Sknbe2

The human SKNBE2, SKNFI and SKNAS cell lines were obtained from the American Type Culture Collection (respectively ATCC CRL‐2271, Cat CRL‐2142 and CRL‐2137); the human KELLY cell lines were obtained from European Collection of Authenticated Cell Cultures (92 110 411) the human MHHNB11 cell line was obtained from the Leibniz Institute DSMZ—German Collection of Microorganisms and Cell Cultures (DSMZ ACC‐157) and the human NB‐1 cell line was donated from Professor Alessandro Quattrone (University of Trento, Italy). We used the in‐house available NB cell lines with rs907187‐CC/CG/GG genotype. SKNAS and SKNFI cell lines were grown in Dulbecco's Modified Eagle's Medium (DMEM; Sigma); SKNBE2 cell line was grown in 1:1 mixture Minimal Essential Eagle's Medium (MEM; Sigma) and Nutrient Mixture F12 (Sigma); KELLY, MHHNB11 and NB1 cell lines were grown in RPMI‐1640 Media (Sigma). The medium was supplemented with 10% heat‐inactivated FBS (Sigma), 1mmol/L L‐glutamine, penicillin (100 U/mL) and streptomycin (100 mg/mL; Invitrogen). The cells were cultured at 37°C, 5% CO₂ in a humidified atmosphere. The cell lines used for all the experiments were re‐authenticated and tested as mycoplasma‐free. Early‐passage cells were used and cumulative culture length was less than 3 months after resuscitation.

HEK293 and MCF7 cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Sigma) supplemented with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA, USA). Human neuroblastoma SH-SY5Y and SK-N-BE(2) cells were maintained in RPMI 1640 (Sigma) supplemented with 10% FBS. HEK293 cells were obtained from the JCRB Cell Bank and MCF7 and SH-SY5Y cells were from ATCC, while SK-N-BE(2) cells were purchased from the European Collection of Authenticated Cell Cultures. Mycoplasma contamination was tested by Mycoplasma Detection Set (Takara), and short tandem repeat analysis was performed for cell authentication (Promega). Transient transfection with C-terminal hemagglutinin (HA) or myc-tagged human NLRR1 plasmids was performed using Fugene HD (Roche) or Lipofectamine2000 (Invitrogen) according to the manufacturer’s instructions. Seventy to eighty percent confluent monolayers of transfected cells were treated with growth factors for the indicated times after 16 h serum starvation, and cell lysates were subjected to western blot analyses. Cell proliferation was determined using a Cell Counting Kit-8 (Dojindo, Japan).

The human NB cell lines (SK-N-SH, SH-SY5Y, SK-N-BE (2), and IMR-32) were obtained from the National Collection of Authenticated Cell Cultures in Shanghai, China.
SK-N-SH and IMR-32 cells were cultured in Dulbecco’s modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) (Gibco) and 1% penicillin–streptomycin mixture (Sigma-Aldrich). SK-N-BE (2) cells were cultured in DMEM/F12 (1:1) with 10% FBS and 1% penicillin–streptomycin mixture (Sigma-Aldrich). SH-SY5Y cells were cultured in a mixture of MEM (44.5%) and Ham’s F12 (44.5%), supplemented with 10% FBS (Gibco), 1% non-essential amino acids (NEAAs) (Gibco), and 1% penicillin–streptomycin mixture (Sigma-Aldrich). All cells were maintained in an incubator at 37°C with a humidified atmosphere containing 5% CO₂.
Gomisin B and ginsenoside Rh2 chemical reagents were procured from MedChemExpress. Gomisin B (purity ≥99.9%) and ginsenoside Rh2 (purity ≥99.9%) were dissolved in dimethyl sulfoxide (DMSO). To ensure minimal impact on cells, the final concentration of DMSO in the cell culture medium was maintained below 0.1%.
Cell viability was assessed using the CellCounting-Lite 2.0 Luminescent Cell Viability Assay (Vazyme, DD1101-03) and measured using a multimode microplate detection system (PerkinElmer EnVision 2015). Aidi injection was donated by Guizhou Ebay Pharmaceutical Co., Ltd.