Reverse transcription reagent

Cells were collected and total cell RNA, cytoplasmic RNA, and nuclear RNA were extracted in strict accordance with the operation instructions of the kit. The expression of HEIH was detected by Takara reverse transcription reagent and qRT-PCR. β-Actin was used as cytoplasmic reference and U6 as nuclear reference.

Real-time fluorescent quantitative PCR and Western Blot were applied to examine transfection efficiency. After 48 h of transfection, total RNA of transfected cells were extracted by Trizol. cDNA was synthetized by reverse transcription reagent (Takara, Japan). Real-time PCR was carried out by using SYBR green PCR master mix (Qiagen, Germany). Primers for fluorescent quantitative real-time PCR consisted of β-actin (F-5′-ACTGGAACGGTGAAGGTGAC-3′ and R-5′-AGAGAAGTGGGGTGGCTTTT-3′) and Annexin A2 (F-5′- ATTGCCTTCGCCTACC-3′ and R-5′-GCTCTTCTACCCTTTGC-3′). 48 h after the transfection, cells were lysed in Radio Immuno Precipitation Assay buffer with protease inhibitor. Protein samples were loaded in 10% SDS-PAGE gel, then transferred onto PVDF membrane and blotted by anti-Annexin A2 antibody followed by the HRP-conjugated secondary antibody. Signals were visualized using ECL Substrates. β-actin served as the control group. The experiment was repeated three times.

TRIzol^® reagent (Invitrogen, Waltham, MA, United States) was used to extract total RNA from the treated chondrocytes. Using reverse transcription reagent (TaKaRa Bio Inc., Kusatsu, Shiga, Japan), cDNA was produced from RNA samples and then the mRNA expression of each gene was determined with qRT-PCR. The target genes’ expression levels were determined using the 2^−ΔΔCT method, normalized with GAPDH. Table 1 contains a list of all primer sequences used in this work.

The cells were cultured for 7 days with the above four types of media with different osmotic pressures. Then, a TRIzol reagent was used to extract total RNA. Total RNA was reverse transcribed into cDNA using a reverse transcription reagent (TaKaRa, Japan). GAPDH was used as an internal reference gene. A SYBR Premix Ex Taq PCR kit (TaKaRa, Japan) and LightCycler system (Roche, Switzerland) were used to analyze the obtained cDNA for real-time quantitative PCR (RT-qPCR). The primers used for RT-qPCR are listed in Table 1 and were synthesized by Sangon Biotech (Shanghai, China). The mRNA expression of target genes was calculated with the 2^−△△Ct method.

Total RNA was extracted from cell and tissue samples by using Trizol reagent (Invitrogen, USA). RNA concentration was measured on the NanoDrop 2000 spectrophotometer (Thermo Scientific, Pittsburgh, PA, USA). Reverse transcription reagent was purchased from TaKaRa Bio Inc (Dalian, China), and cDNA was synthesized following the manufacturer’s protocol. qRT-PCR was performed with QuantStudio™ 5 Real-Time PCR System (Thermo Fisher Scientific Inc., Waltham, MA, USA). β-actin was set as the endogenous control, and the relative expression level of target mRNA was calculated by comparative circulation threshold (CT) (2−ΔΔCT) method. The sequences of gene primers used in this study are shown in Supplementary Table 1.

Total RNA was extracted from passage 3 and passage 10 DPC by using Trizol (Invitrogen, U.S.A.) according to the manufacturer’s instruction. c-DNA was synthesized from 1 µg total RNA using OligodT primers by reverse transcription reagent (Takara, Japan) based on the instruction of the manufacturer. DNA were amplified using Taq DNA Polymerase (Takara, Japan) with the following primers: β-actin (618 bp), 5′-CGG GAC CTG ACT GAC TAC CTC-3′ and 5′-CAA GAA AGG GTG TAA CGC AAC-3′; Annexin A2 (329 bp), 5′-TGAAGTCAGCCTTATCT GGC-3′ and 5′-ATTGACCAAGATGCTCGG-3′. Amplified fragments were separated through electrophoresis on 1% agarose gels and visualized by ethidium bromide staining. The experiment was repeated three times.