Fig 1: Limonene downregulates Akt, mTOR, and NF-κB expression and inhibits IκB phosphorylation in ACHN cells. (A) Evaluating the impact of limonene on mRNA expression of Akt1, mTOR, and NF-κB in ACHN cells. β-Actin served as the internal control (n = 3). (B, C) Western blot analysis to assess the protein levels of NF-κB, IκB-p, and β-Actin in ACHN cells (n = 3). The original, uncropped blots are provided in Supplemental Figures 1–4. (D) ELISA-based quantification of nuclear NF-κB expression in ACHN cells after limonene treatment (n = 3). GAPDH and Lamin B1 levels were also evaluated to confirm the presence of nuclear fraction and the absence of cytoplasmic contamination. (E) Evaluating the impact of limonene on mRNA expression of NF-κB downstream target genes MMP-9, BCL2, TNF-α, COX-2, and IL-6 in ACHN cells (n = 3). Data are reported as mean ± SD. Statistical significance for all comparisons was determined compared with the control group, represented by the asterisk. P value <0.05 is regarded as statistically significant (∗∗∗∗P < 0.0001, ∗∗∗P < 0.001, ∗∗P < 0.01, ∗P < 0.05). ns, not significant.