5036 wn 010 cf

All in utero experiments were performed under protocols approved by the IACUC at Boston Children’s Hospital. Timed-pregnant CD-1 dams were obtained from Charles River Laboratories. At E13.5, dams were anaesthetized by isoflurane inhalation and laparotomy was performed. Recombinant WNT3A protein (R&D Systems, 5036-WN-010/CF, 50 ng (for behavioural studies) and 33 ng (for lower dosage spine density analyses)) and the WNT signalling inhibitor IWP2 (9.34 ng; Sigma Aldrich, 10536) were dissolved in PBS. A volume of 1 µl of PBS, WNT3A or IWP2 were injected very slowly into the lateral ventricle of E13.5 embryos using fine glass capillary pipettes (Drummond Scientific, 21-176-2C) as previously described^{66 (link)}. Meloxicam analgesia was subcutaneously injected following surgery according to the IACUC protocol. All mice that developed a hydrocephalus or other injuries as a consequence of the surgeries were euthanized. To ensure that only healthy mice were part of our dataset, we also inspected mice brains after behavioural studies were completed.

Human normal bronchial epithelial (NHBE) cells were obtained from the American Type Culture Collection (Cat#: CRL‐2078; Manassas, USA), and cultured in RPMI‐1640 media (Gibco, USA) supplemented with 15% foetal bovine serum (FBS; Gibco, USA), 100 U/mL penicillin and 100 U/mL streptomycin at 37^°C in a humidified atmosphere containing 5% CO2. The cells were pretreated metformin (0.8 mmol/L, O0615AS; Meilun Biotech Co., Ltd, Dalian, China), human recombinant Wnt3a (rhWnt3a, 25 ng/mL, 5036‐WN‐010/CF; R&D, USA), or LiCl (10 mmol/L) for 24 hours before treatment with CSE.