Ginsenoside Rh2

supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA) in a humidified chamber with 5% CO2 at 37 °C. GRh2 (Weikeqi Bioscience, China) was prepared in a stock of 100 mg/ml and applied to cultured GBM cells at 0.01 mg/ml, 0.1 mg/ml and 1 mg/ml, respectively .

GRh2 was purchased from Weikeqi Bioscience, Chengdu, China and was exposed to the cultured PC cells at a dose of 0.01, 0.01 and 1 mg/mL.

purchased from Beijing Bailingwei Technology Co., Ltd. (Beijing, China) (purity, 99.7%). Glutathione (reduced) (purity, 99%) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China). Ginsenoside Rh2 (purity ≥ 98%) was purchased from Sichuan Weikeqi Biological Technology Co., Ltd. Dulbecco’s modified eagle medium (DMEM) was obtained from Gibco (Life Technologies Corporation, USA). Fetal bovine serum was purchased from Bioengineering (Shanghai).

GRh2 (Weikeqi Bioscience, China) was prepared in a stock of 100 mg/ml and applied to cultured GBM cells at 0.01, 0.1, and 1 mg/ml, respectively.

Ten mice were analyzed in each experimental condition. Gastric irrigation with GRh2 (Weikeqi Bioscience, China) was performed as has been described previously [14,27], and the frequency is twice per week for 4 weeks, till analyses. GRh2 was given at 0.2 mg/kg body weight, 1 mg/kg body weight, and 5 mg/kg body weight, respectively.

GRh2 (Weikeqi Bioscience, China) was prepared in a stock of 100mg/ml in normal saline and 100µl was injected subcutaneously into the skin grafts at 0.01mg/ml, 0.1mg/ml and 1mg/ml, respectively. sFlt1 (Becton-Dickinson Biosciences, San Jose, CA, USA) was prepared in a stock of

Dulbecco's modified Eagle's medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 15% fetal bovine serum (FBS; Sigma-Aldrich, St Louis, MO, USA) in a humidified chamber with 5% CO2 at 37 °C, as has been described before [34]. GRh2 (Weikeqi Bioscience, China) was prepared in a stock of 100mg/ml and applied to cultured GBM cells at 0.01mg/ml, 0.1mg/ml and 1mg/ml, respectively.

Animals were irradiated with 200 cGy using a RS-2000 irradiator (Rad Source, Suwanee, GA, USA), and immediately received intraperitoneal injection of GRh2 (1mg/kg body weight, Weikeqi Bioscience, China) in 0.3 ml sterile PBS. The control mice received 0.3 ml sterile PBS. Mice were monitored daily and recorded survival twice weekly.

GRh2 (Weikeqi Bioscience, China) was prepared in a stock of 100 mg/ml and applied to cultured GBM cells at 0.01, 0.1, and 1 mg/ml, respectively.

resulted in nearly 100 % infection efficiency based on green fluorescence. The reporter-carrying PC3 cells were termed PC3-luc. GRh2 (Weikeqi Bioscience, China) (0.1 mg/ml) was given to the cultured cells for intervention.

The reporter-carrying glioblastoma cells were termed

GC-luc. Ginsenoside Rh2 (Weikeqi Bioscience, China)

(0.1 mg/ml) alone, or with recombinant EGFR (0.2 μg/ml)

(Millipore, USA), was given to the cultured GC-luc for 24 h

before analysis.

Body weight was measured at 8am in the morning without fasting. Five mice were analyzed in each experimental condition. Gastric irrigation with GRh2 (Weikeqi Bioscience, China) (w/o SB431542 (Santa Cruz, US)) was performed as has been described previously [19, 20].

Cell viability was tested by Cell Counting Kit-8 (CCK-8) assay (Beyotime, Shanghai, China) according to the manufacturer’s instructions. Cells (6 × 103 cells/well) were treated with different concentrations of SMI-4a (0.625–10 μM) (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) or combination of G-Rh2 (Weikeqi Bioscience, China) and SMI-4a in 96-well plates for 24, 48 and 72 h, respectively. After culture, cell viability was evaluated by CCK-8 assay.

The cells were maintained in RPMI 1640 medium

(Thermo Scientific, Waltham, USA) supplemented with

5% foetal bovine serum (Thermo Scientific, Waltham,

USA). G-Rh2 (Weikeqi Bioscience, Chengdu, China), in

varying concentrations (0, 0.01, 0.05, 0.1, 0.5, 1 mg/mL),

was added to the cultured cells according to the following

experimental requirements.

Isomaltose was purchased from Tokyo Chemical Industry (Tokyo, Japan). Protopanaxatriol and protopanaxadiol were obtained from ChromaDex (Irvine, CA). Compound K, ginsenosides Rh1, Rd, F1, F2, Rg3, and Rh2 were obtained from Sichuan Weikeqi Biological Technology (Chengdu, Sichuam, China). Lectin from Phytolacca americana (pokeweed mitogen, PWM), dextran standards certified according to Deutsches Institut für Normung (molecular weight; 668 kDa), Dulbecco’s modified Eagle’s medium

Recombinant human UGT isoforms (UGT1A1, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, UGT2B15) expressed in baculovirus-infected insect cells were obtained from BD Gentest Corp. (Woburn, MA, USA). Ginsenosides Rb1, Rb2, Rc, Rd, Rg3, Rh2, C-K, ppd, ppt, Re, Rg1, Rh1, F1, and F2 were purchased from Sichuan Weikeqi Biotechnology Company (Chengdu, Sichuan, China). The purity of these compounds was above 95%. All other reagents were of HPLC grade or of the highest grade commercially available.

The water layer was further partitioned with water-saturated BuOH three times, yielding the BuOH and water fractions (0.20 and 4.4 g, respectively). Ginsenosides F1, F2, Rd, Rg3, Rh1, Rh2, protopanaxadiol, and protopanaxatriol were obtained from Sichuan Weikeqi Biological Technology (Chengdu, Sichuan, China); ginsenosides Rg1 and Rb1 were obtained from Wako Pure Chemical Industries (Osaka, Japan)

Ginsenosides Rg1 and Rb1 were obtained from Wako Pure Reagents (Osaka). Compound K was obtained from ChromaDex (Irvine, CA, USA). Ginsenosides F1, F2, Rd, Rg3, Rh1, Rh2, protopanaxadiol and protopanaxatriol were obtained from Sichuan Weikeqi Biological Technology (Chengdu, Sichuam, China). Chemical structures of ginsenosides used in the present study are shown in Fig. 1.

The ginsenoside Rb3 (Rb3) was isolated from SCLPN, and its structure was characterized by spectroscopic analysis with both ESI-MS and 13C NMR. 20(S)-Ginsenoside Rg3 (Rg3, Lot No.: 14,197–60-5-15-1), 20(S)-ginsenoside Rh2 (Rh2, Lot No.: 78214-33-2-14-6), compound K (C-K, Lot No.: 39262-14-1-14-9) and 20(S)-protopanaxadiol (PPD, Lot No.: 30636-90-9-14-2) were supplied by Sichuan Weikeqi Biological Technology Co. Ltd. (Chengdu, China). These compounds are white powders with a purity of 98%, which was verified by high