4 oh tamoxifen

Primary chondrocytes were isolated from the condylar cartilage of 4 week-old mice. Dissected tissues with cartilage were first digested with 0.25% trypsinase (Gibco/Life Technologies, Carlsbad, CA, USA) at 37 °C for 15 min, and then remove muscles, ligaments, and bone tissue. Chondrocytes were isolated from TMJ tissues by additional digestion with 0.1% collagenase II (Gibco/Life Technologies) overnight at 37 °C in a CO₂ incubator. Cells were seeded in 12-well plates at a density of 2 × 10⁵ cells/well and cultured in Dulbecco’s Modified Eagle’s Medium/F12 (1:1) supplemented with penicillin/streptomycin (Gibco/Life Technologies) and 10% fetal bovine serum until they reached sub-confluence. On day 3 of culturing, primary chondrocytes from both Cre-negative and Fgfr3 cKO mice were treated with 1 μM 4OH-tamoxifen (Sigma-Aldrich) for 48 h. For IHH signaling inhibitor treatment in vitro, the SMOi GDC-0449 (Selleck Chemicals, Houston, TX, USA) was reconstituted in dimethyl sulfoxide (Sigma-Aldrich) and applied in chondrocytes at a final concentration of 1 μM for 24 h after 4OH-tamoxifen treatment, and in the presence of it.

The Rosa26^Cre-ERT2/+; Taf8^flox/flox mouse embryonic stem cells (mESCs) were generated previously by F. El Saafin^{21 (link)}. Briefly, mice carrying the Taf8^lox allele were bred to mice carrying the Rosa26^Cre-ERT2 allele to produce Rosa26^Cre-ERT2/+;Taf8^flox/flox E3.5 blastocysts and to isolate Rosa26^Cre-ERT2/+;Taf8^flox/flox mouse embryonic stem cells (mESCs)^{21 (link)}. The Rosa26^Cre-ERT2/R;Taf10^flox/flox mESCs were generated previously by P. Bardot^{20 (link)}. Briefly, the ESCs were derived from Rosa26^Cre-ERT2/R;Taf10^lox/lox E3.5 blastocysts^{20 (link)}. mESCs were cultured in DMEM (4.5 g/l glucose) with 2 mM Glutamax-I, 15% ESQ FBS (Gibco), penicillin, streptomycine, 0.1 mM non-essential amino acids, 0.1% ß-mercaptoethanol, 1500 U/mL LIF and two inhibitors (2i; 3 µM CHIR99021 and 1 µM PD0325901, Axon MedChem) on gelatin-coated plates. To induce deletion of Taf8, mESCs were treated with 0.5 µM 4-OH tamoxifen (Sigma) for 5–6 days, and to induce deletion of Taf10, Rosa26^Cre-ERT2/R;Taf10^lox/lox mESCs were treated for 4 days with 0.1 µM 4-OH tamoxifen (Sigma). The above-described mESCs have already been described^{20 (link),21 (link)} and were derived according to animal welfare regulations and guidelines of the French Ministry of Agriculture and French Ministry of Higher Education and Research, and the Australian Animal Welfare Committee, respectively.

mES cells (CreERT2+, EGFP+) were infected with lentiviruses encoding sgEGFP1 and Cas9-p2a-blasti (kind gift from Julian Jude) or sgEGFP1 Pulse-Switch construct. Cells were selected with blasticidin (Gibco, 5 µg/ml final concentration) for 4 days starting at day 1 p.i. Subsequently, the cultures were split into 2 sets, one set was kept under blasticidin selection and in the second set, CreERT2 was induced with 4OH-tamoxifen (Sigma–Aldrich, 0.5 µM final concentration) for 48 h. On day 12 p.i. EGFP loss was measured with flow cytometry (BD Fortessa).
WT NIH 3T3 mouse embryonic fibroblasts were infected with CreERT2-GFP retrovirus and subsequently with a lentivirus encoding EBFP2. Cells were further sorted for GFP and EBFP2 and expanded clonally. Resulting NIH3T3 cells (CreERT2+, GFP+, EBFP2+) were infected with lentiviruses encoding sgEGFP1 and Cas9-p2a-blasti or with sgEGFP1 Pulse-Switch construct in 24-well format. Cells were selected with blasticidin (Gibco, 5 µg/ml final concentration) for 2 days starting at day 1 p.i. Subsequently, the cultures were split into two sets of plates, one set was kept non-induced and in the second set, CreERT2 was induced with 4OH-tamoxifen (Sigma–Aldrich, 0.5 µM final concentration) for 4 days. On day 10 p.i. (day 7 post induction) GFP and EBFP2 loss was measured with flow cytometry (BD Fortessa).

Organ cultures were performed as previously reported [18 (link)]. Cell-injected metanephroi were placed at the air–fluid interface of a polycarbonate filter (3401, Corning, New York, NY, USA; average pore size = 0.4 μm). The Base Medium supplemented with 2 ng/mL of 4OH-tamoxifen (H7904, Sigma-Aldrich, Tokyo, Japan) was changed every day, and the cells were harvested after culturing at 37 °C in 5% CO₂ for 5 days.

Endocrine disrupting compounds (EDCs) such as 17β-estradiol (E2), ethinyl-estradiol(EE2), 4-nonylphenol (4-NP) and 4-OH tamoxifen (TAM) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Stock solutions of all ligands were prepared in methanol; for SPR measurements, the stock solutions were further diluted with methanol into a series of concentrations, and then added to the final working samples in order to obtain the right ligand concentration and a final methanol percentage of 2%. Biotinylated-Dioxa (AEEA) and amine terminated peptides αβ/I (SSNHQSSRLIELLSR) are synthesized by Primm Srl (Milan, Italy). All the other reagents were acquired from general laboratory suppliers with analytical purity. Solutions were prepared in deionized water from water purification system MMilli-Q^® Integral (Millipore-Merck, Burlington, MA, USA).