Mulv rt m mulv reverse transcriptase

5 × 10⁴ /0.2 cm² HUVECs were seeded onto PLA–BG disks and detached with accutase the next day. The cell suspensions were centrifuged at 1,400 rpm for 5 min and the cell pellet was stored at −80°C for future use. Isolation of RNA was performed using PeqGold Total RNA Micro Kit (PeqLab) according to manufacturer’s instruction. Total RNA (1 μg) was reverse-transcribed into cDNA using dNTPs (4you4 dNTPs Mix (10 mM), BIORON GmbH, Ludwigshafen), Random Primers (Promega, Madison, WI, USA), and MuLV RT (M-MuLV Reverse Transcriptase, M0253S New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s instructions. For gene expression analyses, cDNA template underwent PCR amplification (40 cycles) using the SYBR Green (PowerUp™ SYBR® Green master mix, Applied Biosystems, Foster City, CA, USA) and sequence-specific primers (primer sequences listed in Table 1). GAPDH was used for normalization, and results were calculated using the well-established 2^−ΔΔCt method^{[32 (link)]}.

According to the viability assay, collagen gels without and with BSP supplementation (1 μg/mL and 5 μg/mL) were prepared. Cell number was adapted to 1 × 10⁶ cells/well (coculture: 5 × 10⁵ cells/well for each cell type). After 1, 4, and 7 days, the gels were digested using a 1 mg/mL collagenase I/dispase solution. The cell suspensions were centrifuged at 1400 rpm for 5 min and the cell pellet was stored at −80°C until RNA isolation. Isolation of RNA was conducted with the PeqGold Total RNA Micro Kit, according to manufacturer’s instruction. Total RNA (1 μg) was reverse transcribed into cDNA using dNTPs (4you4 dNTPs Mix [10 mM], BIORON GmbH, Ludwigshafen), Random Primers (Promega, Madison, WI, USA), and MuLV RT (M-MuLV Reverse Transcriptase, M0253S New England Biolabs, Ipswich, MA, USA), according to the manufacturer’s instructions. For gene expression analyses, cDNA template underwent PCR amplification (40 cycles) using the SYBR Green (PowerUp™ SYBR® Green Master Mix, Applied Biosystems, Foster City, CA, USA) and sequence specific primers (Primer sequences listed in Table S1). GAPDH was used to normalize gene expression. Results were calculated using the well-established 2^−ΔΔCt method[32 (link)].