4 hydroxytamoxifen 4 ht

All animal studies were performed following guidelines approved by the University of Minnesota Institutional Animal Care and Use Committee (Minneapolis, MN), protocol approval number (1501-32220A). BRAF V600E/PTEN-null mice (B6.Cg-Braf^tm1Mmcm Pten^tm1Hwu Tg(Tyr-cre/ERT2)13Bos/BosJ) were purchased from The Jackson Laboratory. The mice were housed and bred in a virus and antigen-free room. Mice were genotyped by standard PCR analysis according to the Jackson Laboratory genotyping protocol. Mice with the BRAFV600E mutation heterozygote and PTEN loss with Cre were used in this study. For localized melanoma induction on the dorsal skin, adult (6–8 weeks of age) mice were treated topically with 2.5 µL of 1.9 mg/ml (5 mM) 4-hydroxytamoxifen (4-HT; Sigma-Aldrich, H6278) for 3 days. 4HD and XAG were dissolved in PBS with 2.5% dimethylsulfoxide (DMSO), 5% polyethylene glycol 400 (PGE 400) and 5% tween 80. The compounds were administered to mice by oral gavage at a dose of 10 or 50 mg/kg BW. The relevant solvent was administered to control animals in the melanoma prevention studies. For the early treatment study, the compounds or solvent were administered to the mice daily after 4-HT treatment. For the late treatment study, the compounds or solvent were administered to the mice daily beginning at day 23, when the animals had readily measurable melanoma lesions.

Unless otherwise specified, EBV lytic induction in P3HR-1, Akata and AGSiZ cells were induced with 400 nM 4-Hydroxytamoxifen (4HT) (Sigma Aldrich) for 24h, 15 μg/ml anti-IgG crosslinking (Agilent) for 48h and 5 μg/ml doxycycline (Sigma Aldrich) for 24h, respectively. Inhibition of NLRP3 inflammasome in lytic-induced BILF1-depleted P3HR-1 cells was achieved by the addition of 10 μM MCC950 (InvivoGen) simultaneously with the EBV lytic inducer 4HT for 24h. NLRP3 inflammasome activation was induced by the potassium ionophore, Nigericin (InvivoGen) at 10 μM for 4h.

4-hydroxytamoxifen (4HT) (Sigma, St. Louis, MO) was administered at a concentration of 129 nM to Tg(rag2:MYC-ER;lck:EGFP) nacre embryos at 5 days post-fertilization (dpf) (23 (link)) and was replaced in fresh fish water once a week.

4-hydroxytamoxifen (4-HT, Sigma-Aldrich) was prepared as previously described (Badea et al. 2003 (link)). Two weeks after the start of cuprizone feeding, PDGFRα-Cre^ER;Rosa26-eYFP mice were injected with a total of 3 mg of 4-HT over three days to induce recombination (Fig. 1). Recombination was induced in control mice (no cuprizone exposure) at 10 weeks of age.