Trizol

A six-well plate was used to seed 1 × 10⁵ cells per well, which were then cultured in DMEM supplemented with 10% FBS in a CO₂ incubator. Prior to the treatment, the cells underwent 6 h of starvation. Afterward, the culture medium was replaced with fresh medium containing 10% FBS, and the cells were treated with compound concentrations ranging from 5 to 15 µM for 48 h. Following the incubation, the cells were harvested and washed three times with PBS. Total RNA isolation was performed using the TRIZOL (Invitrogen) method, involving the collection and triple washing of approximately 1 × 10⁶ cells. The cells were then lysed with 1 mL of TRIZOL reagent, followed by a 5-min incubation. After adding 200 µL of chloroform and mixing by inversion, the mixture rested at room temperature for 5 min before being centrifuged at 13,000 × g for 15 min. The aqueous phase was carefully transferred to a new tube, mixed with an equal volume of isopropanol, and centrifuged at 13,000 × g for 10 min at 4°C. The resulting pellet was washed with 1 mL of 70% ethanol and centrifuged at 7,500 g for 5 min at 4°C. Once air-dried, the pellet was resuspended in 50 µL of DEPC-treated water. The isolated RNA was treated with DNase (Roche Applied Sciences) to remove any potential genomic DNA contamination. The concentration and purity of the RNA were assessed spectrophotometrically by using the A260/A280 absorbance ratio. The integrity of RNA was verified through agarose gel electrophoresis in MOPS buffer. For cDNA synthesis, 150 ng of RNA was used with the cDNA synthesis kit (BIO-RAD) following the manufacturer’s instructions. RT-PCR was performed using the assay-on-demand method, with primers listed in Table 1 provided by Applied Biosystems. RT-PCR products were verified through 2% agarose gel electrophoresis, and relative mRNA levels were normalised against β-actin.