LPS (E. coli 0111:B4), BN82002, and BAY-11-7082 were purchased from Sigma Chemical Co. (St. Louis, MO). LY294002 was purchased from EMD Millipore (Billerica, MA, USA). PP2 (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine) and staurosporine (STS) were purchased from Merck (Germany). Fetal bovine serum (FBS) and Roswell Park Memorial Institute (RPMI) 1640 were obtained from Gibco (Grand Island, NY). RAW264.7 cells, a BALB/c-derived murine macrophage cell line (ATCC No. TIB-71). Antibodies to phospho-specific and total protein of p50, p65, IκBα, IKKα/β Catalog No.#2694) ERK, JNK, Akt, STAT3, Syk, Src, Lyn, p85, β-actin, and lamin A/C were obtained from Cell Signaling (Beverly, MA, USA).
Staurosporine st
Manufactured by Merck Group
Sourced in United States, Germany, Canada
Staurosporine (STS) is a compound that inhibits a variety of protein kinases. It is commonly used as a laboratory tool in cell biology and biochemical research. STS functions as a broad-spectrum protein kinase inhibitor, affecting the activity of various enzymes involved in cellular signaling pathways.
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Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (4 mentions)
β actin, by Cell Signaling Technology (2 mentions)
Taxol, by Merck Group (2 mentions)
Etoposide eto, by Merck Group (2 mentions)
Bay11 7082, by Merck Group (1 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Ly294002, by Merck Group (1 mentions)
Raw 264.7 cells, by American Type Culture Collection (1 mentions)
Stat3, by Cell Signaling Technology (1 mentions)
Lamin a c, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc apoptosis detection kit, by Immunostep (1 mentions)
Z vad fmk, by Merck Group (1 mentions)
High glucose dmem, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Thermo Fisher Scientific (1 mentions)
Penicillin, by Thermo Fisher Scientific (1 mentions)
Annexin 5 pi kit, by Beyotime (1 mentions)
Dmem glutamax medium, by Thermo Fisher Scientific (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
N acetyl l cysteine nac, by Merck Group (1 mentions)
Mitotempo, by Merck Group (1 mentions)
40 protocols using staurosporine st
1
Inflammatory Signaling Pathway Analysis
2
Annexin V-FITC Apoptosis Detection Assay
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The cell death type induced by compounds was determined with an Annexin V-FITC Apoptosis detection kit (Immunostep, Salamanca, Spain) following the manufacturer’s instructions [56 (link)]. Cells were seeded in 12-well plates at 1 × 106 cells per well and treated with compounds at IC50 concentrations for 6 and 24 h. Then, cells were washed, resuspended in PBS (Phophate-Buffered Saline), and 5 μL of Annexin V-FITC and Propidium Iodide (PI) were added to each tube. Cells were incubated for 15 min in the dark and analyzed by flow cytometry using the ImageStreamMKII instrument (Amnis Corporation, LuminexCorp, Austin, TX, USA). The fluorescence of 10,000 events was analyzed with IDEAS Application 6.0 software (Amnis Corporation, LuminexCorp). The percentages of apoptotic cells, including early apoptotic cells (Annexin-FITC positive and PI negative) and late apoptotic cells (Annexin-FITC and PI-positive), and necrotic cells (Annexin-FITC-negative and PI-positive), were calculated. To further confirm if apoptotic cell death was occurring, SH-SY5Y cells were preincubated with the pan-caspase inhibitor Z-VAD-FMK (Merck) for 24 h. Then, the assay was carried out as described above. Staurosporine (STS) (Merck) was used as a positive control in all the experiments [56 (link)].
3
MCF-7 Cell Culture and Staurosporine Treatment
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MCF-7 was a gift from Professor Jing-Rong Cui (Peking University) and cultured in DMEM high glucose (Life Technologies, USA) supplemented with 100 U/ml penicillin, 100 μg/ml streptomycin, and 10% heat inactivated FBS (Life Technologies, USA), and maintained at 37°C and 5% CO2. Staurosporine (STS) was purchased from Merck (Darmstadt, Germany).
4
Apoptosis Induction Assay Protocol
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Cells were cultured in a 6-wells plate. After being treated with 0.05 μM staurosporine (STS) (Sigma, USA) for 24 h [20 (link)], cells were washed with a cold Phosphate Buffered Saline (PBS) solution followed by digested with trypsin. Digested cells were processed by the Annexin V-PI kit (Beyotime, PRC) according to the manufacturer’s instructions. The result was measured by flow cytometry.
5
Microglia Treatments and Assessments
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BV2 mouse microglia cells were cultivated in DMEM + glutamax medium (Gibco, Fisher Scientific, Waltham, MA, USA) supplemented with 10% FBS (fetal bovine serum) and 1% P/S (Penicillin/Streptomycin) and grown in an incubator at 37 °C and 5% CO2. For treatment conditions, microglia cells were seeded in 6 or 12 well plates the day before the treatment at 200,000 or 100,000 cells per well. 24 h after seeding, cells were treated with either 20 μM Caspase-3 inhibitor (DEVD-fmk, R&D, Minneapolis, MN, USA), 100 ng/mL Lipopolysaccharides (LPS, Sigma Aldrich, Saint Louis, MO, USA), 0.5 μM Staurosporine (STS, Sigma Aldrich) or DMSO used as control for 2 h before harvest.
