Endogenous control 18s rrna

Total RNA was extracted from ScAT tissue using a RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA, USA) and used to measure H3K9 demethylases (KDM4C) mRNA expression by real-time RT-PCR method. PTN primers (forward, 5′-GACCAGAAGAGGGAAGACAAAG, reverse, 5′-ACTCCAGAAGCCTGGTTTATTAC, and probe, /56-FAM/ATACCAAG/ZEN/AAGCACCCAGCCACC/3IABkFQ/) were purchased from Integrated DNA Technology (IDT, Coralville, IA, USA). The Taqman assay reagents and endogenous control (18S rRNA) were purchased from Applied Biosystems (Foster City, CA, USA) and were used to measure Ct values using the ABI Prism 7500 PCR system (Applied Biosystems, Foster City, CA, USA). Normalizations of the target gene expression were done using the 18S rRNA as a reference.

Total RNA was isolated from 3D constructs using TRIzol®Reagent (Thermo Fisher Scientific, USA) and subsequent homogenization. The quality and quantity of the RNA were determined by spectrophotometry. RNA was transcribed to cDNA using the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, USA). Levels of gene expression were detected using commercially available TaqMan® Expression Assays per manufacturer’s instructions and Bio-Rad CFX96 Touch™ Real-Time PCR Detection System (Bio-Rad, Hercules, CA). For amplification, the TaqMan™ Universal PCR Master Mix (4304437, Applied Biosystems, USA) and the following assays were used: human MMP-3: HS00968305_m1, human IL-6: HS00985639_m1, human IL-8: HS00174103_M1, rat MMP-3: RN00591740_m1, rat IL-1β: RN00580432_m1, rat IL-6: RN01410330_m1, rat TNFα: RN01525859_g1 (Applied Biosystems LifeTechnologies, USA). Samples were tested in triplicates. Expression levels of mRNA were normalized to 18S rRNA Endogenous Control (4333760T, Applied Biosystems, USA). For data analysis, the 2^−ΔΔCq (Livak) method was used. Protein expression levels of human MMP-3, IL-6 and IL-8 in response to mechanical overloading were determined with Quantikine ELISAs according to manufacturer’s instructions (R&D Systems, USA).