Sybr green system

The total RNA was isolated with a TRIzol reagent (Invitrogen, Carlsbad, CA, USA) 1 h after UV radiation. The cDNA was synthesized from the total RNA with The PrimeScriptTM RTPCR kit (Takara, Dalian, China) to detect mRNA and lncRNA. Mature miRNAs were converted into cDNA using reverse transcription performed by the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) with miRNA-specific primers. The RT-qPCR reaction was performed in triplicate. Expression levels were measured with RT-qPCR with the SYBR Green system (Roche, Indianapolis, IN, USA). β-Actin served as a reference control. Each sample was analyzed in triplicate. The primer pairs used in this article are listed in Supplementary Table S1.

Genomic DNA was purified from the collected blood 30 and 180 days posttreatment. DNA extraction was performed using the Wizard Genomic DNA purification kit (Promega), with some modifications (33 (link)). qPCRs were performed to amplify T. cruzi DNA using the SYBR green system (Roche Applied Science, Mannheim, Germany) according to the manufacturer's instructions and using the primers TCZ-F (5′-GCTCTTGCCCACAMGGGTGC-3′, where M = A or C) and TCZ R (5′-CCAAGCAGCGGATAGTTCAGG-3′) (Invitrogen) (34 (link)). The internal control, a segment of the murine tumor necrosis factor alpha (TNF-α) gene, was amplified using the primers TNF-5241 (5′-TCCCTCTCATCAGTTCTATGGCCCA-3′) and TNF-5411 (5′-CAGCAAGCATCTATGCACTTAGACCCC-3′) (Invitrogen) (34 (link)). Cycles of amplification were carried out in an ABI 7300 real-time PCR system from Applied Biosystems. The cycles consisted of an initial denaturation hold of 10 min at 95°C followed by 40 cycles of 15 s at 94°C and 1 min at 64.3°C with fluorescence acquisition. Amplification was immediately followed by a melt program with an initial denaturation for 15 s at 95°C, cooling to 60°C for 1 min, and then a stepwise temperature increase of 0.3°C/s from 60 to 95°C. All samples were analyzed in duplicate, and negative samples and reagent controls were processed in parallel for each assay.

For RNA extraction of cartilage tissues and cultured cells, the tissues and cells were homogenized in Trizol reagent (15596-026; Thermo Fisher Scientific) by moderate vortexing. After homogenization, total RNA was extracted following the protocol of Trizol reagent. The complementary DNA (cDNA) from pure RNA (2 μg) was reverse-transcribed using the cDNA Synthesize Kit (K1622; Thermo Fisher Scientific). Fluorescence-based qPCR assay was performed with the SYBR Green system (04913850001, Roche, Shanghai, China) using the qPCR instrument (Agilent Mx3005P, Agilent Tech, Los Angeles, CA USA). The relative gene expression was calculated, normalized to β-ACTIN, and analyzed using the 2^−ΔΔCT method. The primers were synthesized by Sangon Biotech (Supplementary Table S2).

RNA was reverse-transcribed to cDNA using Moloney murine leukemia virus (MMLV) reverse transcriptase (Invitrogen, USA). For real-time PCR analyses of cDNA from different samples, amplifications were performed using the SYBR Green System in a Light Cycler 4800 thermal cycler system (Roche Diagnostics Ltd., Switzerland). The primers used were targeted to HMG-CoA, Ldlr, Srebp2, IL-6, TNF-α, and IL-1β, which are described in S3 Table. The ribosomal protein S18 (RPS18) was used as a housekeeping gene. Expression values were obtained as the relative expression of the target gene minus the constitutively expressed RPS18 gene [relative expression = 2—(Ct, Target gene—Ct, Reference gene)]. Primers for PCR amplification were designed using the Primer3 program (Howard Hughes Medical Institute, USA). Assays for each gene were performed in triplicate.

Total RNA was extracted with TRIzol reagent (Invitrogen), reversely transcribed into cDNA with oligo dT primers and then subjected to real-time PCR assays using a Light cycler 480 and SYBR Green system (Roche) following the manufacturer’s protocol. Expression of target genes was normalized to GAPDH, and quantified using the 2^−ΔΔCt method.

The RPMI-1640 medium was purchased from Hyclone (Logan, UT, USA) and fetal bovine serum from Gibco (Carlsbad, CA, USA). Recombinant human TGF-β1 protein was purchased from the Sino Biological Company (Wayne, PA, USA) and the glucose from Sigma (St. Louis, MO, USA). The Trizol reagent, reverse transcription kit and SYBR Green System were purchased from Roche Company (Basel, Switzerland). The primary antibody against β-actin (CST, 4970) was purchased from Cell Signaling Technology (Danvers, MA, USA), primary antibody against SELENOS (Abcam, Cambridge, UK, 223721) or fibronectin (Abcam, Cambridge, UK, 32419) from Abcam (Cambridge, UK), primary antibody against SELENON (Novus, Saint Charles, MO, USA, 34098) from Novus (Saint Charles, MO, USA). The secondary horseradish peroxidase-coupled anti-rabbit antibodies (CST, 5571) were purchased from Cell Signaling Technology (Danvers, MA, USA).