Quantstudio 1

Cells were collected using the BioFlux Kit (Bioer, China) for total RNA extraction. Total RNA was extracted from tumor tissues by using TriZOL Reagent (Ambion, USA). qRT-PCR analyses were carried out on reverse-transcribed cDNAs with QuantStudio 1 (Applied Biosystems, ThermoFisher Scientific) and analyzed with QuantStudio Design & Analysis Software (version 1.5.1). Expression levels are always normalized to GAPDH. The primers are used as reported [19 (link), 29 (link), 30 (link)].

RT-PCR was performed using a ProFlex PCR System and/or SimpliAmp Thermal Cycler (Applied Biosystems, Foster City, CA, USA). The thermal cycling parameters were as follows: denaturing at 95 °C for 30 s, annealing at 60–65 °C for 30 s, and extension at 72 °C for 30 s. The products were loaded and analyzed on 2% agarose gels. RT-qPCR analysis was performed using a CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA) and/or QuantStudio 1 (Applied Biosystems, Foster City, CA, USA). iQ SYBR Green Supermix (Bio-Rad Laboratories, Hercules, CA, USA) and/or PowerUp SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA, USA) were used for amplification. Cycling conditions for analysis were as follows: denaturation at 95 °C for 15 s, annealing at 60–65 °C (depending on primer conditions) for 15 s, and extension at 72 °C for 20 s (50 cycles). The comparative CT (ΔΔCT) method was used for relative gene expression analysis [41 (link)]. Relative values of gene expression were normalized to the relative expression of RPL7 and/or GAPDH as reference genes. The annealing temperatures for each of the primer sets are shown in Table 1.

Cells were seeded on a six-well plate at a density of 2 × 10⁵ cells per well and incubated for 24 h to allow the cells to adhere and stabilize in the culture dish. Then, chrysophanol at a defined concentration was applied to the cells, cultured for 24 h, and then harvested. The total RNA was prepared using the RNeasy Mini Kit (Qiagen Inc., Valencia, CA, USA). The total RNA concentration was determined with a microplate spectrophotometer using the SpectraMax iD3 micro reader (BioTek, Winousk, VT, USA). cDNA was synthesized using a total RNA (2 μg) with a TOPscript^TM cDNA synthesis kit (Enzynomics, Dajeeon, Republic of Korea) by following the manual. Subsequently, quantitative real-time PCR was executed using the TOPreal^TM qPCR 2× PreMIX (SYBR Green with Low ROX) (Enzynomics, Dajeeon, Republic of Korea) with QuantStudio 1 (Applied Biosystems, Foster City, CA, USA). The relative mRNA levels were normalized using GAPDH as a housekeeping gene. The PCR primer sequences are shown in Table 1.

Total RNA was extracted from cultured cells using a MagaBio plus RNA Purified Reagent Kit (Bioer Technology, Hangzhou, China) and a GenePure Pro Automatic Nucleic Acid Extraction and Purification Instrument according to the manufacturer’s instructions. The quality of the total RNA was evaluated using a NanoDrop One spectrophotometer (Thermo Fisher, USA). cDNA was synthesized by using the PrimeScript RT Master Mix Kit according to the manufacturer’s instructions (TaKaRa, Japan). qRT-PCR was conducted using a TB Green Premix Ex TaqTM PCR kit (TaKaRa, Japan) on a Quant Studio 1 (Applied Biosystems, Thermo Fisher, USA) Real-Time PCR system. Relative gene expression levels were determined using the 2^−△△CT method. GAPDH was used as an internal control to calculate the relative expression of lncRNAs. Each PCR amplification was performed in triplicate. All primer sequences are listed in Supplementary Table S2.

RNA extraction was performed using the RNA-Quick Purification Kit (ES Science, shanghai, China) following the manufacturer’s instructions. Reverse transcription was conducted according to Eco M-MLV RT Premix kit (AG11706, Accurate Biology, Atlanta, Georgia). Supplementary Table S1 shows the primer sequences used for RT-qPCR. Candidate gene expression was normalized to that of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). RT-qPCR was performed using the SYBR Green Premix Pro Tag HS qPCR kit (AG11701, Accurate Biology) by QuantStudio 1 (applied biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Samples were run and analyzed in triplicate.

Total RNA was extracted from all chondrogenic samples at days 2, 10, and 28. Hydrogel-laden hASCs, µmasses, and hASCs in monoculture were treated with 1 mL of Eurogold RnaPure (EuroClone), immediately flash frozen in liquid nitrogen (−196 °C) and stored in a freezer at −80 °C. RNA extraction was performed by homogenizing samples and following the Eurogold manufacturer’s instructions, as previously described [49 (link)]. Reverse transcription was performed using a Super Script^® Vilo™ cDNA synthesis Kit (Life Technologies), according to the manufacturer’s instructions. Real-time polymerase chain reaction (PCR) was performed with QuantStudio1 (Applied Biosystems by Thermo Fisher Scientific, 0706 Singapore 739256) for the quantification of the following genes: Beclin 1 (BECN1), microtubule-associated protein 1 light chain 3 beta (MAP1LC3B), sequestosome 1 (SQSTM1), collagen type 2 alpha 1 chain (COL2A1), aggrecan (ACAN) (Table S1). All primer efficiency was confirmed to be high (>90%) and comparable (Table S1). For each target gene, mRNA levels were calculated, normalized to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) according to the formula 2^−ΔCt, and expressed as a percentage of the reference gene.

The T7–T12 spinal cords of rats were dissected 14 days after trauma and immediately frozen in liquid nitrogen and stored at −80°C. Total RNA was extracted and purified using the RNAeasy™ Animal RNA Isolation Kit with Spin Columns (R0024). Next, we analyzed the quality of RNA using enzyme-labeled instruments, and samples with A260/A280 ratios between 1.9 and 2.2 were used for further experiments. The primers used in quantitative reverse transcription polymerase chain reaction (qRT-PCR) were designed using the Primer3 website¹ and listed in the additional file (Supplementary Table 1). Following the Biomarker One Step SYBR Green RT-qPCR Kit (RK02012, Biomarker Technologies) to construct the reaction system. After centrifugation and blending (D1008, SCILOGEX), qPCR reaction was performed using QuantStudio 1 real-time fluorescence quantitative PCR system (QuantStudio 1, Applied Biosystems). The original CT data were exported and input by Excel, and the data were calculated and analyzed by Delta CT method.