Sybr green

Total RNA was extracted using Trizol reagent following manufacturer’s instructions (9109, Takara, Japan), and reverse-transcribed into cDNA using PrimeScript™ RT Reagent Kit (RR036, Takara). qRT-PCR was performed with SYBR Green (B21703, Bimake, China). The relative expression levels of mRNA were normalized against β-actin (mouse) or GAPDH (human). Specific primers of mouse Ly6G, IL-1β, IL-6, IL-10, TNF-α, EMR1, PPARγ, IFN-γ, ARG1, TGF-β, CD163, CD206, iNOS, Msr1, Scara5, Timd2, Tfrc and human Msr1 were used. Primer sequences were listed in Supplementary Table 2.

Tendon cells were seeded in 6-well plates at 3.0 × 10⁵ cells per well. After treatment, total RNA was extracted from the cells in each group using TRIzol reagent (Thermo Fisher Scientific), according to the manufacturer's instructions. Reverse transcription was performed using the Supermix Kit (Bimake, Houston, TX, USA) and the resulting diluted cDNA was analyzed by qPCR using SYBR Green (Bimake). The qRT-PCR primers used in this study were designed using PrimerBlast (https://www.ncbi.nlm.nih.gov/tools/primer-blast/) and are listed in Table 1.Table 1

Primers used in real-time PCR.

Table 1

Target genes	Forward(5′-3)	Reverse(5′-3)
IL-1β	TGTCTGACCCATGTGAG	GCCACAGGGATTTTGTC
TNF-a	CTGGCGTGTTCATCCGTTCTCTAC	ACTACTTCAGCGTCTCGTGTGTTTC
TNFR1	GAACACCGTGTGTAACTGCC	ATTCCTTCACCCTCCACCTC
Nfkb1	TTCCTGATCCCGACAAGAACTG	CCCCCAGAGACCTCATAGTTGT
Gapdh	GATGCTGGTGCTGAGTATG	GTGGTGCAGGATGCATTGCT

First, the cells were washed with PBS, and TRIzol reagent was used to extract total RNA, which was reverse transcribed to cDNA via a PrimeScript RT‒PCR kit (Takara, Japan) following the instructions provided by the manufacturer. Then, SYBR green (Bimake, USA) was utilized to perform real-time PCR with a 7500 Real-time PCR system (Applied Biosystems, USA). The calculation methodology of △△CT was adopted to confirm the relative expression of specific genes, and GAPDH was the reference gene. The primer sequences are shown in Table 1.
Table 1

Sequences of primers used for real-time PCR

Primer	Sequence 5’-3’
BMP2 Forward	TCATAAAACCTGCAACAGCCAACTCG
BMP2 Reverse	CACCCACAGCGATCATGTCG
CDH1 Forward	ATGAGTGTCCCCCGGTATCTTC
CDH1 Reverse	ACGAGCAGAGAATCATAAGGCG
CDH2 Forward	GTGCATGAAGGACAGCCTCT
CDH2 Reverse	GCCACTTGCCACTTTTCCTG
SLUG Forward	CCTCCATCTGACACCTCC
SLUG Reverse	CCCAGGCTCACATATTCC
SNAIL Forward	GTATCCAGAGCTGTTTGGA
SNAIL Reverse	AACATTTTCCTCCCAGGCC
VIM Forward	GATGGTGTTTGGTCGCATA
VIM Reverse	GATGGTGTTTGGTCGCATA
SERPINB12 Forward	CCAACAGGCTTTATGGAGAGC
SERPINB12 Reverse	CACTTTCAATCGTCGTGTGGTAA
GAPDH Forward	CTGGGCTACACTGAGCACC
GAPDH Reverse	AAGTGGTCGTTGAGGGCAATG

Briefly, total RNA was then extracted using a UNIQ-10 column total RNA purification kit (Sangon, Shanghai, China). The cDNA was then preparated from the total RNAs using an All-in-One cDNA Synthesis SuperMix Kit (Bimake, Shanghai, China). Next, equal amounts of cDNAs were subjected to RT-qPCR with SYBR Green (Bimake, Shanghai, China) using a CFX96 Real-Time PCR System (Bio-Rad, Hercules, USA). The relative expression of the target genes were calculated by 2^−ΔΔCt method using the β-actin gene as a reference gene. The following primers were used: Bcl2 forward primer: 5′-AGT TCG GTG GGG TCA TGT GT-3′, Bcl2 reverse primer: 5′-CCA GGA GAA ATC AAA CAG AGG C-3′; survivin forward primer: 5′-CAT GTA CGT TGC TAT CCA GGC-3′, survivin reverse primer: 5′-CTC CTT AAT GTC ACG CAC GAT-3′; cyclin D1 forward primer: 5′-AAC TAC CTG GAC CGC TTC CT-3′, cyclin D1 reverse primer: 5′-CCA CTT GAG CTT GTT CAC CA-3′; c-Myc forward primer: 5′-TAC AAC ACC CGA GCA AGG AC-3′, c-Myc reverse primer: 5′-TCG TCG CAG TAG AAA TAC GG-3′; SHP1 forward primer: 5′-TGG CGT GGC AGG AGA ACA G-3′, SHP1 reverse primer: 5′-CAG TTG GTC ACA GAG TAG GGC-3′; β-actin forward primer: 5′-CAT GTA CGT TGC TAT CCA GGC-3′, β-actin reverse primer: 5′-CTC CTT AAT GTC ACG CAC GAT-3′.

Total RNA was extracted from rat hearts with Trizol reagent (Invitrogen, Life Technologies, Carlsbad, CA) and reverse-transcribed into cDNA with a specific RT primer (TaKaRa, Dalian, China). Real-time PCR assays were performed using SYBR Green (Bimake, Shanghai, China) with specific primers on a 7900HT FAST real-time PCR system (Life Technologies, Carlsbad, CA). Data analysis was performed by the 2^−ΔΔCt method, as described previously (28 (link)). The primers are listed in Supplementary Table 1.