All cell lines were grown in Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland), supplemented with 6% fetal calf serum (Clontech, Mountain View, CA, USA), 50 μg/ml penicillin–streptomycin (Invitrogen, Waltham, MA, USA) and 2 mM l -glutamine (Lonza). Thymidine (2.5 mM ) and nocodazole (200 ng/ml) were purchased from Sigma (St Louis, MO, USA). HeLa cells stably expressing H2B-YFP were created using retroviral infection with pBabe-H2B-YFP (kind gift from Dr Susanne Lens, University Medical Center Utrecht, The Netherlands). Cell lines were selected with 5 μg/ml blasticidine (Sigma). U2OS cells stably expressing LAP-Mps1 were infected with retrovirus carrying pBabe-LAP-Mps1 and were selected with 5 μg/ml blasticidine. LAP-Mps1 I531M, I598F, C604Y and S611R were created by site-directed mutagenesis PCR. All cell lines were tested negative for Mycoplasma contamination.
Blasticidine
Manufactured by Merck Group
Blasticidine is a laboratory reagent used in cell culture experiments. It functions as a selective antibiotic that inhibits protein synthesis in eukaryotic cells.
Automatically generated - may contain errors
Lab products found in correlation
Doxycycline, by Merck Group (4 mentions)
Puromycin, by Merck Group (4 mentions)
Polybrene, by Merck Group (3 mentions)
Retinoic acid, by Merck Group (2 mentions)
Microvascular endothelial cell growth kit vegf, by Merck Group (2 mentions)
Fetal calf serum (fcs), by Takara Bio (1 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (1 mentions)
Thymidine, by Merck Group (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Dulbecco s modified eagle s medium, by Lonza (1 mentions)
β estradiol, by Merck Group (1 mentions)
Ci 1040, by Merck Group (1 mentions)
Lv810a 1, by System Biosciences (1 mentions)
Pspax2, by Qiagen (1 mentions)
Effectene transfection reagent, by Qiagen (1 mentions)
Pspax2, by Addgene (1 mentions)
Doxycycline dox, by Merck Group (1 mentions)
Ku55933, by Merck Group (1 mentions)
Hoechst 33258, by Merck Group (1 mentions)
Ly2603618, by Selleck Chemicals (1 mentions)
14 protocols using blasticidine
1
Cell Line Establishment and Genetic Manipulation
2
Generation of Fluorescent Cell Lines
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VSV-G pseudotyped retroviral particles were produced as described previously49 (link). In short, HEK293T cells were transfected with 10 μg of indicated pRetroX of pLKO vector, combined with 2.5 μg pMD/p and 7.5 μg pMDg plasmids, expressing the gag/pol and envelop proteins, respectively. The supernatant containing retrovirus was harvested at 48–72 h following transfection, was filtered through a 0.45-μM syringe filter and was subsequently used to infect target cells.
Establishment of HeLa cells stably expressing YFP-H2B cells was described previously27 (link). In short, HeLa cells were retrovirally infected with pBabe-H2B-YFP, and selected with blasticidine (5 μgram ml−1, Sigma). To establish KB2P1.21 and KB2P1.21R1 cell lines expressing H2B-EGFP and α-tubulin-mCherry, cells were first transduced with pRetrox-rTTa virus and selected with Geneticin (400 μg ml−1). Subsequently, cells were infected with a pRetrox-Tight-Pur virus harbouring H2B-EGFP–T2A-α-tubulin-mCherry and selected in puromycin (1 μg ml−1). H2B-EGFP and α-tubulin-mCherry expression was induced by incubation with doxycycline (0.5 μg ml−1, Sigma).
Establishment of HeLa cells stably expressing YFP-H2B cells was described previously27 (link). In short, HeLa cells were retrovirally infected with pBabe-H2B-YFP, and selected with blasticidine (5 μgram ml−1, Sigma). To establish KB2P1.21 and KB2P1.21R1 cell lines expressing H2B-EGFP and α-tubulin-mCherry, cells were first transduced with pRetrox-rTTa virus and selected with Geneticin (400 μg ml−1). Subsequently, cells were infected with a pRetrox-Tight-Pur virus harbouring H2B-EGFP–T2A-α-tubulin-mCherry and selected in puromycin (1 μg ml−1). H2B-EGFP and α-tubulin-mCherry expression was induced by incubation with doxycycline (0.5 μg ml−1, Sigma).
3
MYCN-Inducible SH-SY5Y Cell Line Protocol
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For cell culture conditions and cell sources see Duffy et al. [69 (link)]. MYCN overexpression in the SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) [69 (link), 70 (link)] line was induced by 1μg/ml Doxycycline (Sigma). SY5Y-MYCN media was supplemented with 7.5μg/ml Blasticidine (Sigma) and 200μg/ml G418 (Sigma). Small molecules used were: CI-1040 (PD184352, Sigma), β-estradiol (Sigma) and Retinoic Acid (RA, Sigma). Stock solutions were dissolved in DMSO, except for β-estradiol for which ethanol was used. Compounds were replenished every 24h for any treatment longer than a 24h duration.
