Recombinant human scf

The human MC line LAD2 was a gift from Dr. A. S. Kirshenbaum (National Institutes of Health, Bethesda, MD) [16 (link)]. Cells were cultured at 37 °C in 5% CO₂ and passaged weekly to maintain a 500,000 cells/mL density. Dedicated medium consisted of StemPro-34 serum-free medium (Gibco) supplemented with StemPro-34 Nutrient Supplement (Gibco), 2 mM l-glutamine (Sigma-Aldrich), 100 U/mL penicillin/100 μg/mL streptomycin (Gibco), and 100 ng/mL recombinant human SCF (R&D Systems).

LAD2 cell line was kindly provided by Dr. Kirshenbaum of the National Institutes of Health (Bethesda, MD, USA). LAD2 cells were cultured in StemPro-34 SFM (Thermo Fisher Scientific, MA, USA) with StemPro-34 nutrient supplement (2.5%, Thermo Fisher Scientific, MA, USA), l-glutamine (2 mM, Gibco, CA, USA), penicillin/streptomycin (1%, Hyclone, UT, USA), and recombinant human SCF (100 ng/mL, R&D Systems, MN, USA). Half of the medium was replaced weekly by adding an equal volume of fresh medium containing SCF. The cell density was maintained at 2–5 × 10⁵ cells/mL. The cells were incubated at 37 °C in 5% CO₂ incubator.

The HMC-1(5C6) and LAD2 cells were established at 1996 by Dr. BM Henz [21 (link)] and at 2003 by Dr. AS Kirshenbaum [22 ] separately, since than used widely as human mast cell line.
The HMC-1(5C6) cell line was kindly provided by Dr. T Zuberbier (Universitatsmedizin, Berlin). HMC-1(5C6) cells were grown in Iscove’s Modified Dulbecco’s Medium (GIBCO, Grand Island, NY) containing 10% FBS (GIBCO), 100 U/ml penicillin, and 100 μg/ml streptomycin (GIBCO) [21 (link)].
The human mast cell line LAD-2 was kindly provided by Dr. AS Kirshenbaum (NIAID, Bethesda, MD). LAD2 cells were cultured in StemPro-34 serum-free medium (GIBCO) supplemented with 2 mM l-glutamine (GIBCO) and 100 ng/ml recombinant human SCF (R&D Systems, Inc., Minneapolis, MN) [22 ].

Saracatinib (Cat# S1006), vemurafenib (Cat# S1267), rapamycin (Cat# S1039) and hemin (Cat# S5645) were purchased from Selleck Chemicals (Shanghai, China). Recombinant human SCF (Cat# 255-SC-200) was purchased from R&D Systems (Shanghai, China). Recombinant human IL-3 (Cat# GMP200-03) was purchased from Peprotech (Suzhou, China). Erythropoietin (EPO) was purchased from Darbepoetin alfa Administration (Kyowa Hakko Kirin, Japan). StemSpan SFEM (Cat# 09650) was purchased from STEMCELL Technologies. Propylhydrazine hydrochloride (Cat# 114,715) was purchased from Sigma Aldrich.

The neoplastic huMC lines HMC‐1.1 (KIT V560G) and HMC‐1.2 (KIT V560G, KIT D816V) were kindly provided by Dr. JH Butterfield (Mayo Clinic, Rochester, MN, USA) (Butterfield et al., 1988 (link)). HMC‐1 cells were maintained in a humidified incubator at 37°C and 5% CO₂ in T75 flasks in Iscove's modified Dulbecco's medium (IMDM, Corning) supplemented with 2 mM L‐Glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (Corning) and 10% (v/v) fetal bovine serum (FBS, Gibco, Thermo Fisher Scientific). Cells were passaged every 3–4 days based on cell density. The LAD2 huMC line was maintained in serum‐free StemPro‐34 media supplemented with StemPro‐34 nutrient supplement (Gibco), 2 mM L‐Glutamine, 100 IU/ml penicillin, 100 μg/ml streptomycin (Corning) and 100 ng/ml recombinant human SCF (R&D Systems), as described (Kirshenbaum et al., 2003 (link)). Hemi‐depletions were performed weekly. Cell counts and viability (using acridine orange/propidium iodide stain, Logos Biosystems) were assessed on a Luna‐FL automated brightfield and dual fluorescence cell counter (Logos Biosystems). Cell treatment with 10 μM GW4869 (5 mM stock solution in DMSO; Sigma‐Aldrich), DMSO (vehicle control; Sigma‐Aldrich) or 10 μM amitriptyline (10 mM stock solution in water; Sigma‐Aldrich) was performed for 16 h.

For SCF/c-Kit signaling analysis, the cells were serum starved for 30 min and then pretreated with 4C9 antibody at the indicated concentrations for 30 min. Next, 100 ng/mL recombinant human SCF (R&D Systems, Minneapolis, MN, USA) was added and incubated for an additional 5 min. The cells were washed twice with DPBS and lysed using RIPA buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM Na₂EDTA, 1 mM EGTA, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS, 10 mM β-glycerophosphate, 1 mM Na3OV4, 10 mM NaF, 1 μg/mL leupeptin, 1 mM PMSF, 5 μg/mL aprotinin, and 2 mM 2-mercaptoethanol). The protein extracts were subjected to SDS-PAGE and transferred onto PVDF membranes (Millipore, Burlington, MA, USA). The membranes were blocked with 1 × TBST containing 5% BSA at room temperature (RT) for 1 h and probed with the following antibodies: anti-c-Kit (R&D Systems, Minneapolis, MN, USA), anti-p-c-Kit (Tyr568/570, Tyr703, Tyr719, and Tyr823; Cell Signaling Technology, Beverly, MA, USA), anti-Erk1/2 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-Erk1/2 (Cell Signaling Technology, Beverly, MA, USA), anti-Akt (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-p-Akt (Ser473; Cell Signaling Technology, Beverly, MA, USA), and anti-α-tubulin (generated in our laboratory).