H3122 cells were obtained from the National Cancer Institute. Generation of ceritinib-resistant H3122 cells is described in the Supplemental Experimental Procedures. H2228, HCC827, and HCC4006 were purchased from the American Type Culture Collection (ATCC). PC-9 cells were obtained from Public Health England, and KELLY neuroblastoma cells were obtained from Sigma-Aldrich. Cells were maintained in RPMI-1640 (Cellgro) with 10% fetal bovine serum (Gemini Bioproducts) and penicillin (100 units/mL) / streptomycin (100 µg/mL; Cellgro). MGH006 cells have been previously reported and were maintained in DMEM (Cellgro) with 10% fetal bovine serum, penicillin, and streptomycin (Sequist et al., 2010 (link)). All cell lines were tested to confirm the absence of mycoplasma contamination. Crizotinib, TAE684, ceritinib, erlotinib, lapatinib, and sotrastaurin were purchased from Selleck Chemicals. Blasticidin was obtained from Life Technologies. Phorbol 12-myristate 13-acetate (PMA) was purchased from Santa Cruz Biotechnology.
Sotrastaurin
Manufactured by Selleck Chemicals
Sourced in United States
Sotrastaurin is a laboratory chemical compound used for research purposes. It is a protein kinase C inhibitor. The core function of Sotrastaurin is to inhibit the activity of protein kinase C, an enzyme involved in various cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Sb203580, by Selleck Chemicals (3 mentions)
G 6983, by Selleck Chemicals (2 mentions)
R0 31 8220, by Selleck Chemicals (2 mentions)
G 6976, by Bio-Techne (2 mentions)
Chir99021, by Selleck Chemicals (2 mentions)
Ro 3306, by Selleck Chemicals (2 mentions)
M2 agarose bead, by Merck Group (2 mentions)
Sirna for hfbxl21, by Horizon Discovery (2 mentions)
Ic261, by Selleck Chemicals (2 mentions)
Nu7441, by Selleck Chemicals (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ionomycin, by Merck Group (2 mentions)
Acetylcysteine, by Selleck Chemicals (2 mentions)
Wortmannin, by Cayman Chemical (2 mentions)
Sb600125, by Cayman Chemical (2 mentions)
U0126, by Cayman Chemical (2 mentions)
Penicillin, by Corning (1 mentions)
Streptomycin, by Corning (1 mentions)
Fetal bovine serum (fbs), by Corning (1 mentions)
Crizotinib, by Selleck Chemicals (1 mentions)
21 protocols using sotrastaurin
1
Generation and Maintenance of Cell Lines
2
Comprehensive Reagents and Inhibitors Protocol
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Lysotracker Red DND-99 (L7528) was from ThermoFisher Scientific. Antimycin A (A8674), DFP (379409), DMOG (D3695), SGC-GAK-1 (GAKi, SML2202), SGC-GAK-1N (GAKc, SML2203) and QVD-OPh (SML0063) were from Sigma Aldrich. Bafilomycin A1 (BML-CM110), CCCP (BML-CM124) were from Enzo Life Sciences. Enzastaurin (S1055), oligomycin A (S1478), and sotrastaurin (S2791) were from Selleckchem. MRT68921 (1190379-70-4) and VPS34-IN1 (1383716-33-3) were from Cayman Chemical. IVAP1966 (12 g) and IVAP1966 (12i) were gratefully received from the lab of Prof. Piet Herdewijn29 . HY-19764 was gratefully received from the structural genomics consortium30 (link). Bradford reagent dye (#5000006) was from Bio-Rad. 1,4-dithiothreitol (DTT, #441496P) was from VWR. Complete EDTA-free protease inhibitors (#05056489001) and phosphatase inhibitors (#04906837001) were from Roche.
