Vero E6 and Calu3 2b4 cells were infected with SARS-CoV-2 according to standard protocols described previously [9 (link),47 (link)]. Briefly, growth medium was removed, and cells were subsequently infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 diluted in 100 μl of PBS. Cells were then incubated for 45 minutes at 37°C and 5% CO2. After incubation, the inoculum was removed, cells washed three times with PBS, and fresh growth medium returned. For inhibitor experiments, Calu-3 cells were pretreated with 1–100 μM kenpaullone (Sigma-Aldrich #422000) in growth media for 1 hour at 37°C. Cells were then infected with SARS-CoV-2 at an MOI of 0.01 for 45 min at 37°C and 5% CO2. Inoculum was then removed, cells were then washed three times with PBS, and fresh growth media with kenpaullone added.
Kenpaullone
Manufactured by Merck Group
Kenpaullone is a small molecule that functions as a cyclin-dependent kinase (CDK) inhibitor. It has a molecular formula of C18H15N3O and a molecular weight of 301.35 g/mol. Kenpaullone exhibits inhibitory activity against various CDK enzymes, which are involved in cellular processes such as cell cycle regulation and signal transduction.
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Lab products found in correlation
Roscovitine, by Merck Group (3 mentions)
Torin1, by Bio-Techne (2 mentions)
Epidermal growth factor (egf), by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Sp600125, by Merck Group (2 mentions)
Doxycycline, by Merck Group (2 mentions)
Ly294002, by Cell Signaling Technology (2 mentions)
Calyculin a, by Cell Signaling Technology (2 mentions)
Sb203580, by InvivoGen (2 mentions)
Phorbol myristate acetate (pma), by Cell Signaling Technology (2 mentions)
Olomoucine, by Merck Group (2 mentions)
Horseradish peroxidase coupled secondary antibody, by Cell Signaling Technology (2 mentions)
Tumor necrosis factor (tnf), by Merck Group (2 mentions)
Limulus amoebocyte lysate assay, by Lonza (2 mentions)
M csf, by Merck Group (1 mentions)
Srankl, by Merck Group (1 mentions)
M csf, by Fujifilm (1 mentions)
Soluble rankl srankl, by Thermo Fisher Scientific (1 mentions)
Srankl, by Fujifilm (1 mentions)
Ddy mice, by Japan SLC (1 mentions)
11 protocols using kenpaullone
1
SARS-CoV-2 Infection Assay in Vero E6 and Calu-3 Cells
2
Modulation of Cell Signaling Pathways
3
Osteoclast Differentiation from Murine Precursors
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For the osteoclastogenic culture, RAW264.7 and pNFAT/Luc-RAW cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml soluble RANKL (sRANKL, Peprotech and Oriental Yeast), 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin [10] (link). For osteoclast formation by using primary pre-osteoclast cultures, murine bone marrow cells were obtained from femurs and tibiae of 7-week-old ddY mice (Japan SLC, Inc.). 4.0×105 cells were cultured in α-MEM medium containing 10% fetal bovine serum (FBS), 100 ng/ml sRANKL, 10 ng/ml M-CSF (Wako, JPN), 2 mM l -glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin in 24-well plates. Kenpaullone (Sigma), olomoucine (Sigma), roscovitine (Sigma) are used at a final concentration of 50 nM. The culture medium in each well was replaced by fresh medium containing M-CSF and sRANKL every 2 days. TRAP staining was performed after 5 days of the induction.
4
SARS-CoV-2 Infection of Vero E6 and Calu-3 Cells
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Vero E6 and Calu3 2b4 cells were infected with SARS-CoV-2 according to standard protocols described previously (9 (link), 44 (link)). Briefly, growth medium was removed, and cells were subsequently infected with SARS-CoV-2 at a multiplicity of infection (MOI) of 0.01 diluted in 100 μl of PBS. Cells were then incubated for 45 minutes at 37°C and 5% CO2. After incubation, the inoculum was removed, cells washed three times with PBS, and fresh growth medium returned. For inhibitor experiments, Calu-3 cells were pretreated with 1–100 μM kenpaullone (Sigma-Aldrich #422000) in growth media for 1 hour at 37°C. Cells were then infected with SARS-CoV-2 at an MOI of 0.01 for 45 min at 37°C and 5% CO2. Inoculum was then removed, cells were then washed three times with PBS, and fresh growth media with kenpaullone added.
5
Isolation of GST-p12 Protein Complexes
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GST-p12 proteins were expressed in 293T cells from pCAGGS/GST-derived plasmids by transient transfection using Turbofect (Thermo Fisher Scientific). Approximately 24 h after transfection, cells were changed into fresh media containing 400 ng/ml nocodazole (Sigma) and incubated overnight at 37°C. Cells were then changed into media containing 400 ng/ml nocodazole, 10 μM MG132 (Sigma) and either 40 mM LiCl (Sigma), 75 μM roscovitine (Sigma) or 40 μM kenpaullone (Sigma), for 3.5 h. Cells were immediately washed and lysed for glutathione-sepharose pull-down assays as described above.
