*2.8. Real-Time PCR Analysis*

Quantitative real-time PCR was carried out, using the Applied Biosystems 7300 Real-Time PCR System. The PCR was performed using 3 μL of first strand cDNAs and SYBR Green PCR Master Mix (ThermoScientific®, Waltham, MA, USA) under the following conditions: initial denaturation at 95 ◦C for 1 min; 40 cycles of denaturation at 95 ◦C for 15 s; annealing at 49 ◦C for 15 s; and extension at 72 ◦C for 45 s. Data were normalized to the housekeeping Actin gene.

## *2.9. Statistical Analysis*

All the experiments were carried out in triplicates (*n* = 3). Microsoft Excel 365 software was used for compiling the experimental data, to form a database for further analysis. All the data were evaluated by one-way ANOVA, and for the graphical illustrations and Tukey's HSD test to examine the difference among treatment means (*p* ≤ 0.05), the Origin software (Version 2022, OriginLab Corporation, Northamptom, MA, USA) was used.

#### **3. Results**