4
Lentivirus-Mediated Overexpression and Knockdown
5
Lentiviral Transduction of NMuMG Cells
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Stable populations of NMuMG cells expressing the blasticidine-resistant marker together with TFAP2A-FLAG under a doxycycline-inducible promoter were obtained with the pCLX expression system [26 (link)]. Lentiviral particles were produced in HEK293-LV cells using the helper vectors pMDL, pREV and the envelope-encoding vector pVSV. For infection, viral supernatants were added to target cells in the presence of polybrene (#TR-1003-G, Millipore) (1 μg/ml). Cells were further incubated at 37 °C under 5% CO2 in a tissue culture incubator for 72 h, prior to selection with blasticidine at 10 μg/ml (#15205-25 mg, Sigma-Aldrich).
6
Multimodal Senescence Assessment Protocol
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ATM inhibitor KU-55933 (#SML1109), Hoechst 33258 (#14530), E64d (#516485), bafilomycin A1 (# B1793), 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide, MTT (#M2128), blasticidine (#3513-03-9), Fluoromount (#F4680), puromycin (#58-58-2), Quinoline-Val-Asp-Difluorophenoxymethyl Ketone, QVD-OPH (# SML0063) and doxycycline (Dox) (#D9891) were purchased from Sigma-Aldrich. Pepstatin A methyl ester (#516485) and epoxomycin (# 134381-21-8) were obtained from Calbiochem. Tetramethyl rhodamine methyl ester perchlorate dye (#T-668) and carboxyfluorescein succinimidyl ester (CFSE) (#C34554) were purchased from Molecular Probes. LY2603618 (#S2626) was purchased from Selleckchem. 20A was synthesized as previously described (compound #3 in (20 (link))). Sentragor (#AR8850020) for antibody-enhanced detection of senescence cells was provided by Arriani Pharmaceuticals.
7
Plasmid Transfection and Cell Selection
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The following plasmids were employed: E1A12S- and Harvey-Ras- expressing vectors (kindly provided by Dr. G. Piaggio; [49 (link)]), EGFP-expressing vector (pEGFP-c2; Clontech, Mountain View, CA, USA) and pBabepuro (vector expressing the puromycin-resistance marker), GFP-H2B and GFP-H2BS14D (carrying blastictidine-resistance marker; [13 (link)]). Cells were transfected by using Lipofectamine LTX and Plus reagent (Life Technologies) and selected in 2 μg/mL puromycin (Sigma) and/or in 3 μg/mL blasticidine (Sigma).
8
Immortalized Endothelial Cell Culture
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Human telomerase (hTERT)-immortalized microvascular endothelial cells isolated from human foreskin (TIME cells; ATCC) were grown in ATCC’s vascular cell basal medium supplemented with microvascular endothelial cell growth kit-VEGF and 12.5 μg/ml blasticidine (Sigma-Aldrich) for 48 h at 37°C + 5% CO2 until reaching 80–90% confluence. At confluence, cells were passaged and 5.4 × 104 cells were plated on fibronectin-coated (10 µg/ml; MilliporeSigma), base/acid cleaned, 0.17 mm (#1.5) glass-bottom dishes (14 mm glass diameter; MatTek) for 18 h prior to experiments.
9
Neuroblastoma Cell Culture Conditions
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For cell culture conditions and cell sources see Duffy et al. [1 (link)]. Additionally NBL-S (MYCN single copy, with somewhat elevated MYCN expression [99 (link)]) and BE(2)-C (MYCN-amplified) were used for IC-001 and Wnt Agonist 1 treatment. MYCN overexpression in the SH-SY5Y/6TR(EU)/pTrex-Dest-30/MYCN (SY5Y-MYCN) [1 (link), 67 (link)] line was induced by 1 μg/ml Doxycycline (Sigma-Aldrich). SY5Y-MYCN media was supplemented with 7.5 μg/ml Blasticidine (Sigma- Aldrich) and 200 μg/ml G418 (Sigma-Aldrich). Small molecules used were: 1-azakenpaullone (Azak, Sigma-Aldrich), BIO-acetoxime (BIO, Tocris), Wnt agonist 1 (Merck), ICG-001 (Selleck), iCRT14 (Tocris), and Retinoic Acid (RA, Sigma-Aldrich). Stock solutions were dissolved in DMSO. Compounds were replenished every 24 h for any treatment longer than a 24 h duration.
KCN cells were treated with media containing human recombinant Wnt3a (R&D Systems). The Wnt3a- containing media was replenished 4 times over the course of the 48 h treatment. mβ-catenin/Tcf3 fusion construct (Genbank accession number KM189193) was transfected into the cells using jetPRIME (Polyplus), according to the manufacturer's instructions. Empty or fusion vector was transfected at 100 ng (KCNR) or 200 ng (Kelly) per well of a 96-well plate.
KCN cells were treated with media containing human recombinant Wnt3a (R&D Systems). The Wnt3a- containing media was replenished 4 times over the course of the 48 h treatment. mβ-catenin/Tcf3 fusion construct (Genbank accession number KM189193) was transfected into the cells using jetPRIME (Polyplus), according to the manufacturer's instructions. Empty or fusion vector was transfected at 100 ng (KCNR) or 200 ng (Kelly) per well of a 96-well plate.
10
Sting-/- MEF Reconstitution Assay
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