6
Fluorescent PFK1 Fusion Protein Protocol
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A plasmid expressing PFK1 conjugated with a monomeric enhanced green fluorescent protein (mEGFP) was prepared previously [4 (link)]. Note that mEGFP contains three point mutations, A206K, L221K, and F223R, which prevent oligomerization of EGFP in cells [30 (link),31 (link)]. Small molecules used in this study were dissolved in DMSO (Sigma) and used at the concentrations indicated, that include resveratrol (Sigma), clemastine fumarate (Sigma), ARP101 (Tocris), 1,10-phenanthroline (Acros Organics), triflupromazine hydrochloride (Sigma), kenpaullone (Sigma), calmidazolium chloride (Sigma), PAC-1 (Sigma), SU9516 (Tocris), SL-0101-1 (Tocris), palbociclib (Sigma), BS-181 (Tocris), PF-03814735 (Sigma or Cayman), hesperadin (Tocris), bisindolylmaleimide II (BIM II, Cayman), and BIM X (Cayman).
7
Cytoprotective Screening Against Oxidative Stress
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Cells were exposed to glutamate, H2O2 (Sigma), sodium nitroprusside (SNP, a donor of nitric oxide, Sigma), and ZnCl2 (Sigma) in minimum essential media (Gibco) for 24 h at the indicated concentrations to induce cell death. Anthranilic acid, clotrimazole, dantrolene, flufenamic acid, kenpaullone, NU6027, olomoucine, roscovitine, ruthenium red, SU9516, N,N,N,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), and 2-aminoethyl diphenylborinate (2-APB) were purchased from Sigma. Capsaicin, MK-801, ML204, Pyr3, and (−)-Xestospongin C (XeC) were purchased from Tocris. SB216763 was purchased from Enzo Life Science, and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) was purchased from RBI. All reagents were added 1 h prior to H2O2 exposure, unless otherwise stated.
8
Kinase Inhibition for Cell Signaling
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For stimulation and long-term incubation experiments, TNF from Sigma Aldrich was used. To suppress protein degradation in cell lysates, the complete ethylenediaminetetraacetic acid-free protease inhibitor cocktail (Roche, Rotkreuz, Switzerland) was applied. Inhibitor experiments were performed using the kinase inhibitor Staurosporine (Stauro) and the GSK3α/β inhibitor Kenpaullone (Ken; Sigma Aldrich). For Western blotting, antibodies specific for PKCβ (D3E70), p-PKCα/βII (Thr638/641), GSK3β (27C10), p-GSK3α/β (Ser21/9), p65 (L8F6), p-p65 (Ser536), IKKα, p-IKKα/β (Ser176/180), IκBα (44D4; Cell Signalling, Danvers, USA), B cell lymphoma (Bcl) 3 (150–3.5; Santa Cruz Biotechnology, Santa Cruz, USA), actin, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Sigma-Aldrich) were used. Horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signalling or Santa Cruz. All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza, Basel, Switzerland).
9
Protein Expression and Immunoblotting Protocol
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TNF was purchased from Sigma-Aldrich (Darmstadt, Germany) and PMSF (a protease inhibitor applied for validation experiments) from Roth (Karlsruhe, Germany). Inhibitor experiments were performed using the GSK3α/β inhibitor Kenpaullone (Sigma-Aldrich). For Western blotting, antibodies specific for CD44 (156-3C11), p-C/EBPβ (Thr235), GSK3β (27C10), IDO (D5J4E), MARCKS (D88D11), NFKB1-p105/p50, NFKB2-p100/p52, p65 (L8F6), RELB (C1E4), VIM (5G3F10), p-VIM (Ser56; all from Cell Signalling, Danvers, MA, USA), p-CD44 (Ser706; Santa Cruz Biotechnology, Santa Cruz, CA, USA), C/EBPβ (E299; Abcam, Cambridge, UK), KYNU (Biozol, Eching, Germany), p-MARCKS (Ser159), p-RELB (Ser573; Thermo Fisher), p-GSK3α/β (Tyr279/Tyr216), actin (11C), GAPDH, and TBP (Sigma-Aldrich) were used. Horseradish peroxidase-coupled secondary antibodies were purchased from Cell Signalling or Vector Laboratories (Burlingame, CA, USA). All media and reagents were of the best available grade and routinely tested for endotoxins with the Limulus Amoebocyte Lysate assay (Lonza, Basel, Switzerland).
10
Compounds for Biological Assays
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AT7519 (APE, A5719), TRO 19,622 (Bio-Techne, 2906), AZD5438 (Bio-Techne, 3968), dexpramipexole (Sigma, SML0392), 1-azakenpaullone (Sigma, A3734), alsterpaullone (Sigma, A4847) and kenpaullone (Sigma, K3888) were purchased and dissolved in DMSO as stock. Compound doses were obtained following dose response experiments and at the doses used no significant effect on cell viability was observed.
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