Abemaciclib and lapatinib (Haoyuan Chemexpress) were diluted as previously described6 (link). For in vitro use, palbociclib was diluted in DMSO. Cleaved PARP was measured after 48 hours of treatment with DMSO, lapatinib, or Abemaciclib. Lapatinib was used at 30 nM for BT474 and SKBR3; 500 nM for MDA-MB-453 and MDA-MB-361. Abemaciclib was used at 300 nM for BT474 and SKBR3; 25 nM for MDA-MB-453; 500 nM for MDA-MB-361. For staurosporine experiments, MDA-MB-453 cells were pretreated with DMSO or Abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500nM, Enzo Life Sciences) for 4 hours. For hormonal therapy studies, cells were treated with combinations of DMSO, Abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich). To determine JAK dependency, cells were treated with combinations of DMSO, Abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.
Staurosporine
Manufactured by Enzo Life Sciences
Sourced in United States, United Kingdom, Germany, Switzerland
Staurosporine is a laboratory reagent and a potent broad-spectrum protein kinase inhibitor. It functions by inhibiting a wide range of protein kinases, which are important enzymes involved in cellular signaling pathways.
Automatically generated - may contain errors
Lab products found in correlation
Chloroquine, by Merck Group (3 mentions)
Mg132, by Merck Group (3 mentions)
Sb203580, by Bio-Techne (3 mentions)
Abemaciclib, by Enzo Life Sciences (2 mentions)
Abemaciclib, by Merck Group (2 mentions)
Abemaciclib, by Selleck Chemicals (2 mentions)
Ruxolitinib, by Selleck Chemicals (2 mentions)
Fulvestrant, by Merck Group (2 mentions)
Etoposide, by Enzo Life Sciences (2 mentions)
Cycloheximide (chx), by Enzo Life Sciences (2 mentions)
D4476, by Bio-Techne (2 mentions)
Tunicamycin, by Merck Group (2 mentions)
B27 supplement, by Thermo Fisher Scientific (2 mentions)
Perifosine, by Merck Group (2 mentions)
Tamoxifen, by Enzo Life Sciences (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Cisplatin, by Enzo Life Sciences (1 mentions)
Curcumin, by Enzo Life Sciences (1 mentions)
5 fluorouracil, by Enzo Life Sciences (1 mentions)
Llc1 cells, by American Type Culture Collection (1 mentions)
36 protocols using staurosporine
1
Apoptosis Induction in Breast Cancer Cell Lines
2
Apoptosis Induction in Breast Cancer Cell Lines
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Abemaciclib and lapatinib (Haoyuan Chemexpress) were diluted as previously described6 (link). For in vitro use, palbociclib was diluted in DMSO. Cleaved PARP was measured after 48 hours of treatment with DMSO, lapatinib, or Abemaciclib. Lapatinib was used at 30 nM for BT474 and SKBR3; 500 nM for MDA-MB-453 and MDA-MB-361. Abemaciclib was used at 300 nM for BT474 and SKBR3; 25 nM for MDA-MB-453; 500 nM for MDA-MB-361. For staurosporine experiments, MDA-MB-453 cells were pretreated with DMSO or Abemaciclib (500 nM) for 0, 1, or 7 days prior to exposure to staurosporine (500nM, Enzo Life Sciences) for 4 hours. For hormonal therapy studies, cells were treated with combinations of DMSO, Abemaciclib (100 nM), and fulvestrant (100 nM, Sigma Aldrich). To determine JAK dependency, cells were treated with combinations of DMSO, Abemaciclib (500nM), and ruxolitinib (500nM, Selleckchem) for 7 days.
3
Apoptotic Bodies Isolation Protocol
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Lewis lung carcinoma (LLC1) cells were obtained from the American Type Culture Collection (ATCC, Baltimore, U.S.A.). Cultures were maintained in RPMI-1640 supplemented with 10% FCS. All experiments were carried out within 6 months of resuscitation. LLC1 cells were incubated with etoposide (400 μM), curcumin (160 μM), tamoxifen (40 μM), staurosporine (1 μM), cisplatin (400 μM) or 5-fluorouracil (160 μM) (all from Enzo Life Sciences) for 24 hrs to induce apoptosis. Apoptotic bodies were purified as described by Fransen et al., 2009 (28 (link)); briefly, cells were centrifuged at 1500 g for 10 mins at 20°C. The supernatant was centrifuged at 15700 g for 50 mins at 20°C. Apoptotic bodies were resuspended in PBS.
