The prostate cancer cell lines, PC3 (Sigma-Aldrich, 90112714), VCaP (Sigma-Aldrich, 06020201) and DU145 (ATCC, HTB-81), the normal prostate epithelial PNT2 cell line (ECACC, 95012613), the colorectal cancer cell line HT29 (Sigma-Aldrich, 91072201), and the breast cancer cell line MCF7 (Sigma-Aldrich, 86012803) were cultured in high glucose Dulbecco’s modified Eagle’s medium (DMEM), supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine-penicillin-streptomycin and grown at 37 °C with 5% CO2. Hypoxia was attained in a modular incubator chamber (MIC-101; Billups-Rothenberg, USA) using a gas mixture containing; 1% O2 / 5% CO2 / 94% N2 (BOC, UK). The chamber was flooded with the hypoxic gas mixture for 2 min and then sealed and stored in an incubator for 48 h at 37 °C in 5% CO2. The normoxic control was stored in the same incubator for the same amount of time. CLK1 was inhibited with the benzothiazole TG003 (Sigma-Aldrich) at 10–50 μM; the TG003 stock was dissolved in dimethylsulphoxide (DMSO).
Tg003
Manufactured by Merck Group
Sourced in United States
TG003 is a lab instrument designed for thermal analysis. It is capable of measuring the thermal properties of materials, such as their melting point, glass transition temperature, and thermal stability, through the use of specialized techniques like differential scanning calorimetry (DSC) and thermogravimetric analysis (TGA).
Automatically generated - may contain errors
Lab products found in correlation
Turbofect, by Thermo Fisher Scientific (3 mentions)
Harmine, by Merck Group (2 mentions)
Ucn 01, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Srpin340, by Merck Group (2 mentions)
Htb 81, by American Type Culture Collection (1 mentions)
Mcf 7, by Merck Group (1 mentions)
Ht 29, by Merck Group (1 mentions)
Mic 101, by Embrient Inc (1 mentions)
Du145, by American Type Culture Collection (1 mentions)
Liquid scintillant, by Thermo Fisher Scientific (1 mentions)
Anti srpk1 monoclonal antibody, by BD (1 mentions)
Dnase, by Thermo Fisher Scientific (1 mentions)
Rnase, by Thermo Fisher Scientific (1 mentions)
Phenix imaging film, by Thermo Fisher Scientific (1 mentions)
Protein g agarose, by Thermo Fisher Scientific (1 mentions)
Ni resin, by Thermo Fisher Scientific (1 mentions)
Hybond ecl nitrocellulose blotting membrane, by Cytiva (1 mentions)
The kinasemax kit, by Thermo Fisher Scientific (1 mentions)
Instantblue, by Abcam (1 mentions)
9 protocols using tg003
1
Prostate, Colorectal, and Breast Cancer Cell Lines
2
Screening Antiviral Compounds Against HIV-1 and HCoV-229E
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Screening of compounds (L41 [10 µM], TG003 [10 µM], KH-CB19 [30 µM], and harmine [10 µM]; Sigma-Aldrich) for their effect against HIV-1 was done in HeLa B2 cells. To evaluate the compounds’ (L41 [1 mM], TG003 [50 µM], KH-CB19 [50 µM], harmine [25 µM], 1H3 [250 nM], and 2E3 [250 nM]) effect on seasonal coronavirus HCoV-229E strain, Huh7 cells were used. Compounds were prepared by dissolving in dimethyl sulfoxide, aliquoted, and stored at −20°C. For screening against HIV-1, cells were seeded on a 12-well dish in IMDM complete medium and the following day induced with Dox and treated with DMSO or compounds at the same time. Cells were harvested 24 h after treatment, and lysates were examined for HIV-1 Env gp160, Gag, and Tat p16 and p14 protein levels by Western blotting. For screening against HCov-229E, Huh7 cells were seeded on a 96-well dish in DMEM containing 10% FBS (D10), and the following day, cells were infected with the virus at an multiplicity of infection (MOI) of 2 in serum-free DMEM (D0) for an hour, after which the virus was removed and cells were treated with the above compounds in DMEM supplemented with 2% FBS (D2). Sups were harvested 24 h post-infection (hpi) to quantify viral genomic RNA release in media by RT-qPCR assay.
3
Comprehensive Biochemical Reagents Protocol
4
High-Throughput Screening of Small Molecules
5
NPC Treatment with Inhibitors
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Cell aggregates were dissociated on day 12 with Accumax (Innovative Cell Technologies) and replated onto Corning® Matrigel® Growth Factor Reduced (GFR) Basement Membrane Matrix-coated dishes in DFK 5% medium. NPCs were treated with TG003 (Sigma-Aldrich, 50 μM), SRPIN340 (50 μM) or Okadaic acid (FUJIFILM Wako Pure Chemical Corporation, 10 nM) for 3 h, then harvested, and RNA was isolated as described above. SRPIN340 was used as previously described [24 ].
