T, B, and red blood cells were labeled with 10 μg per spleen of rat monoclonal antibodies against CD4 (GK1.5), CD8 (53.6.72), CD19 (1D3), and Ter119 (Bio-X-cell) and magnetically depleted from total splenocyte suspensions with the use of anti-rat IgG-coupled magnetic beads (QIAGEN). In all experiments, approximately 2 × 105 enriched NK cells were injected intravenously into mice. In adoptive co-transfer experiments, equal numbers of Ly49H+ NK cells from each population (CD45.1+ and CD45.2+) were injected into recipients 1 day before infection. In some experiments, recipient mice were injected intraperitoneally with 1 mg/day of hydroxytamoxifen (4-OHT) dissolved in corn oil or corn oil control (Sigma) for 5 consecutive days starting on day 4 after infection and subsequently injected intraperitoneally with PBS, 200 μg/day metformin (Sigma), 600 μg/kg/day rapamycin (LC Laboratories), or 1.25 mg/day NAC (Sigma) from day 8 to 28 after infection.
4 hydroxytamoxifen oht
Manufactured by Merck Group
Sourced in United States
4-hydroxytamoxifen (OHT) is a metabolite of the breast cancer drug tamoxifen. It is used as a laboratory reagent in research applications.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Thermo Fisher Scientific (2 mentions)
Cycloheximide (chx), by Merck Group (2 mentions)
Metformin, by Merck Group (1 mentions)
N acetyl cysteine (nac), by Merck Group (1 mentions)
Corn oil control, by Merck Group (1 mentions)
Anti rat igg coupled magnetic beads, by Qiagen (1 mentions)
Ter119, by BioXCell (1 mentions)
Rapamycin, by LC Laboratories (1 mentions)
Scgb1a1 creer mice, by Jackson ImmunoResearch (1 mentions)
Foetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Mycoalert, by Lonza (1 mentions)
Antibiotic antimycotic solution, by Thermo Fisher Scientific (1 mentions)
Rotor gene q, by Qiagen (1 mentions)
Kapa sybr fast qpcr master mix, by Roche (1 mentions)
Fastdigest enzymes, by Thermo Fisher Scientific (1 mentions)
Rneasy kit, by Qiagen (1 mentions)
Matrigel, by Corning (1 mentions)
Advanced dmem f12 medium, by Thermo Fisher Scientific (1 mentions)
Ikk 16, by Selleck Chemicals (1 mentions)
Bromodeoxyuridine (brdu), by Merck Group (1 mentions)
20 protocols using 4 hydroxytamoxifen oht
1
Adoptive NK Cell Transfer and Modulation
2
Analyzing Pdcd5 Knockout in Lung Cells
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All animal experiments were conducted in accordance with standard operating guidelines with the approval of Yonsei University College of Medicine Institutional Animal Care and Use Committee (IACUC No. 2015-0047). Six- to 8-week-old C57BL/6 male mice were used for this study (Orient Bio. Korea). We crossbred Pdcd5f/f mice9 (link) with Scgb1a1-CreERTM mice14 (link) (The Jackson Laboratory, Sacramento, CA, USA) or Sftpc-CreERT217 (gifted from Brigid L.M. Hogan, Duke University Medical Center) to generate Pdcd5f/f;Scgb1a1Cre/+ mice (Ccsp-Pdcd5d/d) or Pdcd5f/f;SftpcCre/+ (Spc-Pdcd5d/d) mice. ROSA26-mTmG mouse18 (link) was crossed to Ccsp-Pdcd5d/d or Spc-Pdcd5d/d mice to visualize Cre-mediated excision of Pdcd5 in club cells or AT2 cells. To induce knockout of Pdcd5, 10 mg/kg hydroxytamoxifen (4-OHT; Sigma-Aldrich, St. Louis, MO, USA) was injected into 8-week-old male mice three or four times every other day. Three days after the last injection, mice were injected with saline (vehicle control) or 4 mg/kg BLM (Santa Cruz Biotechnology, Santa Cruz, CA, USA) intratracheally. Mice were sacrificed after receiving bleomycin and BAL fluid and lung tissues were harvested. Mice were housed in a specific pathogen-free animal facility, with controlled temperature (~24 °C) and humidity under 12 h light/dark cycle (lights on at 8 a.m.) and had free access to food and water.
