Psilencer 4.1 cmv neo

The siRNA sequences of LINC01314 and KLK4 were designed using the siRNA design software of Ambion (Austin, Texas, USA). The base sequences of both sides of the Loop (TTCAAGAGA) site were complemented, and the LINC01314 and KLK4 shDNA templates were prepared. Next, HindIII and BamHI sticky restriction sites were added to each end of the shDNA template and were corresponded to restriction sites on both sides of pSilencer 4.1-CMV neo (AM5779, Ambion, Texas, USA). The target genes and the vector were connected by Ligase 4 and then transformed to DH5α competent cell. The genes were marked and positive clones were screened.

MEK inhibitor U0126, transforming growth factor β1 (TGF‐β1), ascorbic acid‐2‐phosphate, dexamethasone, and fibroblast growth factor (bFGF) were obtained Sigma‐Aldrich (St. Louis, MO, USA). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, New York, USA). The mammalian expression vector pcDNA4/Myc was obtained from Invitrogen (Carlsbad, California, USA). siRNA expression vectors pSilencer 4.1‐CMV neo was obtained from Ambion (Austin, Texas, USA). Lipofectamine 2000, G418 and puromycin were purchased from Invitrogen. Other chemicals were purchased from Sigma‐Aldrich. Antibodies against RunX2, phospho‐ERK, ERK2, c‐Myc, and β‐actin were purchased from Cell Signaling Technology Inc (Beverly, Massachusetts, USA). Antibody against NCAM was obtained from Millipore (Billerica, Massachusetts, USA). Antibody against Col X was obtained from Abcam (Cambridge, Massachusetts, USA).

LY294002, β-glycerophosphate, ascorbic acid-2-phosphate, and dexamethasone were obtained Sigma-Aldrich (St. Louis, MO, USA). XAV939 was obtained from EMD Millipore (Darmstadt, Germany). Lithium Chloride (LiCl) was purchased from Nacalai Tesque, Inc. (Kyoto, Japan). Fetal bovine serum (FBS) was purchased from Gibco (Grand Island, NY, USA). The mammalian expression vector pcDNA4/Myc was obtained from Invitrogen (Carlsbad, CA, USA). siRNA expression vectors pSilencer 4.1-CMV neo was obtained from Ambion (Austin, TX, USA). Lipofectamine 2000, G418 and puromycin were purchased from Invitrogen (Carlsbad, CA, USA). Antibodies against RunX2, Osterix, β-catenin, phospho-Akt, and β-actin were purchased from Cell Signaling Technology, Inc. (Beverly, MD, USA). The secondary antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). TRIzol regent was purchased from Invitrogen (Carlsbad, CA, USA). QuantiTect Reverse Transcription kit was purchased from Qiagen (Valencia, CA, USA). SYBR Green Master Mix was purchased from Bio-Rad Laboratories (Richmond, CA, USA). Other chemicals were purchased from Sigma-Aldrich.