Doxycycline hyclate

Neuronal differentiation of ESCs into cortical glutamatergic neurons was carried out as previously described^{92 (link)}. In brief, the differentiation was carried out by adding doxycycline hyclate (2 μg/mL) to N2 supplemented media (Thermo Fisher, 17502048) with patterning factors SB431542 (Tocris, 1614, 10 μM), XAV939 (Stemgent, 04–00046, 2 μM) and LDN-193189 (Stemgent, 04–0074, 100 nM), as described previously^{92 (link)–94 (link)}. Puromycin selection was used (5μg/μL), from days 2 to 6 to remove non-transduced cells. At 4 days post induction, neuronal cells were resuspended into Neurobasal media (Gibco, 21103049) that was supplemented with B27 (Gibco, 17504044, 50X), doxycycline (2 μg/mL), brain-derived neurotrophic factor (BDNF), ciliary neurotrophic factor (CTNF), and glial cell-derived neurotrophic factor (GDNF) (R&D Systems 248-BD/CF, 257–33 NT/CF, and 212-GD/CF at 10 ng/mL each). From this point onwards the neurons were either co-cultured with murine glial cells that were derived from early postnatal (P1-P3) mouse brains as described previously^{95 (link)} or were left to grow as monocultures (mouse strain https://www.jax.org/strain/100012; animal ethical committee approval by Harvard University: Animal Experimentation Protocol (AEP) # 93–15).

Eight-week-old female athymic nu/nu mice (Charles River laboratories, Wilmington, MA, USA) were used for xenograft studies. Mice were housed four/cage in sterile cages, kept under light/dark cycles (12 h:12 h), and were acclimated for 7–10 days before the experiments.
Vector transfected (control) and doxycycline-inducible MDA-MB-468 cells (clone 2) were suspended 1:1 in PBS/Matrigel (BD biosciences, San Jose, CA, USA) and inoculated into the inguinal mammary fat pad (2 × 10⁶ cells/100 μL). After surgery, mice were provided water containing 4% sucrose with or without 1 mg/mL doxycycline-hyclate (Thermo Fisher, Pittburgh, PA, USA). Tumor dimensions were measured twice/week and tumor volume was calculated as length × width² × 0.52. In half of the mice, when tumors were about 150 mm³ in volume (controls) or 250 mm³ in volume (mice inoculated with clone 2), Alzet osmotic mini-pumps (model 1004, Durect Corporation, Cupertinoo, CA, USA), were implanted sc in the dorsal neck. These pumps with a 100-μL reservoir are rated for a continuous delivery at 0.11 μL/h for 4 weeks. The pumps delivered a 37% solution of hydroxypropyl-β-cyclodextrin (HPCD) (CTD, Gainesville, FL, USA) in PBS (control), or 50 mM SMI-6-HPCD complex in PBS. After 3 weeks, animals were euthanized and the tumors were weighed.