D4627

The RCMB32 and ICb-984 cell lines were generated in the laboratories of R.J.W.-R. and X.-N.L., respectively. The BT084 cell line was provided by T. Milde (Heidelberg University Hospital). The primary tumor sample (ST01) (Fig. 5, B to D) obtained from the operating room was minced and then placed in a digestion buffer consisting of 1× HBSS (14185, Thermo Fisher Scientific), papain (10 U/ml; LSOO3126, Worthington Biochemical), and DNase (250 U/ml; D4627, Sigma). Papain solution was aspirated and replaced with 1× HBSS containing ovomucoid (8 mg/ml; LK003182, Worthington Biochemical), BSA (8 mg/ml; Sigma), and DNase (250 U/ml) and then triturated using a Pasteur pipette to obtain a single-cell suspension. Cells were pelleted and resuspended in 0.02% HBSS-BSA. Cells were passed through a 70-μm nylon cell strainer (21008–952, VWR International). Of 20 tumors implanted, 5 grew to a sufficient size to resect. Cell viability and cell cycle stage were assayed by high-throughput single-cell immunofluorescence imaging, as described above; 12 individual cultures were assessed at each dose. Gli1 transcript expression was assessed by qRT-PCR, as described above, and three cultures were assessed at each dose. Final pathological report is provided in table S4.

Wild-type CD1-Elite mice (strain 482, Charles River) were sacrificed at P1, P7, and P14. Thirty-five to 40 mice were used per P1 and P14 biological replicate, and 14 to 17 mice were used per P7 biological replicate. Cerebella were dissected, and GNPs were isolated and purified using a Percoll gradient, as previously described (58 ,59 ). This approach results in a population that is 95 to 99% GNPs (58 ). Briefly, cerebella were minced and then placed in a digestion buffer consisting of 1× Hanks’ balanced salt solution (HBSS) (14185, Thermo Fisher Scientific), papain (10 U/ml; LSOO3126, Worthington Biochemical), and deoxyribonuclease (DNase) (250 U/ml; D4627, Sigma). Papain solution was aspirated and replaced with 1× HBSS containing ovomucoid (8 mg/ml; LK003182, Worthington Biochemical), bovine serum albumin (BSA) (8 mg/ml; Sigma), and DNase (250 U/ml) and was then triturated using a Pasteur pipette to obtain a single-cell suspension. Cells were passed through a 70-μm nylon cell strainer (21008–952, VWR International) and purified using a step gradient of 35 and 65% Percoll (P4937, Sigma). Pellets were flash-frozen in liquid nitrogen and kept at −80°C until all samples were collected for MS.