Fig 2: AGR2 acts through the AKT signaling pathway during CIS escape. (A) Cell emergence was measured by colony counting after iAKT treatment (100 µM; n=3 ± standard deviation; **P-value=0.0011) and Torin treatment (10 nM; n=3 ± standard deviation; *P-value=0.0231) (B) The phosphorylation of AKT protein on emergent cells transfected with siAGR2 or treated with soluble AGR2 (CM and rAGR2) was assessed after two days of emergence using western blotting (n=3). (C) The phosphorylation of S6 and RICTOR after AGR2 suppression or eAGR2 and rAGR2 treatment in emergent cells was evaluated using western blotting following two days of emergence (n=3). AGR2, anterior gradient 2; CIS, chemotherapy-induced senescence; si, small interfering; iAKT, inhibitor of AKT; eAGR2, extracellular AGR2; rAGR2, recombinant AGR2; NT, non-treated; p-, phosphorylated.

Fig 3: Mean and standard deviation of the relative expression of the genes coding for the same of the proteins that constitute the mTOR complexes, normalised to Saline/Withdrawal 1 (VhSA) values. The rats that self-administered heroin showed higher mRNA levels of Rictor than their saline-treated controls after 1 day of withdrawal (F1,28 = 5.094, p = 0.032, ?2 = 0.182). Also, among the groups that self-administered saline solution, the rats that underwent 30 days of withdrawal showed higher Rictor mRNA levels than the rats that only had one day of withdrawal (F1,28 = 7.104, p = 0.013, ?2 = 0.254). *Significant effect of the simple effect analyses (p < 0.05)

Fig 4: Representative western blot images and densitometry bar graphs normalised to total protein loaded (Rictor and Phospho-Rictor (thr1135) or to Rictor levels (p-Rictor/Rictor)). The rats that self-administered heroin showed higher phosphorylation rates at thr1135 than their saline-treated controls (F1,22 = 5.247, p = 0.032, ?2 = 0.155). $Significant effect of the treatment factor (p < 0.05)

Fig 5: mTOR signalling. (A) mTOR phosphorylation relative to total mTOR expression. (B) Raptor phosphorylation relative to total Raptor expression. (C) Rictor phosphorylation relative to total Rictor expression, following 16 h with no treatment or with TGFß1 (1 ng/ml or 5 ng/ml) and 30 min insulin stimulation (0 nM or 100 nM). Data are reported as Mean ± SEM. AU, arbitrary units (defined as band density values). Phospho (P) and Total (T). Healthy: N = 5 participants (clear bars), PCOS: N= 5 participants (filled bars).