4 x 106 LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.
4 x 106 LLC1 cells were incubated with 100 µl RIPA buffer containing a protease inhibitor cocktail (20 µl/ml; Calbiochem), phosphatase inhibitor (Thermo Scientific) and 1% Triton X-100 for 20 mins at 4°C. Supernatants, collected after centrifugation at 18000 g for 30 mins at 4°C, were employed as cell lysate.
4
Cell Cycle and Apoptosis Analysis
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For cell cycle analysis, cells were serum-starved for 24 h and stimulated with HRG (20 ng/ml) for other 24 h. Cells were then harvested with 1 mM EDTA, washed with PBS, and fixed with ice-cold 100% ethanol. After washing again with PBS, cells were resuspended in Propidium Iodide (PI)/RNase Staining Solution (Cell Signaling Technology). Samples were analyzed on a BD FACSCalibur (BD Biosciences) and data were examined with FlowJo V10 software. Cyclin D1 antibody was detected by Western blot using an antibody from BD Biosciences.
For induction of apoptosis by anoikis, cell culture plates were coated with 6 mg/ml poly-HEMA (Sigma-Aldrich) in 95% ethanol and incubated at 37°C for several hours until they dry, and then washed with PBS. For single cell isolation, cells in suspension were passed through a 40 µm cell strainer (BD Biosciences) that retained the clumped cells, and then grown at low confluency (1 × 104 cells/ml) to avoid cell-cell contact. Apoptosis was also induced by staurosporine (1 µM, 3–6 h, Enzo Life Science, Farmingdale, NY, USA). Total and cleaved PARP were detected by Western blot using an antibody from Cell Signaling Technology.
For induction of apoptosis by anoikis, cell culture plates were coated with 6 mg/ml poly-HEMA (Sigma-Aldrich) in 95% ethanol and incubated at 37°C for several hours until they dry, and then washed with PBS. For single cell isolation, cells in suspension were passed through a 40 µm cell strainer (BD Biosciences) that retained the clumped cells, and then grown at low confluency (1 × 104 cells/ml) to avoid cell-cell contact. Apoptosis was also induced by staurosporine (1 µM, 3–6 h, Enzo Life Science, Farmingdale, NY, USA). Total and cleaved PARP were detected by Western blot using an antibody from Cell Signaling Technology.
5
Immune Cell Activation Assay
6
Erlotinib Cytotoxicity Assay in Fibroblasts
7
Apoptosis in T cell subsets
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Blood from healthy donors at different ages was obtained from the Asturias Transfusion Centre and the Internal Medicine Department of the Hospital Central de Asturias, Spain, after obtaining informed consent in accordance with the Declaration of Helsinki and following approval by the hospital's Ethics Committee. Healthy status was defined as the absence of autoimmune diseases, cancer, infectious diseases, dementia, and cardiovascular diseases that could interfere with the study. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation. CD28null and CD28+ T cells were purified using a CD4 Multisort kit and CD28 MicroBead kit (Miltenyi Biotec, Bergisch Gladbach, Germany) or sorted with a BD FACSARIA II cytometer (BD Bioscience, San José, CA, USA) after staining with CD4‐APC and CD28‐FITC monoclonal antibodies (Abs; BioLegend, San Diego, CA, USA). Purity was > 95% for all samples. Staurosporine (50 nm ; Enzo Life Science, Lausen, Switzerland) was added to isolated PBMCs for 18 h to induce apoptosis.
8
Synthesis and Characterization of (+)-Betulin
9
Evaluation of Apoptosis Inducers
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Sodium azide, anisomycin and doxycycline (dox) were purchased from Sigma (St Louis, MO, USA). zVAD-fmk, SP600125 and staurosporine were purchased from Enzo Life Sciences (Farmingdale, NY, USA). Etoposide was purchased from EBEWE Pharma (Kundl, Austria) and cisplatin was purchased from TEVA ABIC (Netanya, Israel).
10
Virus Infection and Apoptosis Induction
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