3
Induction and Maintenance of Cellular Senescence
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IMR90 (CCL-186) female human foetal lung fibroblasts and A549 (CCL-185) human lung adenocarcinoma cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The cell lines were confirmed to be mycoplasma negative (Lonza MycoAlert, cat #LT07-118). IMR90 ER:RAS is a derivative of IMR90 cells expressing a switchable version of oncogenic H-Ras84 (link). IMR90 ER:RAS and A549 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, ThermoFisher) supplemented with foetal bovine serum (10%), L-glutamine (2 mM, ThermoFisher), and antibiotic-antimycotic solution (1%, ThermoFisher) and incubated under standard tissue culture conditions (37 °C and 5% CO2). For induction of the senescent phenotype, IMR90-ER:RAS cells were cultured in hydroxytamoxifen (4-OHT) (Sigma) added media at 100 nM final concentration. IMR90 were kept in culture for over 27 passages for replicative senescence. Cells were tested for mycoplasma on a regular basis.
4
Measuring Single-Stranded DNA at Double-Strand Breaks
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DIvA cells were treated for 4 h with 300 nM hydroxytamoxifen (4-OHT, Sigma) to induce the translocation of the AsiSI endonuclease into the cell nucleus. Genomic DNA was isolated with phenol:chloroform and digested with 1 U of Fast Digest enzymes (ThermoFisher) BsrGI and HindIII37 (link). Mock-digested samples were processed in parallel. The percentage of ssDNA adjacent to the DSB was measured by quantitative PCR (qPCR) using KAPA SYBR Fast qPCR Master mix (Kapa Biosystems) with the primers indicated in the Supplementary Table 1 in a QIAGEN Rotor-Gene Q. The following equation was used to calculate the percentage of ssDNA: For each sample, the ΔCt was calculated by subtracting the Ct value of the mock-digested sample from the Ct of the digested sample.
5
Intestinal Organoid Generation and Manipulation
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Intestinal organoids were generated from the colon of HDAC3FF and HDAC3ΔIEC-IND mice as previously described (38 (link), 112 (link)). Briefly, the colon was opened, cut into small pieces, and incubated in chelation buffer (2 mM EDTA in PBS) for 30 minutes at 4°C with rotation. Tissue was then transferred to shaking buffer (PBS, 43.3 mM sucrose, 54.9 mM sorbitol) and shaken by hand for 2–5 minutes. Colonic crypts were resuspended and plated in Matrigel (Corning) with organoid culture medium (60% Advanced DMEM/F12 medium [Gibco, Thermo Fisher Scientific] supplemented with 10 mM HEPES, 2 mM l -glutamate, 40% L-WRN conditioned medium, 1× N2 supplement, 1× B27 supplement, 50 ng/mL murine EGF, and 10 μM Y-27632 ROCK inhibitor). Culture medium was refreshed every 2–3 days. To induce HDAC3 deletion, organoids were treated with 1 μM hydroxytamoxifen (4-OHT, MilliporeSigma) for 24 hours. Organoids were treated with vehicle (DMSO) or 5 μM IKK-16 (Selleckchem) for 24 hours. For IFN-γ stimulation, organoids were treated with 100 U/mL of mouse recombinant IFN-γ (BioLegend) for 24 hours. After incubation, organoids were washed 3 times in PBS and lysed using the RNeasy kit (Qiagen).
6
Induction of Recombination in Nestin-Cre/ADAM10 Mice
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For induction of recombination, Nestin-CreER/ADAM10fl/fl mice (4–6 weeks old) were administered tamoxifen (TAM) at 100 mg/kg per day for 3 days with 48 hours apart (intraperitoneally (IP); dissolved in 10% EtOH/90% sunflower oil). Littermate mice WT for ADAM10 or negative for Nestin-Cre were used as controls and were also treated with tamoxifen concurrent with ADAM10fl/fl mice. For in vitro experiments, hydroxy-tamoxifen (4-OHT; MilliporeSigma, Burlington, MA, USA), a TAM metabolite, was added at 1 μM to the stem cell media (SCM; 1:1 DMEM:F12 medium (MilliporeSigma) 1× B27 supplement) for the duration of the experiment unless otherwise indicated.
For the bromodeoxyuridine (BrdU)-label-retaining cell (LRC-BrdU) assay, BrdU (10 mg/mL, MilliporeSigma) was injected IP at 100 mg/kg five times every 3 hours into P45 ADAM10fl/fl and WT control mice following TAM administration to label dividing cells. Mice were euthanized 30 days after the final BrdU injection. In vitro short-term BrdU analysis involved BrdU administration (10 μM) 2 hours before fixation.
