Ea hy926 cells

GBM 8401 (BCRC, CVCL_B051), U87 MG (ATCC^® HTB14), T98G (ATCC^® CRL-1690), and U138 MG (ATCC^® HTB-16) were the commercial GBM cell lines of Bioresource Collection and Research Center (Hsinchu, Taiwan) or American Type Culture Collection (Manassas, VA, USA). Alpha-MEM medium (Gibco BRL, Rockville, MD, USA) was used for the maintenance of U87 MG, T98G, and U138 MG, while GBM 8401 cells were cultured in RPMI-1640 medium (Gibco). These cells were supplemented with 10% fetal bovine serum, 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco) and cultured under a humidified atmosphere of 5% CO₂ and 95% room air at 37 °C. EA.hy926 cells (ATCC^® CRL2922™) was an established human fusion cell line by fusing primary human umbilical vein cells with a thioguanine-resistant clone A549. The fusion cells were cultured in Dulbecco’s Modified Eagle’s Medium with 10% of fetal bovine serum and 100 U/mL of penicillin and streptomycin and cultured under the same atmosphere as for the GBM cells.

HEK293T and HeLa cells were maintained in Dulbecco’s modified eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Pan-Biotech, Aidenbach, Germany), 100 µg/mL Penicillin (Pan-Biotech, Aidenbach, Germany), and 100 U/mL Streptomycin (Pan-Biotech, Aidenbach, Germany). EA.hy926 cells (ATCC, CRL-2922, University Blvd, VA, USA) were cultured under the same conditions as HEK293T and HeLa cells but additionally supplemented with 100 µg/mL Normocin (InvivoGen, San Diego, CA, USA) and 2% HAT ((Sodium Hypoxanthine (5 mM), Aminopterin (20 µM), and Thymidine (0.8 mM)) (ATCC, USA). Cells were maintained in a humidified CO₂ incubator (5% CO₂, 37 °C). Stable EA.hy926 ] and HEK293T cell lines expressing O-geNOps-NES [24 (link)] used in this study were developed in previous studies [25 (link)]. The same protocols were applied to generate stable HeLa cells, as described previously [29 (link)]. Cells with Mito-C-geNOps, stably O-geNOps-NES expressing HeLa, were seeded on 30 mm glass coverslips for transient transfection. 16–24 h later, 1 µg of each purified plasmid was transfected using a 2.5 µL PolyJet transfection reagent according to the manufacturer’s instructions.

The murine melanoma cells (B16-F10), human foreskin fibroblasts (Hs68), and human umbilical vein cell line (EA.hy926) were separately cultured on 96-well plates [28 (link)]. B16-F10 cells (No. CRL-6475) were obtained from the American Type Culture Collection (ATCC: Manassas, VA, USA); Hs68 cells (No. CRL-1635) were purchased from ATCC. Hs68 is one of a series of human foreskin fibroblast lines developed at the Naval Biosciences Laboratory (NBL, Oakland, CA, USA); and EA.hy926 cells (No. CRL-2922) were also acquired from ATCC. The human umbilical vein cell line, EA.hy926, was established by fusing primary human umbilical vein cells with a thioguanine-resistant clone of A549 cell (human lung cancer) by exposure to polyethylene glycol. The testing compound at suitable doses was added to each of the three cell cultures. The cells were cultured in DMEM medium within a 5% CO₂ atmosphere humidified incubator at 37 °C for 24 h. After 24 h incubation, 10% alamarBlue^® (Biosource, CA, USA) was aseptically added to measure cell viability, according to commercial kit protocols. The vehicle control group (without 4-(phenylsulfanyl)butan-2-one) was defined as 100%. The optical absorbance values (A) of the supernatants were quantified at 570 nm, and the cell viabilities were analyzed according to the following formula:
$\text{Cell viability }\left(\text{\%}\right)= \frac{\left(A_{\text{sample}} -A_{\text{blank}}\right)}{\left(A_{\text{control}} -A_{\text{blank}}\right)}\times 100\text{\%}$

Cardiac fibroblasts were isolated from 1–3 days old Neonatal Swiss albino mice by explant culture technique followed by differential trypsinization and BMSCs were isolated from 6–8 week old Swiss albino mice (∼20 g) by modification of Soleimani & Nadri protocol [18] (link). All procedures, approved by the Institutional Animal Ethics Committee (IIT Madras, India) and the Committee for the Purpose of Control and Supervision of Experiments on Animals, Government of India, were performed in accordance with the Rule 5(a) of the Breeding and Experiments on Animals (Control and Supervision; 1998). EA.hy926 cells (ATCC CRL-2922) were a kind gift from Dr. Madhulika Dixit, Vascular Biology Lab, Department of Biotechnology, Indian Institute of Technology Madras (IITM), India. The cells were incubated at 37°C in a humidified atmosphere containing 5% CO₂ with medium containing DMEM/F12 (1∶1) medium supplemented with FBS (20% for cardiac fibroblasts; 10% for BMSCs and EA.hy926 cells), 4.00 mM L-glutamine, 1000 mg/l glucose, 110 mg/l sodium pyruvate, 100 U/ml penicillin, 100 mg/ml streptomycin and 0.25 mg/ml amphotericin (Life Technologies, USA). The isolated cardiac fibroblasts and BMSCs were characterized using Immunocytochemistry (ICC).

Shenfu injection, Red ginseng extraction injection and Prepared aconite extraction injection were donated by China Resources Sanjiu Medical & Pharmaceutical Co., Ltd. The main components of shenfu injection include ginsenosides (>0.8 mg/ml) and aconitine (<0.1 mg/ml). The main components of Red ginseng extraction injection include ginsenosides (>0.8 mg/ml). The main components of Prepared aconite extraction injection include aconitine (<0.1 mg/ml). 9, 11-dideoxy-1a, 9a-epoxymethanoprostaglandin F2a (U46619), N-omega-nitro-L-arginine methyl ester hydrochloride (L-NAME),tetraethylammonium chloride (TEA), and 5-chloro-N-[4-(cyclohexylureidosulfonyl) phenethyl]-2-methoxybenzamine (glybenclamide, glyburide) were obtained from Sigma. EA.hy926 cells were commercially available from ATCC. Fetal bovine serum (FBS) and Dulbecco's modified Eagle medium (DMEM) were purchased from Gibco. Antibody against eNOS was bought from Cell Signaling Technology, China (#9586). RIPA buffer was bought from Sigma-Aldrich