Tri regent

Total RNA was extracted from Canine osteoblast cell line (CO), Abrams, D17 and Moresco cell lines with Tri Regent (Sigma) and chloroform-ethanol method. Then determined the quantity and quality of RNA with the help of Biophotometer (eppendorf). Total 2 µg of total RNA was converted to cDNA by using High Capacity RNA to cDNA Kit (Applied Biosystem). Transcript expression (mRNA) level of GLI1, GLI2, PTCH1, and PAX6 was determined by using the StepOne Plus Real-Time PCR system (Applied Biosystem). Taq-Man assays (Applied Biosystem) GLI1 (Assay ID # Cf04230663_m1) GLI2 (Assay ID#4331348), PTCH1 (Assay ID #Cf02690587_m1), and PAX6 (Assay ID#Cf02675240_m1) were used to determine the expression. All transcripts expression was equilibrated with endogenous control HPRT1 (Assay ID#Cf02626256_m1) expression (Figure S1, S2, S3). The real time expression calculation is based on Delta-delta (ΔΔC) method by PE Applied Biosystem (Perkin Elmer, Forster City, CA)[42] (link).

Post 24 hours exposure of ADMSCs with 0 and 0.5 ng/ml of astaxanthin, the total RNA was isolated using TRI regent (Sigma-Aldrich, MO, IL, USA). The total RNA concentration was measured using UV/Vis-spectrophotometry (BioTek, Winooski, VT, USA) at 260 nm. cDNA was synthesized from the RNA using the reverse transcription method PrimeScript 1^st strand cDNA Synthesis Kit (Takara, Shiga, Japan). Template DNA was then used in gene-specific PCR, wherein synthesized cDNA using oligo-dT primer was amplified by 40 cycles (initial denaturation, denaturation, annealing, and extension: 98 °C, 1 min, 98 °C, 10 sec; 55–60 °C, 30 sec; 72 °C, 1 min). The expression of stemness-related genes (SOX2 and KLF4 (Bioneer, Alameda, California, USA)) and proliferation-related genes (Rex1, c-MYC, and Wnt3a, (Bioneer, Alameda, California, USA)) were studied, wherein Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was utilized as a housekeeping gene. Details of the primers are listed in Table 1. Further, aliquots of PCR product were electrophoresed on 1.5% agarose gels, PCR fragments were stained by loading STAR dye (Dynebio, Gyeonggi-do, South Korea) and detected by the gel documentation system (Daihan Scientific, Seoul, South Korea). All gene expression experiments were performed in triplicates.

RNA was extracted from HEK293 and HeLa cells using TRI regent (Sigma-Aldrich) according to the manufacturer’s instructions. For northern blotting, 3 μg of total RNA was separated by electrophoresis on a 1.2% agarose-glyoxal gel (RNAs >300 nt) or a denaturing (7 M urea) 10% polyacrylamide gel (RNAs <300 nt) and transferred to a nylon membrane. Probes used were 5ʹ-[³²P]-labelled DNA oligonucleotides hybridising to the 5ʹ end of ITS1 and between sites 2a and 2 in ITS1, as described previously [23]. Random prime [³²P]-labelled DNA probes for the detection of the U3 snoRNA, the U1 snRNA and ETS3-containing pre-rRNAs, were prepared as described in [54]. RNAs were detected using a phosphorimager and signals were quantified using ImageQuant software. S1 nuclease mapping was performed as previously described [41] using a 5ʹ-[³²P]-labelled probe hybridising across the A’ cleavage site. Cleaved and uncleaved RNA fragments were separated by denaturing polyacrylamide gel electrophoresis and detected using a phosphorimager.