Rneasy preparation kit

Total RNA was prepared using the RNeasy preparation kit (Qiagen, MD, USA) according to the manufacturer’s instructions. Total RNA (1000 ng) was analyzed using the PrimeScript^TM RT reagent Kit (Takara, Shiga, Japan) according to standard protocols. qRT-PCR was performed using TB Green Premix Ex Taq (Takara, Shiga, Japan) and a CFX96 real-time PCR detection system (BioRad, CA, USA). The reproducibility of the quantitative measurements was evaluated by three independent cDNA syntheses and PCR amplification from each preparation of RNA. For mRNA analysis, data were normalized to GAPDH as an endogenous loading control, and fold changes were calculated via relative quantification (2^−ΔΔCt). A detailed list of qRT-PCR primer sequence information is provided in Table S3.

Total RNA was extracted using an RNeasy preparation kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s instructions. Quantitative real-time PCR was performed using 1 μg of RNA and SuperScript VILO Master Mix (Thermo Fisher Scientific, Waltham, MA, USA). The expression of each gene was quantified using the StepOnePlus real-time PCR system (Thermo Fisher Scientific) and Fast SYBR Green Master Mix (Thermo Fisher Scientific) with the following primers: CDKN1A, forward 5′-TGTCCGTCAGAACCCATGC-3′, reverse 5′-CCAGTTGGTAACAATGCCATGT-3′ [57 (link)]; TP53, forward 5′-TAACAGTTCCTGCATGGGCGGC-3′, reverse 5′-AGGACAGGCACAAACACGCACC-3′ [58 (link)]; ACTA2, forward 5′-GTGTTGCCCCTGAAGAGCAT-3′; reverse 5′-GCTGGGACATTGAAAGTCTCA-3′; COL1A1, forward 5′-GAGGGCCAAGACGAAGACATC-3′, reverse 5′-CAGATCACGTCATCGCACAAC-3′; COL4A1, forward 5′-GGACTACCTGGAACAAAAGGG-3′, reverse 5′-GCCAAGTATCTCACCTGGATCA-3′; COL5A1, forward 5′-GCCCGGATGTCGCTTACAG-3′, reverse 5′-AAATGCAGACGCAGGGTACAG-3′; CXCL1, forward 5′-GATTGTGCCTAATGTGTT-3′, reverse 5′-ATCCAGATTGAACTAACTTG-3′; CXCL2, forward 5′-GGGCAGAAAGCTTGTCTCAA-3′, reverse 5′-GCTTCCTCCTTCCTTCTGGT-3′ [59 (link)]; CXCL3, forward 5′-CGCCCAAACCGAAGTCATAG-3′, reverse 5′-GCTCCCCTTGTTCAGTATCTTTT-3′ [60 (link)]; and GAPDH, forward 5′-GGCGTCTTCACCACCATGGAG-3′, reverse 5′-AAGTTGTCATGGATGACCTTGGC-3′ [12 (link)]. The mRNA expression levels were normalized to GAPDH.