6
Cell culture and treatment protocols
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Human colon carcinoma BE cells (a gift from S. Wilkinson and C. Marshall, Institute of Cancer Research, London, UK; Petsalaki et al., 2018 (link)) and cervical carcinoma HeLa cells stably expressing LAP2b fused to RFP (a gift from D. Gerlich, Institute of Molecular Biotechnology, Vienna, Austria; Steigemann et al., 2009 (link)) were grown in DMEM (Gibco) containing 10% FBS at 37°C in 5% CO2. Cells were treated with 300 nM UCN-01 (U6508; Sigma-Aldrich), 10 µM PP2 (1407; Tocris Bioscience), or 1 µM TG003 (T5575; Sigma-Aldrich) as appropriate. Negative siRNA or siRNA duplexes designed to repress human Chk1, Src, or Aurora B were transfected into BE cells 24 h before analysis using Lipofectamine 2000 (Invitrogen). For expression of GFP proteins, plasmids were transfected into cells in the absence or presence of appropriate siRNA duplexes 24 h before analysis or further drug treatment using Turbofect (Thermo Fisher Scientific). All cell lines used exhibited consistent morphology and growth properties and were negative for mycoplasma contamination.
7
Neonatal Rat Cardiomyocyte Isolation and SRPK1/CLK1 Inhibition
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NRCMs were isolated from one-day-old rats, using the neonatal cardiomyocyte isolation system, as described previously [43 (link),44 (link)]. The cells were re-suspended in complete media (M199/DMEM media supplemented with 20% fetal calf serum (FCS) and 1% penicillin/streptomycin), plated at a density of 1 × 106 cells per cm2, and maintained in 5% CO2 at 37 °C. Cells were cultured for two days in complete media; supplemented with either the SRPK1 inhibitor SRPIN340 (25 μM) (Sigma-Aldrich, St. Louis, MO, USA, cat#SML1088) or the CLK1 inhibitor TG003 (10 μM) (Sigma-Aldrich, cat#T5575); and collected after 0 min, 5 min, 1 h, 6 h, 12 h, 24 h, or 48 h. Treatment with vehicle (DMSO) was used as a control. Harvested cells were lysed for protein and RNA preparation.
8
Imaging Carcinoma Cell Lines with Genetic Markers
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Human colon carcinoma BE cells (a gift from Simon Wilkinson and Christopher Marshall, Institute of Cancer Research, London, UK) and cervical carcinoma HeLa cells stably expressing LAP2b fused to RFP (a kind gift from Daniel Gerlich, Institute of Molecular Biotechnology, Vienna, Austria)5 (link) were grown in DMEM medium (Gibco) containing 10% fetal bovine serum at 37 °C in 5% CO2. BE cells stably expressing H2B fused to GFP were as described11 (link). Cells were treated with 1 μM TG003 (Sigma) or 300 nM UCN-01 (Sigma) for 5 h before analysis, unless otherwise stated. Negative siRNA or siRNA duplexes designed to repress human Aurora B, Chk2, Clk1, Clk2, Clk4, Chmp4c, CENP-A or Nup153 were transfected into BE cells 24 h before analysis using Lipofectamine 2000 (Invitrogen). For expression of GFP proteins, plasmids were transfected into cells in the absence or presence of appropriate siRNA duplexes 24 h before analysis or further drug treatment using Turbofect (Life Technologies).
9
Neonatal Rat Cardiomyocyte Inhibitor Assay
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Primary cultures of NRCMs were prepared from one-day old rats using the neonatal cardiomyocyte isolation system as described previously [35] [36] . The cells were re-suspended in complete medium (M199/DMEM media and 20% fetal calf serum (FCS) supplemented with 1% penicillin/streptomycin), plated at a density of 1x10 6 cells per cm 2 , and maintained in 5% CO2 at 37 °C. Cells were cultured for two days in complete media, and then supplemented with SRPK1 inhibitor SRPIN340 (25 μM) (Sigma-Aldrich, St. Louis, MO, Cat#SML1088) and CLK1 inhibitor TG003 (10 μM) (Sigma-Aldrich, St. Louis, MO, Cat#T5575) respectively for 0 min, 5 min, 1h, 6h, 12h, 24h, and 48h. The vehicle DMSO treatment was used as control. Harvested cells were lysed for protein and RNA preparation.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
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