3
PKCα and NF-κB Signaling Pathway Analysis
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Rabbit monoclonal antibody against PKCα (Phospho T638) (1:500 dilution) and rabbit polyclonal antibodies against PKCα (1:2000 dilution), NF-κB p65 (1:2000 dilution), and Histone H3 (1:3000 dilution) were purchased from Abcam (Cambridge, MA, USA). The rabbit polyclonal antibody against α-Tubulin (1:5000 dilution) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Tumor necrosis factor (TNF) -α was purchased from R&D systems (Minneapolis, MN, USA). It was reconstituted at 100 μg/ml in sterile PBS and stored at −80 °C; the TNF-α solution was diluted in serum-free medium to a concentration of 10 ng/ml when added to the cells. BAY 11–7082, Gö6976 and Sotrastaurin were purchased from Selleckchem (Houston, TX, USA). They were reconstituted in DMSO, and when added to the cells, 10 μL of DMSO was added per 1.0 ml of media as the control. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
Tumor necrosis factor (TNF) -α was purchased from R&D systems (Minneapolis, MN, USA). It was reconstituted at 100 μg/ml in sterile PBS and stored at −80 °C; the TNF-α solution was diluted in serum-free medium to a concentration of 10 ng/ml when added to the cells. BAY 11–7082, Gö6976 and Sotrastaurin were purchased from Selleckchem (Houston, TX, USA). They were reconstituted in DMSO, and when added to the cells, 10 μL of DMSO was added per 1.0 ml of media as the control. Phorbol 12-myristate 13-acetate (PMA) was purchased from Sigma-Aldrich (St. Louis, MO, USA).
4
Synergistic Anticancer Compound Screening
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Cells were seeded in triplicate, in 96-well format. Next day, compounds were added and cells were incubated for 72 h. Cell survival was determined using CellTitre-Blue Cell Viability assay (Promega); fluorescence was measured in a microplate reader (Victor3, Perkin Elmer, San Jose, CA, USA). Synergism between Sotrastaurin and Nutlin-3 was calculated using Compusyn software (Paramus, NJ, USA). Sotrastaurin and Nutlin-3 were obtained from Selleck Chemicals (Houston, TX, USA) and Cayman Chemical (Ann Arbor, MI, USA), respectively.
5
Stimulation and Inhibition of Jurkat Cells
6
Stimulation and Inhibition of Jurkat Cells
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For stimulation of Jurkat cells, we set up independent replicates by diluting cells to ~3.5 × 105 cells/mL and treating with 20 ng/mL phorbol 12-myristate 13-acetate (PMA) for 48 h prior to cell harvest. Unstimulated JSL1 cells were cultured in parallel for each replicate at a seeding concentration of ~2.5 × 105 cells/mL. For inhibition studies, cells were pre-treated with inhibitors SP600125 (JNK, 50 μM, Calbiochem, 420,119), Gö6983 (PKC, 5 μM, Selleck, S2911), R0-31-8220 (PKC, 1 μM, Selleck, S7207), Sotrastaurin (PKC, 5 μM, Selleck, S2791), MK2206 (AKT, 5 μM, Selleck, S1078), or GDC-0068 (AKT, 1 μM, Selleck, S2808) for 1 h before stimulating as described above. Transcription inhibition was achieved by incubation of cells with actinomycin D for 0–12 h (5 μg/mL final concentration). Translation inhibition was achieved by incubation of cells with cycloheximide for 0–12 h (5 μg/mL final concentration). Expression of short hairpin RNAs (shRNAs) and cDNA expression plasmids (see Cloning and CRISPR designs and Transfections below) was achieved by pre-treating cells with doxycycline for 24 h (1 mg/mL final concentration) prior to setting up unstimulated/stimulated cultures as described above. For PMA time course experiments, cells were diluted to ~3.5 × 105 cells/mL, treated with PMA and collected at 0, 3, 6, 9, 12, 24, 36, and 48 h post-PMA treatment.
7
Osteoblast and Osteocyte Cell Culture
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Cell culture and experiments with UMR106 rat osteoblast-like cells (purchased from ATCC, Manassas, VA, USA) were conducted as described before [39 (link)]. Briefly, cells were cultured in DMEM high-glucose medium containing 10% FBS and penicillin-streptomycin at 37°C and 5% CO2.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
IDG-SW3 bone cells (purchased from Kerafast, Boston, MA, USA) were cultured as described earlier [40 (link)]. Briefly, non-differentiated cells were kept at 33°C in AlphaMEM medium (with L-glutamine and deoxyribonucleosides) containing 10% FBS, penicillin-streptomycin and interferon-gamma (INF-γ; 50 U/ml). For differentiation, cells were plated on collagen-coated dishes at 37°C in medium with 50 μg/ml ascorbic acid and 4 mM β-glycerophosphate but without INF-γ. All reagents were from ThermoFisher unless indicated.
