Rnaiso plus system

LS-8 cells were seeded on Ti samples at 1×10⁵ cells/well and cultured for 24 h to allow cell attachment. Ti samples were then cultured in standard or RA medium for 48 h. LS-8 cells on prepared Ti surfaces were resuspended using 0.25% trypsin (Cellgro). The apoptosis/necrosis of LS-8 cells was assessed using the Annexin V/propidium iodide (PI) kit (Seven Seas, People’s Republic of China) as described by the manufacturer. Using the FACS Aria II system (BD Biosciences, San Jose, CA, USA), Annexin V⁻/PI⁻ cells were considered viable, annexin V⁺/PI⁻ cells were considered apoptotic cells, and Annexin V⁺/PI⁺ cells were considered necrotic. Quantitative polymerase chain reaction (qPCR) was used to detect the expression of the apoptosis-related genes – B-cell lymphoma-2 (BCL-2); BCL2-associated X protein (BAX); and caspase 3 (CASP3). Total RNA was isolated from cells using the RNAiso Plus system (Takara, Japan). Aliquots of 1 μg total RNA were translated to cDNA using the Prime-Script^TM RT reagent kit (Takara). qPCR was conducted using FastStart Universal SYBER Green Master (Hoffman-La Roche Ltd.) on a CFX96™ PCR System (Bio-Rad Laboratories Inc., Hercules, CA, USA). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene. The primers used are listed in Table 1.

Following RNA extraction using the RNAiso Plus system (TaKaRa), the total cellular RNA was reverse-transcribed into complementary DNA using an RT kit (TaKaRa). β-Actin was used as an internal reference gene. The primer details are provided in Table S8.