Nanodrop device

Total cell extracts were prepared by sonicating, in 2 mL of PBS 0.5% IGEPAL CA-630 containing protease and phosphatase inhibitors (HALT cocktail, Pierce), a pellet of ~300 µL of U2OS cells treated for 2 hr with 2 µM DAC (S)–9, collected by scrapping in cold PBS, followed by centrifugation 30 min at 15,000 RPM at 4 °C. Protein concentration in the supernatant was evaluated using a Nanodrop device (Thermo Fisher Scientific) and the extracts diluted at 1 mg/mL with PBS containing protease and phosphatase inhibitors. One mg of proteins were then precipitated overnight at –20 °C by addition of 4 volumes of cold acetone followed by centrifugation at 16,000 g for 15 min at 4 °C. The precipitates were further processed as described for the detection of the DACone-amino acid selectivity. The data presented correspond to two independent experiments (Exp.1 and 2 in Supplementary file 4). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE (Perez-Riverol et al., 2019 (link)) partner repository with the data set identifier PXD033059.

A real-time PCR test was used to detect the expression of odontoblastic markers, including DMP1 and DSPP, at the mRNA level. Hence, 2.5 × 10⁵ cells were seeded and differentiated in each well of a 6-well plate (SPL, Pocheon-si, Korea). RNA was extracted using a kit (Cinnagen, Tehran, Iran). The concentration and purity of the extracted RNA were measured with a nanodrop device (Thermo, Waltham, MA, USA). cDNA was synthesized using the SuperScript First-Strand Synthesis kit (SinaClon, Tehran, Iran). A real-time PCR test was performed using the Amplicon kit and RUSH device (Rush, Heidelberg, Germany), and the Actinβ gene was used as an internal control. Primer sequences were also designed using the NCBI reference site (Table 1).

Total RNA were extracted from brain hemispheres, ipsilateral to ligature at 3, 6, 12, or 24 h after HI procedure at P5 or P10 in sex-matched groups and from the right brain hemispheres of age- and time-matched naive animals (details in Supplementary Material). RNA samples at P2 and P15 required for the developmental study were collected in 8 separate pools each of 6 sex-matched animals at both stages (③ in Figure 1). Brain tissues were defrosted in 350 μl lysis buffer from NucleoSpin RNA Plus extraction kit (Macherey-Nagel, Hoerdt, France) and homogeneized using ceramic beads (1.4 mm Ozyme, Montigny le Bretonneaux) in a tissue lyser^® (Qiagen, Courtaboeuf, France) for 20 s at 50 hz. Total RNAs extraction was performed using Nucleospin RNA plus kit^® according to manufacturer instructions (Macherey-Nagel) in 350 μL and frozen at −80°C. The integrity and quantity of isolated mRNAs were assessed using the 2100 Bio-analyzer (Agilent Technologies, Santa Clara, CA, United States) and the Nanodrop device (Thermo Scientific, Wilmington, United States). See details in Supplementary Material. Pooled samples for microarray study were made with 5 μg of total RNA extracted from 6 ipsilateral hemispheres of sex matched injured or control mice at both ages and all time points.

The levels of PPARγ and KI67 proteins were determined through ELISA after a 48 h of treatment with 1 µM Les236 or co-treatment with Les-236 and 10 µM rosiglitazone or Les-236 and GW9662. These proteins were specifically detected with ELISA and subsequently subjected to quantitative sandwich enzyme immunoassay, which was conducted according to the manufacturer’s instructions (Elabscience Biotechnology, Wuhan, China). Briefly, a 96-well plate was pre-coated with monoclonal antibodies specific to PPARγ and KI67. Standards and the collected cell extracts were added to the wells and incubated for 90 min at 37 °C. Next, the liquid was removed, and biotinylated detection antibodies (100 µL) were added to the cultures for 60 min. We washed the cells three times to remove any unbound substances, and then added horseradish peroxidase-conjugated avidin. The cells were washed again, and 90 µL of substrate solution was added to the wells for 15 min. Then, 50 µL stop solution was added to terminate the reaction and the absorbance was measured at 450 nm; the value obtained was proportional to the amount of PPARγ and KI67, respectively. The total protein concentration was determined in triplicate in each sample by using a Thermo Fisher NanoDrop device.

Prior to RNA isolation blood samples were thawed on ice, then circulating RNA was isolated from 500 µL plasma samples using the miRNeasy Serum/Plasma RNA isolation kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol. The quality of the RNA was analyzed using the Nanodrop device (Thermo Scientific, Waltham, MA, USA).