Mg 63

The human osteosarcoma cell lines MNNG/HOS (CRL-1547TM, ATCC), Saos-2 (HTB-85TM, ATCC), MG-63 (CRL-1427TM, ATCC), U-2OS (HTB-96TM, ATCC), HUVEC (CRL-1730TM, ATCC) were obtained from Cell Bank of Shanghai Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences (Shanghai, China). According to the ATCC instructions, MNNG/HOS cells were cultured in EMEM, with MG-63 in MEM, Saos-2 in McCoy’s 5A medium, U-2OS in RPMI 1640 and HUVECs in F-12K medium. All media included 10% FBS, 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were incubated at 37 °C in a humidified incubator with 5% CO₂.

Canine OSA cell line D17 and human OSA cell line (SAOS2, U2OS, and MG63) were purchased from ATCC. The Abrams canine OSA cell line was provided by Dr. Elizabeth McNeil and originally established and shared by Dr. Doug Thamm. The BZ canine OSA cell line was established by our laboratory from a German shepherd dog. D17 was derived from a lung metastasis of a poodle; the canine Abrams cell line was derived from a metastatic nodule and the canine Gracie cell line was derived from a primary tumor sample. As indicated in the ATCC repository, the SAOS2 cell line was derived from a primary tumor in a Caucasian female, U2OS was from a tibia biopsy sample from a Caucasian female, and MG63 from a Caucasian male.
For cell culture maintenance, human OSA cell lines were incubated with DMEM medium and canine OSA cell lines were maintained with a-MEM medium, and all cells were supplemented with 10% fetal bovine serum and incubated in a humidified incubator at 37 °C with 5% CO₂.

Human OS cell lines U2OS, MG-63, HOS, SJSA-1, and 143B (ATCC, USA) and normal osteoblast cells hFOB1.19 (ATCC, USA) were included in this study. MG-63, HOS, hFOB1.19 and 143B cell lines and SJSA-1 cells were cultured in EMEM (ATCC, USA) with 10% FBS and RPMI-1640 medium (ATCC, USA) with 10% FBS, respectively, at 37 °C (or at 33.5 °C for hFOB1.19 cells) in an incubator with 5% CO₂ and 95% humidity.

A human osteoblastic cell line, OB3, and the human osteosarcoma cell lines Saos-2, MG-63, and U2OS were purchased from the American Type Culture Collection (ATCC, Rockville, USA). Saos-2 and U2OS cells were cultured in McCoy’s 5a medium modified (ATCC, USA), but 15% fetal bovine serum was added to Saos-2 growth medium, and 10% fetal bovine serum was added to MG-63 growth medium. MG-63 cells were cultured in Eagle’s minimum essential medium (ATCC, USA) containing 10% heat-inactivated fetal bovine serum. The circ-03955 interference vectors sh-circ-03955-1 and sh-circ-03955-2 and the circ-03955 overexpression vector pCDNA3.1-circ-03955 (OE-circr-03955) were obtained from GenePharma (Shanghai, China). A non-targeting shRNA (shNC) or the empty pcDNA3.1vector (OE-NC) were used as negative controls. The miR-3662 mimics and miR-3662 inhibitor were purchased from GeneCopoeia (Guangzhou, China). The MTDH mRNA interference vector sh-MTDH was obtained from GenePharma (Shanghai, China). All transfections were performed using Lipofectamine 6000 reagent (Life Technologies, USA) according to the manufacturer’s protocols.