Cells were treated for 48 h with 50 μM sirtinol (Tocris), with camptothecin 30 nM for 24 h or pre‐treated with sirtinol for 24 h and then incubated with camptothecin and sirtinol for another 24 h as indicated. Cells were then washed three times with PBS and left to form colonies for 7–14 days. Colonies were stained with 0.1% (w/v) crystal violet in 20% (v/v) ethanol for counting. Results were normalised to plating efficiencies of untreated cells.
Sirtinol
Manufactured by Bio-Techne
Sourced in United Kingdom
Sirtinol is a small molecule inhibitor that selectively targets SIRT1, a member of the sirtuin family of NAD+-dependent protein deacetylases. It inhibits SIRT1 activity in a dose-dependent manner.
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Lab products found in correlation
Dorsomorphin, by Merck Group (1 mentions)
Wst 1, by Roche (1 mentions)
Lys c, by Fujifilm (1 mentions)
Trypsin, by Worthington (1 mentions)
Penicillin streptomycin, by Harvard Bioscience (1 mentions)
Fetal bovine serum (fbs), by Harvard Bioscience (1 mentions)
Ultrapure lps from e coli 0111 b4, by InvivoGen (1 mentions)
L glutamine, by Harvard Bioscience (1 mentions)
5 protocols using sirtinol
1
Cell Colony Formation Assay
2
Sirtuin and AMPK Modulation of Oxidative Stress
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After 24 h, to allow cell attachment, the medium was replaced and both the cell lines were incubated for 1 h with Sirtinol (Sirt, a sirtuins inhibitor; Tocris Bio-Techne, Minneapolis, MN, USA) or Compound C (CC, also known as dorsomorphin, an AMPK inhibitor; Merck KGaA, Darmstadt, Germany) at the concentration of 10 μM, or both, or their vehicle. After 1 h of incubation, Taurisolo® 100 μg/mL or vehicle (culture medium) was added and incubated for 1 h. After 1 h, the cells were incubated for 2 h with the pro-oxidant agent represented by H2O2 (200 μM for HASMCs and 100 μM for HUVECs). Cell viability was assessed using an aqueous solution of the cell proliferation reagent WST-1 (Roche, Basilea, Switzerland), (see Supplemental Materials ).
3
Fly Head Acetylome Enrichment Protocol
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Fly samples were prepared as previously described 8 , 57 with the following modifications. Briefly, 100 mg of male fly heads was homogenized in 1.4 ml homogenizing buffer (50 mM Tris–HCl pH 7.5, 500 mM NaCl, 1 mM EDTA, 0.1% NP‐40, and 20% glycerol) in the presence of 15 mM sodium butyrate and 60 μM of sirtinol (Tocris) and then added with 200 μl of 6 M urea/2 M thiourea. The proteins were digested for 4 h with 10 μg of Lys‐C (Wako) at 25°C, followed by overnight incubation (ON) with 300 μg trypsin (Worthington). Following the peptide isolation, peptide amount was measured by a nanodrop. Equal amount of peptides was enriched for acetylated peptides with 45 μl Ac‐K beads (ICP0388, Immunechem) overnight. Beads were washed 4× in PBS–Tween 0.1% followed by 4× in PBS. Finally, peptides were eluted with 125 μl of TFA 0.1% and were concentrated to a final volume of 17 μl. As input, 15 μl of each sample was taken before the enrichment for acetylated lysines and was diluted in TFA 0.1% and around 30 μg was desalted. From the resulting desalted samples, 3 μg was analyzed by mass spectrometry as described below.
4
Melatonin and ATP Modulate Inflammatory Response
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Melatonin and adenosine 5′-triphosphate (ATP) disodium hydrate were obtained from Sigma-Aldrich (St. Louis, USA) and Ultra-Pure LPS (from E. coli 0111: B4) was purchased from InvivoGen (San Diego, USA). Sirtinol was purchased from Tocris (Bristol, UK). Fetal bovine serum (FBS), RPMI 1640 cell culture media, L-Glutamine, penicillin/streptomycin, phosphate buffered saline (PBS), trypsin/EDTA were purchased from Biochrom (Berlin, Germany). All the antibodies used in experiments are given in Supplementary Table 1 .
5
Inhibitors of Oncogenic Signaling Pathways
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Inhibitors of Skp2 (compound 25, alias SZL-P1-41; range 1.25–20 μM; Tocris, Bristol, UK), histone deacetylases (sirtinol; range 1.56–20 μM; Tocris), and neddylation (MLN4924, pevonedistat; range 0.04–2 μM; Active Biochem, Hong Kong) were first tested by MTT cell viability assay, as described previously [42 (link)]. PC3 and PC3 DR12 cells were treated with a concentration scale (10–40 μM) of compound 25 and incubated for 48 h. After 24 h of incubation of cells with 25 μM sirtinol, cycloheximide (50 μg/mL) was added for 2 and 4 h. PC3, PC3 DR12, and C4-2 and 22rv1 cells were also treated with 2 μM of MLN4924 (pevonedistat) and incubated for 24 h. LAPC4 cells were treated in the same way but with 1 μM of MLN4924. All experiments were performed at least three times.
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