Scrambled control sirna

The siRNA for DR3 and GATA3 were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Scrambled siRNA control was purchased from GenePharma (Shanghai, China). The sense and antisense sequences of the scrambled siRNA were as follows: 5′-UUCUCCGAAC GUGUCACGUT T-3′ and 5′-ACGUGACACG UUCGGAGAAT T-3. Cells were transfected with siRNAs using Lipofectamine 2000 reagent (Invitrogen, Carlsbad, CA, USA). Messenger RNA and protein levels of the target gene products were determined 24 hrs post transfection or as indicated in the text.

The specific siRNA of MAP3K1 and scrambled siRNA control were designed and synthesized by GenePharma (Shanghai, China). The shMAP3K1 plasmid was constructed and synthesized using the pSIH vector by GenScript Company (Nanjing, China). Lentivirus was packaged by cotransfection of psPAX2, pMD2.G, and shMAP3K1 plasmids into HEK293T cells. PBMCs were cultured in a 96-well plate in the presence of polybrene (1 : 1,000, Sigma-Aldrich), with the addition of 30 μL lentivirus for 3 days.

The mimics control, miR-106b mimics, inhibitor control, anti-miR-106b inhibitor, scrambled siRNA control, Tp53inp1 siRNA, and Cdkn1a siRNA were purchased from GenePharma (GenePharma Co., Ltd., Shanghai). Transfection of miRNA mimics/inhibitor or siRNAs was performed using the Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instruction.

Cells at a density of 3 × 10⁵ cells/well in six-well plates were used for siRNA transfection. Briefly, 50 nM siRNA against the NF-κB p65 subunit or scrambled siRNA control (GenePharma Company, Shanghai, China) was mixed with lipofectamine 3000 (Life Technologies, Grand Island, NY, USA) and then added to each well. The effects of siRNA on NF-κB p65 subunit mRNA and protein levels were examined 48 and 72 h after transfection, respectively.

At 24 hr before transfection, chondrocytes were plated onto six-well culture plates and transfected with siRNAs at 70–80% confluence. siRNAs for β-catenin was purchased from Santa Cruz Biotechnology (sc-29210). siRNAs for Intigrin β1, Intigrin α1, Intigrin α10, FAK, ILK, YAP/TAZ and control scrambled siRNA were purchased from GenePharma. Chondrocytes were washed in PBS and resuspended in resuspension buffer R included with Neon™ kit (Invitrogen). Transfections of siRNA into chondrocytes with Neon Transfection System (Invitrogen) were performed according to the manufacturer’s protocol with two pulses of 1400 V and 20 ms. Transfection efficiency was assessed using qPCR and western blot (Data not shown). Eletroporated cells were resuspended in culture medium containing serum and supplements without antibiotics and incubated for 24 hr at 37 °C in a humidified 5% CO₂ incubator.

Small interfering RNA (siRNA) specific for GDF11 gene expression (siRNA^GDF11) and control scrambled siRNA were synthesized by GenePharma Co., Ltd (Shanghai, China). Cells were seeded at 1x10⁵ per cell into 12‐well plates one day before transfection. Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) (2 μL/mL) was mixed with siRNAs (final 50 nmol/L) in reduced serum medium OptiMEM (Invitrogen) and then added into each well (1ml per well). Medium was changed to DMEM containing 10% FBS 8 hours after transfection and cultured for further analysis. Expression of GDF11 at levels of mRNA and protein was detected by RT‐PCR and Western blot, respectively. For the experiment of differentiation that lasted 14 days, the cells were transfected with siRNA again 7 days after the first transfection to maintain GDF11 being knocked down. VEGF165 was added to the culture 24 hours after either transfection to induce differentiation of MSCs to ECs.