U20S were retrieved from ATCC and modified with a plasmid encoding for the restriction enzyme (pBABE-AsiSIER and pAID-AsiSIER)29 (link),30 (link). U2OS, DIvA (AsiSI-ER-U20S), and AID-DIvA (AID-AsiSI-ER-U20S) cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with antibiotics, 10% FCS (InVitrogen) and either 1 µg/mL puromycin (DIvA cells) or 800 µg/mL G418 (AID-DIvA cells) at 37 °C under a humidified atmosphere with 5% CO2. The cell lines were regularly checked for mycoplasma contamination. For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. When indicated, 4OHT-treated cells were washed three times in pre-warmed PBS and further incubated with 500 µg/mL auxin (IAA) (Sigma; I5148) for the indicated time. For transcriptional inhibition, DRB (Sigma, 100 μM) or cordycepin (Sigma, 50 µM) was added to the medium 1 h prior to 4OHT (4 h) and auxin (2 h) treatments. Cells were arrested in G1 using a 48 h treatment with 40 μM lovastatin (Mevinolin from LKT Laboratories) and in G2 with a 24 h treatment with 40 μM Ro-3306 (CDK1 inhibitor, Calbiochem). For clonogenic assays in U20S cells, DSBs were induced either by increasing doses of etoposide (Sigma) for 16 h as indicated or by irradiation with a Cs137 source (Biobeam 8000).
4 oht
Manufactured by Merck Group
Sourced in United States, United Kingdom, Germany, China
4-OHT is a laboratory reagent used in cell biology research. It is a selective estrogen receptor modulator (SERM) that can be used to induce gene expression in cell lines engineered with the CreERT2 system. The core function of 4-OHT is to enable temporal control of Cre recombinase activity in such cell lines.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin, by Thermo Fisher Scientific (23 mentions)
Corn oil, by Merck Group (17 mentions)
Tamoxifen, by Merck Group (16 mentions)
4 hydroxytamoxifen 4 oht, by Merck Group (14 mentions)
Glutamax, by Thermo Fisher Scientific (13 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (12 mentions)
4 hydroxytamoxifen, by Merck Group (11 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (10 mentions)
Shield1, by Takara Bio (9 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (7 mentions)
Doxycycline, by Merck Group (6 mentions)
Progesterone, by Merck Group (6 mentions)
L glutamine, by Thermo Fisher Scientific (6 mentions)
Dmem f12, by Thermo Fisher Scientific (5 mentions)
β estradiol, by Merck Group (5 mentions)
Fetal bovine serum (fbs), by Merck Group (5 mentions)
Etoposide, by Merck Group (4 mentions)
Ici 182 780, by Bio-Techne (4 mentions)
Interleukin 6 (il 6), by Thermo Fisher Scientific (4 mentions)
Matrigel, by Corning (4 mentions)
320 protocols using 4 oht
1
Genome Editing with AsiSI Endonuclease
2
Induction and Inhibition of AsiSI-Dependent DSBs
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For AsiSI-dependent DSB induction, cells were treated with 300 nM 4OHT (Sigma, H7904) for 4 h. For transcriptional inhibition, cells were pre-treated with DRB (20µM or 100µM as indicated) for 1h prior 4h of 4OHT, and left during the entire 4OHT treatment (Fig. S8d ). For cell synchronization in G1, cells were incubated with 2 mM thymidine for 16 h, released for 14 h and subjected to the second thymidine treatment for 19 h. G1 and G2 cells were treated with 4OHT respectively after 11h and 4 hours and fixed 4 hours later (Fig.4b ). For BLESS experiment (Fig. 5b ) cells were treated with 4OHT (4h) 12h after release, followed by an auxin treatment of 2h before harvesting. For cell arrest in G1 (Fig. 5a ), cells were treated with 40µM Lovastatin for 48 hours. siRNA transfections were performed with the Cell Line Nucleofactor kit V (Amaxa) according to the manufacturer's instructions or using interferin (PolyPlus). Sequences for siRNAs are shown in Supplemental table 4 . To assess the efficiency of siRNAs, mRNA were extracted using the Qiagen RNeasy kit (#69504) and reverse transcribed using the AMV reverse transcriptase (Promega, M510F). cDNA were quantified by RT-qPCR (primer sequences are shown in Supplementary Table 3 ) and normalized to P0 cDNA levels.
3
FosTRAP Mice: Targeted Recombination
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FosTRAP (Fos the targeted recombination in active populations) mice were a gift of Dr. Fan Wang (MIT) and used as recently reported [9 ; 10 (link)]. The FosTRAP mice were kept in single cages with at least two environmental enrichments for a minimum of 10 days prior the day of 4-Hydroxytamoxifen (4-OHT) injection. The 4-OHT (4-OHT; Sigma, MO, USA Cat# H6278) was dissolved in ethanol to create a 20mg/ml stock solution, which was then divided and frozen in −20°C. On the day of administration, the 4-OHT was added to a sunflower seed oil solution (Sigma, MO, USA Cat # S5007) and heated to 50°C to remove the ethanol, resulting in a final 4-OHT solution with a concentration of 10 mg/ml. Any unused solution was discarded at the end of the day. The mice received an intraperitoneal 4-OHT injection 30 minutes before undergoing 30 minutes of pVNS treatment or sham. Both pVNS and sham group received sevoflurane (UPS, Covetrus North America, OH, USA) anesthesia. The mice were returned to the same single-housing cage for 10 days to minimize any sources of distress before termination.
