3.8.4. mRNA Expression

The bone cells were treated with collagen as mentioned above and the total mRNA was isolated using the TRIzol method as per our earlier protocol [13]. Briefly, a Trizol lysed cell monolayer was mixed with chloroform to obtain RNA and the RNA was precipitated using isopropanol. After thorough washing with 70% ethanol, the RNA was converted to first strand cDNA using an Invitrogen kit as per the manufacturer's instructions. The percentage of osteogenic mRNA expression (ALP, Runx2, osteocalcin, Col1a1, and beta-actin) (Table 2) of blue shark collagen treated bone cells was determined using an ABI 7500 Fast Real-Time PCR System (Applied Biosystems, Shanghai, China) [13]. The cDNA template was mixed with SYBR Green Fast qPCR RT Master Mix (Invitrogen, Shanghai,

China) and target primers as per the manufacture's instructions. The relative mRNA expression of the target gene was calculated by subtracting the mRNA expression of a house-keeping gene, beta-actin.


**Table 2.** List of primers and its sequence used in this study.

### 3.8.5. Immunocytochemistry

For immunocytochemistry, cells were grown in the confocal disc (Cat no. 150682, ThermoFisher Scientific, Shanghai, China) with collagen, washed with ice-cold PBS, fixed with 4% paraformaldehyde for 15 min and permeabilized at room temperature with 0.1% Triton X-100 for 15 min. Then, the cells were incubated with fluorescence stains such as fluorescein isothiocyanate (FITC) and 4 ,6-diamidino-2-phenylindole (DAPI). The images were captured using a confocal laser scanning microscope (Leica TCS SP8, Leica Microsystems CMS GmbH, Wetzlar, Germany).

### *3.9. Western Blot Analysis*

The osteogenic effect of the ASC and PSC was further tested in dMBMS cells using the western blot method, as an empirical study. In brief, the cells were seeded at a cell density of 1 × 105 in 6 well plates and cultured with 1 mL culture medium for control. For collagen treatment, the samples (50 ng/mL) were mixed with 1 mL cultured medium and cultured for three days. Then, the treated and untreated cells were harvested using a lysis buffer with 30 s sonication and centrifuged at 12,000 rpm at 4 ◦C for 15 min for the extraction of total cellular protein. The protein content was quantified using a BCA kit as per the manufacturer's instructions (Thermo Fisher Scientific, Shanghai, China) and then was separated by 12% SDS-PAGE and transferred to PVDF nitrocellulose membrane (Invitrogen, Shanghai, China) using iblot-2 dry blotting system (Invitrogen, Shanghai, China). A protein transferred membrane blocked with 5% BSA-PBST were incubated with primary antibodies such as anti-GAPDH, anti-Col1α2 and anti-Runx2 (ABcam, Shanghai, China) overnight at 4 ◦C. Then the membrane was incubated with secondary goat anti-rabbit IgG-HRP (Abcam) for 1 h at 37 ◦C and exposed to an enhanced chemiluminescent reagent. Images were captured with a Universal Hood II Gel Doc System (Bio-Rad, Rochester, NY, USA).
