Trypsin edta

Neural stem cells (NSCs) were isolated from C57BL/6 mouse brains at embryonic day 14. The forebrain was separated and dissected out and digested in 0.25% trypsin‐EDTA (Thermo Fisher) for 10 minutes at 37°C. Dissected tissues were resuspended briefly by pipetting and filtering them through a 40‐μm strainer after inactivation of trypsin‐EDTA by FBS (Thermo Fisher). Cell suspensions were plated on Matrigel (Thermo Fisher)‐coated dishes in an NSC culture medium (DMEM/F12 [Thermo Fisher]) supplement with 1% N2 (Thermo Fisher), 2% B27 (Thermo Fisher), 1% penicillin/streptomycin (Thermo Fisher), 1% GlutaMAX (Thermo Fisher), and 10 ng/mL of bFGF and EGF (PeproTech, USA). Before using them in experiments, NSCs were processed multiple times (>3).

Cells were dissociated using Accutase (Thermo Fisher Scientific, 00-4555-56) (for NPCs), 0.05% Trypsin-EDTA (Thermo Fisher Scientific, 25300054) (for ESCs) or 0.25% Trypsin-EDTA (Thermo Fisher Scientific, 25200056) (for iPSCs) and then stained with SSEA-1 eFluor 660 (Invitrogen, 50-8813-42, eBioscience eBioMC-480 (MC-480)) at a dilution of 1/100 for 15 min at 4 °C. Cells were washed once with 0.5% BSA in PBS and then FACS sorted using a BD FACSAria II SORP or a BD Influx. Gating strategy is exemplified in Supplementary Fig. 7.

HEK cells were maintained in T75 flasks with 15 ml medium. Culture conditions were 37 °C and 5% CO₂. In order to passage cells, old medium was aspirated and cell layer was washed with 10 ml DPBS (37 °C). PBS was aspirated and 3 ml Trypsin-EDTA (ThermoFisher; 25300-054; 37 °C) were added for approximately 1 min until the cell layer detached. Trypsinization was stopped with 10 ml medium (37 °C). Cells were pelleted at Trypsin-EDTA (ThermoFisher; 25300-054; 37 °C). Supernatant was aspirated and pelleted cells were resuspended thoroughly in 10 ml medium (37 °C). Cells were counted and 0.5–0.75 million cells were transferred to a new culture flask within a final volume of 15 ml medium (37 °C). Cells were passaged every 3–4 days when they reached approximately 80% confluency.
HMC3 cells were maintained in 10 cm dishes (Sigma, CLS430167) with 10 ml medium. Culture conditions were 37 °C and 5% CO₂. For passaging, old medium was aspirated and cell layer was washed with 10 ml DPBS (37 °C). PBS was aspirated and 3 ml Trypsin-EDTA were added for approximately 5–15 min until the cell layer detached. Trypsinization was stopped with 10 ml medium (37 °C). Cells were counted from this suspension and 0.25–0.5 million cells were transferred to a new culture dish within a final volume of 10 ml medium (37 °C). Cells were passaged every 3–4 days when they reached approximately 80% confluency.

D16-BO-QD cell line (QD for short) was established in our laboratory from a transgenic HER-2-positive mammary carcinoma of a Delta16 female mouse. QD cell line was cultured in MammoCult medium (STEMCELL Technologies, Vancouver, Canada) supplemented with 1% fetal bovine serum (FBS), 100 U/mL penicillin and 10 μg/mL streptomycin (all from Thermo Fisher Scientific).
Human HER-2⁺ breast cancer cell line BT-474 (HER-2⁺⁺⁺ cell line) and its trastuzumab-resistant clone C5 [24 (link)] were routinely cultured in RPMI (Thermo Fisher Scientific) supplemented with 10% FBS, 100 U/mL penicillin and 10 μg/mL streptomycin.
Cells were cultured at 37 °C in a humidified 5% CO₂ atmosphere and were split once or twice a week according to density using 0.05% trypsin EDTA (Thermo Fisher Scientific).
HER-2 expression of all cell lines is shown in Supplementary Figure S1.

Lumbar DRG were harvested from adult ADAM17^ex/ex and ADAM17^+/+ mice and prepared as previously described (14 (link), 30 (link)). Briefly, ganglia were incubated in Liberase Blendzyme 1 (9 mg/100 ml DMEM; Roche, Basel, Switzerland) for 2 times 30 min, washed, and incubated with Trypsin-EDTA (Thermo Fisher Scientific) for 15 min. After washing with supplemented TNB medium (Biochrom, Berlin, Germany) with l-glutamine (Thermo Fisher Scientific), penicillin G sodium, streptomycin sulfate (Thermo Fisher Scientific), and Protein-Lipid-Komplex (Biochrom AG), DRG were dissociated and centrifuged through a 3.5% bovine serum albumin gradient. The sensory neurons were resuspended and plated on coverslips coated with a mixture of poly-L-Lysine/laminin (MilliporeSigma) and kept in TNB medium containing protein lipid complex, supplemented with 25 ng/ml nerve growth factor (NGF) (Alomone Labs, Jerusalem, Israel) at 37°C in a humidified incubator with 5% CO₂.

MEM-α, glutamine, HEPES, penicillin-streptomycin, trypsin-EDTA, DPBS, sodium pyruvate, and HBSS were purchased from Thermo Fisher Scientific (Waltham, MA, USA; Invitrogen brand). FBS was purchased from HyClone Laboratories, Inc. (Logan, UT, USA). Dabrafenib was purchased from LC Laboratories (Woburn, MA, USA). 3-aminopropylphosphonate (3-APP) was purchased from AmBeed, Inc. (Arlington Heights, IL, USA). Trimethylsilyl propanoic acid (TSP) was purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA).