Hct116 cells

HCT116 cells were purchased from American Type Culture Collection (authenticated by STR DNA profiling) and were tested negative for Mycoplasma contamination.
HCT116 cells were cultured in McCoy’s 5A (modified) Media (Gibco) supplemented with 10% fetal bovine serum (FBS) (Gibco) and Pen/Strep at 37 °C in humidified incubator with 5% CO₂. Cells were passaged every 2–3 days.
HCT116 cells in 6-well plates were transfected by Lipofectamine LTX (15338100, Thermo Fisher) with 2.5 μg of pX330-puro plasmid containing gRNA (5′-TCCAGTAAGTACAGAAAATG-3′) and Cas9 for RHBDL4 knockout. Twenty-four hours after transfection, cells were trypsinized and plated in a 10 cm petri dish. After 24 h, the cells were treated with puromycin (1 μg ml⁻¹). The puromycin selection stopped after 48 h. Cells were trypsinized and limited dilution was performed to generate single clones, which were expanded and analysed by western blot (anti-RHBDL4) and genotyped by sequencing the genomic DNA region targeted by the gRNA.
To detect the proteolytic fragments from endogenous substrates generated by endogenous RHBDL4, 10 million wild-type or RHBDL4 knockout HCT116 cells were cultured in hybridoma serum free medium for 40 h. The medium was collected, filtered and concentrated by a 30 kDa cut-off concentrator. Proteins were precipitated by TCA and dissolved in 1× LDS loading buffer for immunoblotting analysis.

LoVo and HCT116 cells were purchased from the Type Culture Collection of the Chinese Academy of Science (Shanghai, China). LoVo cells were cultured in RPMI 1640 cell culture (Gibco) containing 10% fetal bovine serum, and HCT116 cells were cultured in McCoy’s 5A (Gibco) containing 10% fetal bovine serum and cultured in a 5% CO₂ incubator at 37°C. The proteasome inhibitor MG132 (MedChemExpress, HY-13259) was used to treat HCT116 cells for 6 h, the YAP inhibitor verteporfin (TargetMol, T3112) was used to treat HCT116 cells for 16 h, and the YAP agonist PY-60 (TargetMol, T9566) was used to treat HCT116 cells for 24 h.

HCT-116 human colon carcinoma cells and human HaCaT keratinocyte cells were purchased from the American Type Culture Collection (Manassas, VA, USA). The HCT-116 cells were cultured in RPMI-1640 medium (Gibco-BRL, Carlsbad, CA, USA) and HaCaT cells were cultured in Dulbecco’s modified Eagle’s medium (Gibco-BRL) supplemented with 10% fetal bovine serum (Gibco-BRL) and 1% penicillin-streptomycin (Gibco-BRL) at 37°C in a humidified atmosphere containing 5% CO₂ (Forma 311 S/N29035 CO₂ incubator; Forma Therapeutics, Inc., Watertown, MA, USA). The medium was changed two-three times a week.

CD34⁺ peripheral blood progenitors from human blood and bone marrow aspirates from patients with SM were obtained following informed consent under protocols approved by the NIAID Institutional Review Board (98-I-0027 and 02-I-0277). The characteristics of these patients are specified in Table S1 in Supplementary Material. Primary HuMC cultures were derived from CD34⁺ progenitors as described (32 (link), 33 (link)); and mononuclear cells from marrow aspirates were separated in a Ficoll gradient and cultured for 5 days in StemPro media supplemented with 100 ng/mL SCF (34 (link)).
HMC-1.1 and HMC-1.2 were kindly provided by Dr. Butterfield at the Mayo Clinic. HMC-1 cells, LAD-2 cells, and murine bone marrow-derived mast cells (BMMCs) from Sphk1-, Sphk2-deficient, and WT mice were cultured as described (28 (link), 35 (link), 36 (link)). P815 mastocytoma murine cells with a mutation homologous to the human D816V mutation were from ATCC (Manassas, VA, USA). HCT116 cells with or without the D816V-KIT introduced by CRISPR were from Thermo Fisher Scientific. P815 and HCT116 cells were cultured as specified by the respective providers. Cell lysates for Western blots were prepared from 1 × 10⁶ cells and processed as described (37 (link)).