I taq

DNA was extracted from soil microcosms using a modified protocol for the NucleoSpin Soil kit (Macherey-Nagel) (Houghton and Stewart, 2019 (link)). DNA was amplified using universal primers for the V4 region (515F, 806R) of the 16S rRNA gene (Caporaso et al., 2011 (link)). PCR was carried out in 50 μL reaction volumes containing 100 μM dNTPs, 0.5 μM primers, 1 U i-Taq (iNtRON Biotechnology) and 7 μL of an enhancer solution (2.7 M betaine, 0.2 M trehalose, 6.7 mM DTT, 0.06 mg ml⁻¹ BSA and 0.07% DMSO). The final concentration of MgCl₂ was 1.5 mM. DNA templates from microcosms were used at final concentrations of 10–50 ng reaction⁻¹. Three PCR amplicons (~300 bp) for each sample were pooled. The amplicons were then purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel) and Agencourt AmPure XP (Beckman Coulter). Amplicon libraries using the PCR products were prepared and sequenced by Macrogen Inc. Sequencing data was deposited in the NCBI BioProject database (PRJNA766707 and PRJNA546003).

Cells were seeded in 6-well plates at a density of 1 × 10⁶ cells/well and incubated for 24 h at 37 °C in a humidified 5% CO₂ atmosphere. After 24 h, the medium was replaced with a serum-free medium and incubated for another 24 h. Each cell line was treated with ALC at the IC₅₀ and incubated for 48 h. TRIzol reagent (Invitrogen) was used to extract total RNA according to the manufacturer’s instructions. The QuantiNova reverse transcription kit (Qiagen, Hilden, Germany) was used to obtain cDNA from extracted RNA according to the manufacturer’s instructions. A 2x PCR master solution (i-Taq, iNtRON Biotechnology, Seoul, Republic of Korea) was used according to the manufacturer’s instructions with the following primers: MMP9 sense: 5′-TTGACAGCGACAAGAAGTGG-3′, antisense: 5′-GCCATTCACGTCGTCCTTAT-3′; VEGF: sense 5′-CCCACTGAGGAGTCCAACAT-3′, antisense: 5′-TTTCTTGCGCTTTCGTTTTT-3′; β-actin: sense 5′-GCTCTTTTCCAGCCTTCCTT-3′, antisense: 5′-GAGCCAGAGCAGTGATCTC-3′. The mRNA expression levels were adjusted to the β-actin expression level and compared with the mRNA expression in the control cells.

Total RNA was isolated from articular chondrocytes using TRIzol (Molecular Research Center Inc, Cincinnati, OH, USA). Total RNA was reverse transcribed and the resulting cDNA was amplified by PCR (iTaq, Intron Biotechnology, Gyeonggi‐do, South Korea). The PCR primers are summarized in Table S1. The transcript levels of target genes were quantified by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR) using SYBR^® premix Ex Taq (TaKaRa Bio, Shiga, Japan). For each target gene, the transcript level was normalized to that of Gapdh and expressed as a fold change relative to the indicated control.