For the bromodeoxyuridine (BrdU)-label-retaining cell (LRC-BrdU) assay, BrdU (10 mg/mL, MilliporeSigma) was injected IP at 100 mg/kg five times every 3 hours into P45 ADAM10fl/fl and WT control mice following TAM administration to label dividing cells. Mice were euthanized 30 days after the final BrdU injection. In vitro short-term BrdU analysis involved BrdU administration (10 μM) 2 hours before fixation.
7
Osteosarcoma and Lung Cancer Cell Culture
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U2OS and SAOS-2 osteosarcoma cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 5% fetal bovine serum (FBS). Early passage WI38 human embryonic lung fibroblasts were grown in minimal essential medium supplemented with 10% fetal bovine serum, 1 mM L-glutamine, 1 mM sodium pyruvate and non-essential amino acids. H1299 human lung adenocarcinoma cells were grown in RPMI 1640 medium supplemented with 5% fetal bovine serum. Cells were maintained at 37°C in a humidified atmosphere containing 8% CO2. To induce activation of ER-E2F1 or ER-E2F3, cells were treated with 100 nM 4-hydroxytamoxifen (OHT, Sigma) for the times indicated. Where indicated, cycloheximide (CHX, Sigma) was administered for 8 hr at 10 μg/ml. Nocodazole (Sigma) was used at 60 ng/ml for 18 hr.
8
Cell Culture and ER-E2F1 Induction Protocol
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HeLa (ATCC, obtained from Ilana Keshet, the Hebrew University in Jerusalem), MCF7 (ATCC, obtained from Mary-Claire King, University of Washington, Seattle), and U2OS (ATCC) cells were cultured in DMEM, supplemented with 10% Fetal Bovine Serum (FBS), 2mM L-glutamine, 10U penicillin, 10μg streptomycin/ml (Biological Industries, Beit Ha'emek, Israel). Media for U2OS ER-E2F1 stable lines[42 (link)] also contained 0.5mg/ml G418 (Alexis Biochemicals, San Diego, CA, USA). Cell lines were maintained at 37°C with 5% CO2 and subcultured 2–3 times weekly.
ER-E2F1 induction of stably transfected U2OS cells, employed 300nM 4-hydroxytamoxifen (OHT) (Sigma-Aldrich, St Louis, MO, USA) following 48 hr. serum starvation (0.1% FBS containing medium).
ER-E2F1 induction of stably transfected U2OS cells, employed 300nM 4-hydroxytamoxifen (OHT) (Sigma-Aldrich, St Louis, MO, USA) following 48 hr. serum starvation (0.1% FBS containing medium).
9
Estrogen and Tamoxifen Exposure Protocol
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17β-estradiol (E2; CAS #50-28-2) and 17α-ethinyl estradiol (EE; CAS #57-63-6) were purchased from Tocris Bioscience (Bristol, UK); tamoxifen (TAM; CAS #10540-29-1) and 4-Hydroxytamoxifen (OHT; CAS #68392-35-8) from Sigma (St. Louis, MO); HEPES 1 M solution, L-Glutamine 200mM solution, Trypsin 2.5%, Penicillin—Streptomycin solution, phenol red-free DMEM/F-12 (1:1) and DPBS/Modified from Hyclone Laboratories, Inc. (Logan, Utah); MEM NEAA (100x) from Gibco® by Life Technologies (Grand Island, NY). Fetal bovine serum (FBS) and charcoal stripped FBS (CSS) were from Atlanta Biologicals (Flowery Branch, GA).
10
Conditional Knockout and Degradation of ESC Regulators
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All mouse ESC lines used in this study were grown at 37 °C and 5% CO2 on gelatinised plates. Dulbecco’s Modified Eagle Medium (Gibco) was supplemented with 15% foetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukaemia inhibitory factor. Cells were passaged using trypsin-EDTA (0.25%, Gibco) with 2% chicken serum. Cells were regularly tested for the presence of mycoplasma.
To induce conditional knockout, PRC1CKO cells were treated with 800 nM 4-hydroxytamoxifen (OHT) (Sigma) for 72 h. To induce and maintain degradation and depletion of SUZ12, dTAG-SUZ12 were treated with 100 nM dTAG-13101 (link) for 96 h.
To induce conditional knockout, PRC1CKO cells were treated with 800 nM 4-hydroxytamoxifen (OHT) (Sigma) for 72 h. To induce and maintain degradation and depletion of SUZ12, dTAG-SUZ12 were treated with 100 nM dTAG-13101 (link) for 96 h.
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