Hfob1

Tumor tissue specimens (n = 13) and corresponding adjacent normal tissues (n = 13) were obtained from OS patients at the First Affiliated Hospital of Zhengzhou University. Informed written consent was provided by all the participants, and all procedures used in this study were approved by the Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The clinical information of the 13 OS patients is listed in Table 1. Five human OS cell lines (SOSP-9607, Saos-2, HOS, SW1353, and U2OS) and a normal osteoblast cell line (hFOB1.19) were purchased from ATCC (Manassas, VA, USA). The hFOB1.19 cell line was cultured in a Dulbecco's modified Eagle’s medium (DMEM), U2OS and Saos-2 cells in a McCoy’s 5A medium, SW1353 cells in an L-15 medium, HOS cells in MEM, and SOSP-9607 cells in an RPMI 1640 medium. All of the culture media were supplemented with 10% fetal bovine serum (FBS), and all the cell lines were maintained in a space containing 5% CO₂ at 37°C. All reagents were purchased from Life Technologies (Gibco, Waltham, MA, USA).

Human MDA PCa 2b, RWPE-1 and hFOB1.19 cells were obtained from ATCC (Manassas, VA, USA). MDA PCa 2b cells, RWPE-1 cells were cultured in F12K nutrient mixture supplemented with L-Glutamine (Gibco BRL Co. Ltd., USA), and hFOB1.19 cells were cultured in DMEM/F12 (HyClone, Logan, UT, USA); all culture media were supplemented with 100 units of penicillin/mL and 100 mg of streptomycin/mL (HyClone, Logan, UT, USA) and 10% FBS (Gibco BRL Co. Ltd., USA). The cells tested negative for mycoplasma contamination, and this testing was completed every 3 months and after the initiation of cell culture.

Normal osteoblast cell hFOB1.19 and human OS cell lines, including 143B, SJSA-1, HOS, MG-63, U2OS were purchased from ATCC. All the cell lines were grown in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS). All the cells were identified to be mycoplasma-free and were kept at 37°C with 5% CO₂.

Osteosarcoma cell lines (U2OS, MG-63, HOS, and SAOS-02) and human osteoblast cell line (hFOB1.19) were purchased from ATCC. All the cells were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum with 1% penicillin and streptomycin (containing 100 u/ml penicillin and 100 μg/ml streptomycin) (Gibco, USA). All the cells were cultured at 37°C and 5% CO₂.

The human osteoblasts cell line hFOB 1.19 (ATCC CRL-11372) was cultivated, following the manufacturer’s indications, and it was infected with different S. aureus strains (Table S5). Briefly, osteoblasts cells were infected with a MOI (multiplicity of infection) of 50. After 1.5 hr, cells were washed with PBS, and lysostaphin (20 μg/mL) was added for 30 min, to lyse all extracellular or adherent staphylococci, and then fresh culture medium with Penicillin and streptomycin were added to the cells. The washing, the lysostaphin, and medium-exchange steps were repeated every two days, to remove all of the extracellular staphylococci. To detect live intracellular bacteria at different time points post-infection (p.i.), host cells were lysed in H₂O, and the number of colony-forming units (CFU) was determined by serial dilutions on blood agar. The colony phenotypes were analyzed by a Colony Counter Shuett (Biosys, Karben, Germany). SCVs were colonies with a diameter < 0.6 mm.