Sybr1 premix

Chromatin immunoprecipitation analysis in C₃H₁₀ cells was performed using an Enzymatic Chromatin Immunoprecipitation Kit (EZ ChIPTM #17‐371, Merck‐Millipore) following the manufacturers' instructions. Briefly, C₃H₁₀ cells were cross‐linked with 1% formaldehyde for 10 minutes at room temperature followed by quenching with glycine. Chromatin digestion was performed by micrococcal nuclease to obtain DNA fragments from 150 bp to 900 bp. Immunoprecipitation was performed with STAT3 (#1264, Cell Signaling Technology), and IgG was used as a negative control. Precipitated DNA was detected by qPCR with specific primers. Primers for the STAT3 binding site in the Ocn promoter were 5′GGATACCCCATGTTCCCAGC3′ and 5′TGCAGCCCGTCTACTGGAGC3′.
Real‐time PCR was conducted with a Roche LC 480 system using SYBR1 Premix (TaKaRa Bio Inc) on the basis of the manufacturer's instructions. All samples were analysed in triplicate, and β‐actin was used as an internal control. The primer sequences used in this study are listed in Table 1.

Total RNA was obtained from NP cells and disc tissues by TRIzol reagent (TaKaRa, Inc., Dalian, China). Reverse transcription was performed to gain the first strand cDNA by using the PrimeScript RT Master Mix cDNA Synthesis Kit (TaKaRa, Inc., Dalian, China). The real-time PCR was operated on ABI 7500 system Applied Biosystems, Foster City, CA, USA) using SYBR1 Premix (TaKaRa, Inc., Dalian, China) according to manufacturer's protocol. The relative expression of target genes was quantified the 2^-ΔΔCt methods after normalization to GAPDH. The sequence of specific primers was listed as Table 4.

Total RNA weighing about 1 ug was isolated using TRIzol reagent (Invitrogen) and reverse transcribed using the PrimeScript RT Master Mix cDNA Synthesis Kit (Takara, Japan) to obtain first strand cDNA. Real-time PCR was performed with a Roche LC 480 system using SYBR1 Premix (TaKaRa, Inc., Dalian, China) according to the instructions of the manufacturer. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data were analyzed using the comparison Ct (2^−ΔΔCt) method and expressed as the fold change relative to GAPDH. Each sample was presented in mean with the standard error of triplicate.
Primer sequences were as follows: GAPDH: forward, 5′-AGGTCGGTGTGAACGGATTTG-3′; reverse, 5′-GGGGTCGTTGATGG CAACA-3′; CHRDL1: forward, 5′- CCTGGAACCTTATGGGTTGGT-3′; reverse, 5′-AACATTTGGACATCTGACTCGG-3′; ALP: forward, 5′-ACCACCACGAGAGTGAACCA-3′; reverse, 5′-CGTTGTCTGAGTACCAGTCCC; COL1A1: forward, 5′-GAGGGCCAAGACGAAGACATC-3′; reverse, 5′-CAGATCACGTCATCGCACAAC-3′; osteopontin (OPN): forward, 5′-CTGTGTTGGTGGAGGATGTCTGC-3′; reverse, 5′-GTCGGCGTTTGGCTGAGAAGG−3′; OCN: forward, 5′-GACAAGTCC CACACAGCAACT-3′; reverse, 5′-GGACATGAAGGCTTTGTCAGA-3′.