Sant 1

Co-injected embryos with 3.8 ng of control, ATG, or E14 MO with either 1 ng control or p53^ATG MO (Chen et al., 2005 (link)) were treated with final concentrations of 0.15%v/v DMSO, 20 µM smoothened agonist (Sigma Aldrich), or 20 µM smoothened antagonist-1 (SANT-1) (Sigma, Dorset, UK, S4572) from 4 hpf until 48 hpf. Embryos were dechorionated and put in fresh treatment solution at 24 hpf. Images were taken with a fluorescence stereomicroscope (Leica Microsystems, Mannheim, Germany).
For cell experiments, hTERT-RPE1 cells were subjected to two rounds of transfection. Six hours after the second round, cells were serum starved in un-supplemented DMEM F12 HAM overnight, then treated with either 500 ng/ml recombinant Sonic Hedgehog (Shh) (C42II, N-terminus, R&D systems, Abingdon, UK, 1845-SH-025), 500 ng/ml SANT-1 (Sigma, Dorset, UK, S4572) or an equivalent volume of drug vehicle control for 24 h before methanol fixation and immunolabelling.

After PBS rinse, cells are exposed to DMEM with B27 (100×), RA (2 µM, Sigma), Noggin (100 ng/ml, Peprotech), SANT1 (0.25 µM, Sigma), Vc (0.25 mM, Sigma), Pen/Strep (100×), and Glutamax (100×). Cells are fed with fresh medium every other day.

After PBS rinse, cells are exposed to DMEM with B27 (100×), EGF (100 ng/ml, Peprotech) + NICO (Sigma, N0636, 10 mM) + Noggin (100 ng/ml) + Vc (0.25 mM). Cells are fed with fresh medium every other day.
V-bottom plate-based aggregation: stage-4 cells were rinsed with PBS and then incubated with Accutase (Millipore) for 10–15 min at 37 °C. Dissociated single cells were rinsed twice with DMEM/F12 and spun at 300 × g for 3 min. The resulting cells were re-suspended in aggregation medium (5a-Medium supplied with 10 μM Y27632). Cell solution with 0.1–0.4 (according to experimental design) million cells was added into each well of V-bottom 96-well plate, followed by a spun at 300 × g for 3 min. The plate was put into 37 °C incubator for 8–12 h to form clusters. 5a-Medium is made of V4b-Medium + heparin (Sigma, H3149, 10 µg/ml) + ZnSO₄ (10 µM, Sigma, Z0251) + LDN (Selleck Chemical, S2618, 100 nM) + T3 (1 µM, Sigma, T6397) + RA (0.05 µM) + SANT1 (0.25 µM; Tocris) + GABA (1 mM, Sigma) + human EGF (100 ng/ml, Peprotech) + NICO (10 mM) + Vc (0.25 mM). V4b-Medium (800 ml) is made of 340 ml MCDB 131 medium + 170 ml F12 medium + 170 ml KO-DMEM medium + 3.9 ml Glucose (45%, Sigma) + 80 ml 20% FF-BSA + 16 ml Sodium Bicarbonate (7.5%) + 4 ml ITX + 8 ml Pen/Strep + 8 ml Glutamax; all items are from Thermo Fisher Scientific unless indicated.