6
Inducing Apoptosis in Cervical Cancer Cells
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In a preliminary experiment to determine the optimal T. vaginalis/SiHa cell ratio for inducing apoptosis, SiHa cell monolayers (1×106) were washed with PBS (pH 7.4), and live T. vaginalis trophozoites were incubated in mixed-medium (DMEM/TYM = 2∶1) at multiplicities of infection (MOIs) of 0.5, 1, and 2 for 12, 16, and 24 h. The optimal conditions for inducing apoptosis were found to be an MOI of 2 and incubation for 16 h (Fig. S1 in File S1 ).
Next, 100 µg/mL T. vaginalis ESP or lysate was used to induce apoptosis in SiHa cells and MS74 cells. As a positive control, apoptosis was induced by treatment with staurosporine (STS, 1 µM) (Sigma-Aldrich) under identical conditions.
Next, 100 µg/mL T. vaginalis ESP or lysate was used to induce apoptosis in SiHa cells and MS74 cells. As a positive control, apoptosis was induced by treatment with staurosporine (STS, 1 µM) (Sigma-Aldrich) under identical conditions.
7
Investigating Cytotoxic Agents and Apoptosis
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Cells were treated with PST, PST Analogs, Taxol (Sigma-Aldrich Canada, Cat. No. T7402, Mississauga, ON, Canada), Staurosporine (STS) (Sigma-Aldrich Canada, Cat. No. S4400, Mississauga, ON, Canada), Doxorubicin (DOX) (Sigma-Aldrich Canada, Cat. No. D1515, Mississauga, ON, Canada), Gemcitabine (GEM) (Sigma-Aldrich Canada, Cat. No. G6423, Mississauga, ON, Canada), piperlongumine (PL) (INDOFINE Chemical Company, Inc., Cat. No. P-004, Hillsborough, NJ, USA), the broad spectrum caspase inhibitor, Z-VAD-FMK (EMD Chemicals, Gibbstown, NJ, USA), Antimycin A (AMA) (Sigma-Aldrich Canada, Cat. No. A8674, Mississauga, ON, Canada), Thenoyltrifluoroacetone (TTFA) (Sigma-Aldrich Canada, Cat. No. T27006, Mississauga, ON, Canada), rotenone (ROT) (Sigma-Aldrich Canada, Cat. No. R8875, Mississauga, ON, Canada), carbonyl cyanide-4-(trifluoromethoxy)phenylhydrazone (FCCP) (Sigma-Aldrich Canada, Cat. No. C2920, Mississauga, ON, Canada), mitoTEMPO (Sigma-Aldrich Canada, Cat. No. SML0737, Mississauga, ON, Canada) and EM20-25 (Sigma-Aldrich Canada, Cat. No. SML0183, Mississauga, ON, Canada) dissolved in DMSO stock solutions. N-Acetyl-L-cysteine (NAC) (Sigma-Aldrich Canada, Cat. No. A7250) was dissolved in double distilled water. PST analogs were produced by synthesis from bromobenzene24 (link)55 (link).
8
Apoptosis Induction in MSCs and MEFs
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MSCs were treated with 1.25 μM of ABT199 (BCL-2 inhibitor, iBCL2), A1331852 (BCL-XL inhibitor, iBCLxL) and S63845 (MCL-1 inhibitor, iMCL1) in MSC media for 2 h, 24 h or 72 h at 37 °C, 5% CO2. Both adherent and detached cells were collected, washed and stained with AnnexinV and PI to detect apoptosis by flow cytometry. In some experiments, as indicated, MSCs were treated with 0.5 μM staurosporine (STS) (Sigma-Aldrich) in MSC medium for 6 h. MEFs were treated with 10 μM of iBCL2, iBCLxL and iMCL1 for up to 20 h.
9
ZnO Nanoparticles Cytotoxicity Evaluation
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For treatments with ZnO nanoparticles, after 24 h from seeding, CTC medium was substituted with RPMI supplemented with 1% non-essential amino acids, 2 mM glutamine, 50 IU/ml penicillin and 50 µg/ml streptomycin, 1% water without FBS (Serum-Free Culture, SFC, medium). In all the experiments cells were treated with ZnO NPs (1, 5 and 20 μg/cm2), for 24, 48, 72 and 144 h. We used 1 μM Staurosporine (STS, Sigma-Aldrich, Saint Lois, Missouri, USA) as positive control of apoptosis induction and 10 μM Rapamycin (Rap, Sigma-Aldrich) as an autophagic positive control for 24, 48, 72 and 144 h.
10
Differentiation of 661W cells
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The 661W cells were cultured in 10% fetal bovine serum supplemented Dulbecco's modified Eagle medium, and cells were incubated at 37°C and 5% CO2 until they reached 70% to 80% confluence before experiments. Cell identity was confirmed as Mus musculus origin by PCR of the mitochondria d-loop40 (link) and their ability to be differentiated.41 (link),42 (link) Differentiation of wild type and BAX-edited 661W cells (line Sg1, described below) was achieved by addition of 316 nM staurosporine (STS; Sigma-Aldrich, St. Louis, MO, USA) dissolved in DMSO and brought up in full media and incubated for 0, 0.5, 1, 6, 12, or 24 hours. Differentiation was carried out 24 hours before transfection for the initial BAX recruitment assay, or differentiation was initiated 24 hours after nucleofection in subsequent experiments. The differentiated state of cells was confirmed by visualization of neurite extensions using differential interference contrast microscopy.
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