Recombinant shh

The human PC cell lines Panc‐1 and BxPc‐3 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotic/antimycotic (Life Technologies, Carlsbad, CA, USA). The cells were maintained at 37°C in a humidified 5% CO₂ atmosphere. Antibodies against sHH (ab53281), SMO (ab5694), PTCH (ab53715), Gli‐1 (ab49314), GAPDH (ab8245) and EIF5A (ab32443) were purchased from Abcam (Cambridge, MA, USA). Recombinant sHH was obtained from R&D Systems (Minneapolis, MN, USA).

Human peripheral blood mononuclear cells (PBMCs) were freshly isolated using Ficoll-Paque (MilliporeSigma) from randomly selected unknown leukocyte-cone donors from the United Kingdom NHS Blood and Transplant Centre. After magnetic separation (EasySep Isolation Kit, StemCell), naive CD4⁺ T cells (CD3⁺CD4⁺CD45RA⁺CD45RO^–) were differentiated under iTreg conditions at 1 × 10⁶ cells/mL. Cells were stimulated with plate-bound anti-CD3 (5 μg/mL, UCHT1; eBioscience), soluble anti-CD28 (1 μg/mL, eBioscience), IL-2 (100 IU/mL, eBioscience) and TGF-β1 (5 ng/mL, Peprotech). Recombinant Shh (0.5 μg/mL, R&D Systems) was added, cells were incubated for 5 days and analyzed for intracellular FOXP3 expression.

Differentiation into retinal photoreceptors was performed as previously described [26 (link)], using iPSCs cultured in feeder-free conditions on Matrigel in mTESR^TM medium. The cells were dissociated enzymatically and plated onto Matrigel-covered dishes in neural differentiation medium containing N2 and B27 supplements (Life Technologies). After settling for an hour, adhered cells were covered in a 2% Matrigel solution. The following day, and thereafter every second day, the medium was replaced with neural differentiation medium without Matrigel. From day 10 the medium was supplemented with 3nM recombinant SHH (R&D Systems), 50ng/μl acidic fibroblast growth factor (αFGF) (R&D Systems), 10ng/μl basic fibroblast growth factor (bFGF) (Miltenyi), 1mM taurine and 500nM retinoic acid (both Sigma Aldrich). After 30 days of culture characterisation was performed by immunocytochemistry and qRT-PCR (for antibodies and primers, see S1 and S2 Tables). Each patient and control line underwent two rounds of differentiation, with each time point analysed in biological duplicate.

Lck-Gli2ΔC2 (C2, Gli2R [8 (link)]), GFP-Gli reporter mice (Gli binding site-GFP transgenic, GBS-GFP Tg [16 (link)]), and littermate/age-matched controls, all C57BL/6 background, and C57BL/6 and BALB/c WT mice were bred and maintained at UCL under UK Home Office regulations. AAD was induced as described [5 (link)]. HDM allergen was given 3 times per week for the number of weeks stated in the figures. BAL, lung lobes, and draining LNs were harvested. Lung tissue was cryopreserved for sectioning or homogenization or minced and digested with 1.5 mg/ml Liberase (Roche Diagnostics, Burgess Hill, UK) and 0.5 mg/ml DNAse (Roche Diagnostics), subjected to erythrocyte lysis and prepared for flow cytometry, or subjected to lysis for RNA extraction. To obtain SiglecF⁺ cells (eosinophils), cells were purified from pooled BALB/c lung and spleen leukocytes by streptavidin-based magnetic bead positive selection (Thermo Fisher Scientific, Waltham, MA, USA) using anti-SiglecF-biotin (Miltenyi, Bergisch-Gladbach, Germany). Cells were then cultured at 2.5 × 10⁵/ml in AIM V medium (Thermo Fisher Scientific) for 24 h in the presence or absence of 500 ng/ml recombinant Shh (R&D Systems, Minneapolis, MN, USA) before lysis for RNA extraction.

Adipose, normal ovary and CA-MSCs plated at 1×10⁵ cells/6well in serum free supplemented MEBM media were treated with recombinant SHH (10-100ng/ml) (R&D systems, Pittsburg, PA) for 24hrs then processed for qRT-PCR as described above. For TCM, media was collected from tumor cell lines grown to 60% confluence after 3 days and centrifuged for 15min at 3500rpm to remove cellular debris. MSCs were treated with TCM ± IPI-926 (10nM) for 3 days then cells were processed for qRT-PCR or immunoblotting.