IDG-SW3 osteocytes were used after 35 days of differentiation, and UMR106 cells were pretreated with 100 nM 1,25(OH)2D3 (Tocris, Wiesbaden-Nordenstadt, Germany) for 24h before the experiment. Cells were then incubated with activator phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA; Sigma, Schnelldorf, Germany; 0.1 μM; 6 h) with or without 1 μM PKC inhibitors calphostin C (Tocris), Gö6976 (Tocris), sotrastaurin (Selleckchem, München, Germany), ruboxistaurin (Selleckchem), or NFκB inhibitor withaferin A (Tocris; 0.5 μM), or with vehicle only for another 24 h.
8
Inhibition Strategies for Cellular Signaling
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Inhibitors used in this study were as follows: CHIR 99021 (5, 15, or 30 μM, Stemgent) and LiCl (40 mM, Sigma) for GSK3β inhibition; LY294002 (50 μM, Cell Signaling) for PI3K inhibition; MK2206 (20 μM, Selleckchem) and perifosine (25 μM, Selleckchem) for AKT inhibition; KU-60019 (10 or 20 μM, Selleckchem) for ATM inhibition; sotrastaurin (10 μM, Selleckchem) for pan-PKC inhibition; z-VAD-fmk (100 μM, BD Pharmingen) for pan-caspase inhibition; calyculin A (5 or 10 nM, Melck Millipore) and okadaic acid (1 μM, Melck Millipore) for protein phosphatase inhibition.
9
Quantifying TCAP Degradation Dynamics
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293T (ATCC CRL-3216) cells and C2C12 (ATCC CRL-1772) were cultured in DMEM supplemented with 10% fetal bovine serum (GenDEPOT). For immunoprecipitations, 2 × 106 cells were plated into 60 mm dishes 1 day before transfection, and iMFectin (GenDEPOT) was used to transfect DNA according to the manufacturer’s protocol. M2 agarose bead (Sigma) was used to pull down flag tagged proteins. TCAP degradation assays were performed by transfecting pCMV10-3XFlag-TCAP with or without Fbxl3 and Fbxl21 expression constructs into 2 × 105 293T cells in a 12-well plate. Thirty-six hours after transfection 100 μg/ml cycloheximide (CHX) was added and cells collected at the indicated times. The incubation time length was chosen to avoid significant cytotoxicity while still sufficient to distinguish effects. Half-life was determined by using nonlinear, one-phase decay analysis (GraphPad Prism). siRNA for hFbxl21 was purchased from Dharmacon and transfection was performed by using Lipofectamine 2000 (Invitrogen). CHIR99021, Ro3306, NU7441, Sotrastaurin, and IC261 were purchased from Selleckchem.
10
Automated Quantification of Neutrophil Extracellular Traps
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WT iNeutrophils, GATA1 KO and primary neutrophils were plated in IMDM (Gibco, 21056023) at a density of 5 x 104 cells per well in ultra-low base 384 well dishes (Aurora, ABD241001A) and stimulated using 50 nM PMA (Sigma-Aldrich, P1585), 25 μg/ml LPS O128:B12, (Sigma-Aldrich, L2887) or 5 μM A23187 (Sigma-Aldrich, C7522) for 3 hours at 37°C. For inhibition studies, cells were pre-treated with 20 μM sotrastaurin (Selleck Chemicals, S2791), 20 μM DPI (Sigma-Aldrich, D2926), 100 μM 4-ABAH (Sigma-Aldrich, A41909) or 20 μM disulfiram (Tocris, 3807) for 1 hour followed by 3 hours of stimulation using 50 nM PMA. After the 3 hour stimulation, a fix/perm/stain solution was added for a final concentration of 2% paraformaldehyde (Electron Microscopy Services, 15710), 0.1% Triton X100 (Sigma-Aldrich, X100-100ML) and 50 nM Sytox Green (Invitrogen, S7020). Nine fields per well were imaged using the Yokogawa CV8000 automated microscope at 20x magnification. Image features were extracted using CellProfiler followed by analysis using custom supervised machine learning software to classify NET versus non-NET nuclei based on nuclei features including size, shape, intensity, etc. Experiments were performed on at least three independent differentiations and three independent donors in at least technical triplicates.
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