Reverse transcriptase

Total RNA was isolated from MG-63 cells cells by RNeasy kit (QIAGEN, Valencia, CA). MG-63 cells were then placed in serum-free DMEM with 100 μg/ml EMD protein for 12 h. After denaturation of total RNA at 70°C for 10 min, cDNA was synthesized with oligo-dT primer by incubating with reverse transcriptase (Qiagen) at 50°C for 30 min. The primers for MMP-2 were 5′-TGGTTTTCCTCCATCCAGTGG-3′ (forward) and 5′-CAGGTTGTCTGAAGTCACTGC-3′ (reverse). The primers for GAPDH were 5′-ACC ACA GTC CAT GCC ATC AC-3′ (forward) and 5′-TCC ACC ACC TTG CTG CTG TA-3′ (reverse). Polymerase chain reactions were performed with Pfu polymerase (Qiagen) initiated by 1 cycle at 95°C for 15 min followed by 30 cycles at 94°C for 45 sec, 55°C for 45 sec, 72°C for 1 min and 1 cycle at 72°C for 10 min for final extension. PCR products were loaded to agarose gel, and stained with ethidium bromide. The bands were analyzed using AlphaImager™ IS-3400 software. Briefly, IDV was measured as the sum of all the pixel values after background correction in each band. The values (AVG) of each band were calculated as IND/AREA, where AREA is the size of the region that was measured. The results are shown the values of AVG_MMP1/AVG_GAPDH at each time point.

To detect the expression of endogenous Igfn1 in C2C12 and Igfn1 KO cell lines, mRNA was extracted from C2C12 myoblasts using GenElute DIRECT mRNA MINIPREP KIT (SIGMA), and the cDNA was converted from mRNA by Reverse transcriptase (QIAGEN). The cDNA concentration of each sample was measured, diluted to the same concentration and mixed with primers and SYBR Green Master Mix (Applied Biosystems) to form RT-PCR reactions on an optical 96 well plate. The reactions for Igfn1 and housekeeping reference gene were carried out using the following primer sets: RT-PCR IGFN1-Forward: GGT ATC GTC GAC TTC CGGG; RT-PCR IGFN1-Reverse: GTC AAA CGT AGC GAC CCC T; RT-PCR HPRT-Forward: GTT GGA TACA GGC CAG ACT TTG TTG; RT-PCR HPRT-Reverse: GAT TCA ACT TGC GCT CAT CTT AGG C.

To determine the expression levels of target transposase genes and oxidative stress related genes, we performed qRT-PCR, as previously reported (Kim et al., 2017 (link)). After cells were harvested at OD₆₀₀ 4.0, cells were diluted to OD₆₀₀ 2.0, and total RNAs were extracted using a phenol-based RNA extraction procedure. cDNAs were synthesized by PCR using reverse transcriptase (Qiagen, Germany), and the synthesized cDNAs were quantified by DeNovix (United States), normalized, and stored at −70°C, until real time PCR performance. Quantitative PCR was conducted using the RT-PCR machine (model CFX96^TM Optics Module, Bio-Rad, United States). Relative gene expression levels were calculated using the comparative threshold cycle (ΔΔC_T) method and normalized to the expressed level of the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), such as a control for stable expression level on H₂O₂ treatment (Livak and Schmittgen, 2001 (link))². The two-way ANOVA was used to test difference between the samples which were represented the means and standard deviations (SD) of three replicate experiments and it was considered to be significant at p < 0.05.

Pterostilbene (>97% high-performance liquid chromatography (HPLC)-pure) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Pterostilbene was dissolved in dimethyl sulfoxide (DMSO) and further diluted in a sterile culture medium before use. DMSO, Sulforhodamine B dye, Dulbecco’s minimum essential medium (DMEM), trypsin/EDTA, phosphate-buffered saline (PBS), TRIS base, and acetic acid were also procured from Sigma-Aldrich (St. Louis, MO, USA). Alexa Fluor 488 and Alexa Fluor 647 donkey anti-rabbit IgG antibodies were purchased from Invitrogen (Grand Island, NY, USA). A TRIzol Plus RNA isolation and purification kit was obtained from Life Technologies (Invitrogen, Rockville, MD, USA), and real-time polymerase chain reaction (rt-PCR) primers, dNTP, reverse transcriptase, and Taq polymerase were obtained from Qiagen (Valencia, CA, USA).

RNA was extracted from cells using TRIzol-chloroform (Invitrogen) according to the manufacturers instructions, precipitated using 100% isopropanol then re-suspended in RNase-free water. Samples were DNase-treated and reverse transcribed using random hexamers, Reverse Transcriptase and RNase inhibitor (all Qiagen). Amplification of specific cDNAs was performed on an Mx3000P qPCR system (Agilent Technologies) using SYBR green (Qiagen) and the following primers: MULE (5′-GGGGTTATGACCCAAGAGGT-3′, 5′-CCCATCTCGAGACTCCTCTG-3′); GAPDH (5′-CCACCCATGGCAAATTCCATGGCA-3′, 5′-TCTAGACGGCAGGTCAGGTCCACC-3′). Analysis was performed using the delta CT method.