4
Investigating B Cell Class Switching
5
Tamoxifen-Induced KIR6.1AAA Expression In Mice
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4-OHT (Catalog number H7904, Sigma), the active metabolite of tamoxifen, was dissolved in a corn oil:ethanol solution (90:10% v/v) at a concentration of 2 mg/ml.103 (link)Cspg4-Cre-KIR6.1AAA mice were given either 4-OHT (10 mg/kg, intraperitoneal injection; KIR6.1AAA induction) or vehicle (corn oil:ethanol; vehicle control), and control KIR6.1AAA mice were given 4-OHT (10mg/kg; Cre control) once a day for 5 consecutive days. 4 weeks after the last injection, mice were imaged in vivo as described below.
6
Preparation of 4-OHT Stock Solution
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4-OHT (H6278, Sigma) was dissolved at 20 mg/mL in ethanol by shaking at 37°C for 15 min (aliquots were stored at −20°C for up to several weeks) (Allen et al., 2017 (link)). On the day of the experiment, 4-OHT was redissolved in ethanol by shaking at 37°C for 15 min with a 1:4 mixture of castor oil (259853, Sigma); sunflower seed oil (S5007, Sigma) was added to obtain a final concentration of 10 mg/mL. 4-OHT, and the ethanol was evaporated under vacuum centrifugation.
7
Exploring Tamoxifen Resistance in Breast Cancer
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Human breast cancer MCF7 cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences) were cultured in DMEM: F-12 (HyClone; Cytiva) containing 10% FBS (HyClone; Cytiva), 100 U/ml penicillin (Thermo Fisher Scientific, Inc.), 100 mg/ml streptomycin (Thermo Fisher Scientific, Inc.), 2 mM GlutaMax (HyClone; Cytiva) and 6 ng/ml insulin (HyClone; Cytiva) at 37°C and 5% CO2. MCF7/TAMR-7 cells (MCF7/R; Ximbio), which are tamoxifen-resistant, were cultured in the presence of 1 µM 4-OHT (Sigma-Aldrich; Merck KGaA) under the same conditions as aforementioned.
MCF7 and MCF7/R cells were divided into control, 4-OHT and 4-OHT+3-methyladenine (3MA) groups. Cells in the 4-OHT group were treated with 10 µM 4-OHT with DMSO as the vehicle. Cells in 4-OHT+3-MA group were treated with 10 µM 4-OHT and 1 mM 3MA (Sigma-Aldrich; Merck KGaA) with DMSO as the vehicle. MCF7/R cells in rapamycin group were treated with 10 nM rapamycin (Selleck Chemicals) with DMSO as the vehicle. Cells in control group were treated with DMSO in follow up experiments.
MCF7 and MCF7/R cells were divided into control, 4-OHT and 4-OHT+3-methyladenine (3MA) groups. Cells in the 4-OHT group were treated with 10 µM 4-OHT with DMSO as the vehicle. Cells in 4-OHT+3-MA group were treated with 10 µM 4-OHT and 1 mM 3MA (Sigma-Aldrich; Merck KGaA) with DMSO as the vehicle. MCF7/R cells in rapamycin group were treated with 10 nM rapamycin (Selleck Chemicals) with DMSO as the vehicle. Cells in control group were treated with DMSO in follow up experiments.
8
Induction of Cellular Senescence Pathways
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4-OHT (Sigma) was reconstituted in ethanol and then diluted in filter-sterilized corn oil (Sigma) to a concentration of 1 mg/ml 4-OHT, 12.5% ethanol. DMSO (Sigma) was added, constituting up to 5% of the final volume, to improve 4-OHT solubility. Resuspension of 4-OHT was performed in sterile conditions in the dark. The 4-OHT solution was divided into aliquots and kept frozen in the dark until injection. Before injection, the aliquots were heated to 37 °C. In reactivation experiments, G0 R26CreER/CreER/Tert+/+, G0 R26CreER/CreER/TertLSL/+, or G5/G6 R26CreER/CreER/TertLSL/LSL mice were injected with 1 mg of 4-OHT solution every other day for a total of four injections.
Polyinosinic-polycytidylic acid (pI:pC; Thermo Fisher Scientific) was reconstituted in sterile PBS to a concentration of 10 mg/ml. Mice were injected with one dose of pI:pC at a concentration of 10 mg/kg.
The control 5′-gctagatgttagcgt-3′ ODN and the A151 5′-ttagggttagggttagggttaggg-3′ ODN were custom synthesized by WuXi AppTec. Bases were phosphorothioate-linked to increase nuclease resistance. ODNs were reconstituted in sterile PBS to a concentration of 1 mg/ml and injected at a dose of 300 μg/mouse every 3 days for 3 weeks.
Polyinosinic-polycytidylic acid (pI:pC; Thermo Fisher Scientific) was reconstituted in sterile PBS to a concentration of 10 mg/ml. Mice were injected with one dose of pI:pC at a concentration of 10 mg/kg.
The control 5′-gctagatgttagcgt-3′ ODN and the A151 5′-ttagggttagggttagggttaggg-3′ ODN were custom synthesized by WuXi AppTec. Bases were phosphorothioate-linked to increase nuclease resistance. ODNs were reconstituted in sterile PBS to a concentration of 1 mg/ml and injected at a dose of 300 μg/mouse every 3 days for 3 weeks.
9
Induction and Inhibition of AsiSI-Dependent DSBs
10
Tamoxifen-Induced KIR6.1AAA Expression In Mice
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