Ttnpb

BMS compounds (BMS493 [32 (link)], BMS614 [20 (link)], BMS411 [20 (link)], BMS948, BMS961 [9 (link)]) were provided by Bristol-Myers Squibb. TTNPB was purchased from Sigma. Am580 was kindly provided by Reinhold Tacke (University of Würzburg). pSG5-based RAR expression vectors were described previously (19). All ligands are in ethanol solutions. (RARE)_3x-tk-luc was a kindly gift of Patrick Balaguer (INSERM, Montpellier). hRARβL₂₉₈F was generated into pSG5-hRARβ by PCR-assisted site-directed mutagenesis with Deep Vent DNA polymerase (New England Biolabs). The construct was verified by DNA sequencing.

Vhl^loxP/loxP MEFs were generated from 13.5 days post-coitum (d.p.c.) embryos of Vhl^loxP/loxP mice (JAX Labs). These MEF cells were immortalized by serial passaging in high glucose DMEM (Life Technologies) supplemented with 10% FBS (Sigma-Aldrich) and penicillin-streptomycin (Life Technologies). Immortalized MEF cells were transfected with a tamoxifen-inducible ER-Cre vector expressing Cre recombinase fused with a mutated ligand-binding domain for the human estrogen receptor (ER-Cre). Stable MEF cell lines expressing ER-Cre were generated by culturing in selection media containing 5 μg/ml blasticidin (Thermo Fisher Scientific, Waltham, MA). Vhl^flox/flox MEFs were treated with 3 μM 4-hydroxytamoxifen (Sigma-Aldrich) for 2 days for efficient Vhl knockout.
Compounds used for the primary screen (Table S1) were solubilized in DMSO and used at a final concentration of 10 µM. Bexarotene, was obtained from Sigma-Aldrich and Selleck Chem (Houston, TX); LG100268 and TTNPB were obtained from Sigma-Aldrich and used in the secondary validation studies in vitro at final concentrations of 0.1 µM, 0.3 µM and 1.0 µM doses (solubilized in DMSO) for 48 h as indicated for each individual treatment.

Sieved AGX1-2 beads (150 or 200 μm in diameter, Sigma) were soaked in a stable form of all-trans-retinoic acid, TTNPB (Sigma, 0.05 mg/ml dissolved in DMSO, Sigma) or AGN193109 (Sigma, 1 mg/ml dissolved in DMSO, Sigma) for 1 hour and then washed in DMEM before being grafted to the middle of wing buds using a sharp tungsten needle. TTNPB has been shown to diffuse from AGX1-2 beads over an approximate 12-20-hour period and can be used to model RA distribution in chick wing buds due to comparable patterning effects, kinetics and diffusion constants ((Eichele et al., 1984 (link)), (Eichele et al., 1985 (link)), (Eichele and Thaller, 1987 (link)).

1α,25-Dihydroxyvitamin D₃ (1,25D) was obtained from Cayman Europe (Tallinn, Estonia), while ATRA and TTNPB were from Sigma (St Louis, MO, USA). The compounds were dissolved in absolute ethanol at a concentration of 10 µM and stored at −20 °C. The synthetic retinoids AGN195183, AGN194310, AGN196996, and AGN205728 were synthesized at the Shanghai Institute of Materia Medica. Their synthesis, development, and specificities have been described previously (Hughes et al. 2006 (link); Johnson et al. 1995 (link); Klein et al. 1996 (link); Nagpal et al. 1995 (link); Nagpal and Chandraratna 1996 , 2000 (link); Teng et al. 1996 (link)). Retinoids were dissolved in 50 % methanol/50 % dimethylsulphoxide (DMSO) at a concentration of 10 mM (stored at −20 °C), and this stock was diluted using culture medium to the required concentration. Rabbit polyclonal antibodies to RARα (sc-550), RARβ (sc-552), and to actin (sc-1616), and a mouse monoclonal antibody to HDAC1 (sc-7872) were from Santa Cruz Biotechnology Inc.