159

Figure Lengend Snippet: SS-mediated anti-mammosphere activity depends on Notch expression. Vector control and intracellular Notch1-overexpressing (N1IC) MDA-MB-231 cells (10,000) were grown in Mammocult media and treated with SS (10 or 50 μM) for one week (twice/week). Following incubation, mammospheres were counted using a Nikon microscope. Representative photographs and average mammospheres sizes (areas) are presented in (A, B) respectively. Data are means ± SD; P-values: *** P < 0.001; **** P < 0.0001, student t-test, using GraphPad Prism. ns, not significant.

Article Snippet: Membranes were incubated with primary antibodies against Notch1 (D1E11), Notch1-IC (Val1744:D3B8) (Cell Signaling), GAPDH, and β-tubulin (Santa Cruz Biotechnology).

Techniques: Activity Assay, Expressing, Plasmid Preparation, Control, Incubation, Microscopy

Journal: Frontiers in Immunology

Article Title: Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer

doi: 10.3389/fimmu.2023.1244159

Figure Lengend Snippet: SS monotherapy inhibits the growth of a syngeneic TNBC model (C0321). Mouse TNBC C0321 cells (1 million) were injected into the mammary fat pads of syngeneic immunocompetent FVB (female) mice with 1:1 ratio of Matrigel. Palpable tumors were treated with vehicle or SS (60mg/kg, daily, PO) for another two weeks. Tumor volumes and weights were measured twice per week, and three weeks after tumor inoculation, tumors were harvested, weighed, and analyzed by H&E and immunohistochemistry for Notch1 and Jagged1 (A, B) . Fresh tumor specimens were dissociated by Liberase digestion, and single-cell suspensions were analyzed for tumor-infiltrating T-Cells (CD4 and CD8), Dendritic cells (CD11c), MDSC, and TAM (C) by Flow cytometer. All cells were gated on pan-leukocyte marker CD45. Data are means ± SD; P-values: * P < 0.05; **** P < 0.0001, Student t-test, using GraphPad Prism.

Article Snippet: Membranes were incubated with primary antibodies against Notch1 (D1E11), Notch1-IC (Val1744:D3B8) (Cell Signaling), GAPDH, and β-tubulin (Santa Cruz Biotechnology).

Techniques: Injection, Immunohistochemistry, Flow Cytometry, Marker

Journal: Frontiers in Immunology

Article Title: Sulindac sulfide as a non-immune suppressive γ-secretase modulator to target triple-negative breast cancer

doi: 10.3389/fimmu.2023.1244159

Figure Lengend Snippet: SS does not suppress T-cell proliferation, but blocks BM-MDSC-mediated immune-suppressive activity. T-cells (CD3 + ) were isolated from naïve FVB (female) mice using a negative T cells isolation kit (Stemcell Technologies). Isolated T cells were then labeled with 1 μM CFSE and plated on 24-well culture plates coated with α-CD3 and α-CD28 (1 μg/ml each). T cells were treated with SS (5, 25 or 50 μM SS) at the beginning of incubation, and T-cells proliferation was measured after 72 hours by CFSE dilution using Flow Cytometry (A) . Isolated T-cells from naïve FVB (female) were cultured with plate-bound anti-CD3 and anti-CD28 and were treated with SS (5, 25 or 50 μM SS). Following 72 hours of culture, the expression of Cleaved Notch1 (CN1) and Notch1 was measured by Western Blotting (B) , and IL-2 production was assessed by ELISA (C) . Bone marrow cells were harvested from FVB mice and cultured with GCSF, GM-CSG, and IL-6 (20 ng/ml each) for four days to generate bone marrow-derived MDSC (BM-MDSC) in the presence or absence of SS (5, 25 or 50 μM). CFSE labeled T-cells were co-culture with BM-MDSC at a 4:1 (T-cells: MDSC ratio) with SS (5, 25 or 50 μM) on plate-bound anti-CD3 and anti-CD28 (1 μg/ml each) plate. T-cell proliferation was measured after 72 hours by CFSE dilution using Flow Cytometry (D) . Data are means ± SD; P-values: **** P < 0.0001, one-way ANOVA for multiple comparisons, using GraphPad Prism.

Article Snippet: Membranes were incubated with primary antibodies against Notch1 (D1E11), Notch1-IC (Val1744:D3B8) (Cell Signaling), GAPDH, and β-tubulin (Santa Cruz Biotechnology).

Techniques: Activity Assay, Isolation, Labeling, Incubation, Flow Cytometry, Cell Culture, Expressing, Western Blot, Enzyme-linked Immunosorbent Assay, Derivative Assay, Co-Culture Assay