Fig 2: APAP-induced Sestrin2/AMPK activation is enhanced in the liver of mice treated with cajaninstilbene acid (CSA). (A–C) Animal experiment was identical to that in Figure 1, and liver tissues were collected at 6 h after APAP overdose. Immunoblot analysis of phosphorylated (p-) or total protein in lysates of total liver extracts. The graphs show results of densitometric analyses of p-AMPK relative to total AMPK, CAMKK2, Sestrin2, LKB1 relative to GAPDH respectively (n = 6/group). (D,E) Compound C (10 mg/kg) was injected to mice 0.5 h prior to APAP, followed by CSA (75 mg/kg) administration. Liver tissues were collected at 6 h after APAP overdose. (D) Immunoblot analysis of whole liver homogenate. The graphs show results of densitometric analyses of p-JNK relative to total JNK, PGC-1α, TOM20 relative to GAPDH respectively. (E) Immunoblot analysis of mitochondrial LC3 II and the corresponding densitometry normalized to VDAC1. * p < 0.05, ** p < 0.001, *** p < 0.001 compared with the vehicle control; # p < 0.05, ## p < 0.01, ### p < 0.001 compared with the corresponding APAP-treated group. N.S, nonsignificant.

Fig 3: Cajaninstilbene acid (CSA) inhibits JNK signaling pathway in APAP-induced liver injury. Animal experiment was identical to that in Figure 1, and liver tissues were excised at 6 h after APAP overdose. (A) Western blot analysis of whole liver homogenate for p-JNK, JNK, 3-Nitrotyrosine (left panel) and the corresponding densitometry normalized to GAPDH (right panel) (n = 6–7/group). (B) Representative immunostaining of 3-Nitrotyrosine in liver tissues (scale bar = 100 µm). (C) Quantification with ImageJ of 3-Nitrotyrosine positive areas (n = 6/group). (D) Mouse liver ATP levels. (E) Enzyme activity of mitochondrial respiratory complex I. ** p < 0.01, *** p < 0.001 compared with the vehicle control group; ## p < 0.01, ### p < 0.001 compared with the corresponding APAP-treated alone group.

Fig 4: Effects of caffeine (0–100 μM) on P-glycoprotein (P-gp) expression, cortisol transport, and related pathway in placental trophoblasts. A, B Relative mRNA (n=6) and protein (n=3) expression levels of P-gp in primary placental trophoblasts; C, D relative mRNA (n=6) and protein (n=3) expression levels of P-gp in BeWo cells; E Rho 123 accumulation was analyzed by the fluorescence plate reader, n=6; F Effect on cortisol efflux in BeWo cells by caffeine treatment, n=6; G relative mRNA expression levels of RYRs, n=6; H protein levels of JNK, p-JNK, and E1A-binding protein P300 (P300), n=3; I Protein-protein interactions between p-JNK and YB-1 and between YB-1 and P300; J Relative protein levels of the nucleus and cytosol YB-1, n=3; K binding level of YB-1 on the ABCB1 promoter region; binding level of YB-1 and P300 on the ABCB1 promoter region; L histone 3 Lysine 9 (H3K9) and histone 3 Lysine 14 (H3K14) acetylation levels on the ABCB1 promoter region, n=3. Mean ± S.E.M. *P<0.05, **P<0.01 vs. control

Fig 5: Expression of placental C-Jun N-terminal kinase (JNK)/Y-box protein 1(YB-1) pathway in intrauterine growth retardation (IUGR) and the gestational age-matched control neonates. Relative mRNA or protein expression levels of p-JNK (A), YB-1 (B), and P300 (C) in the female and male placentas, n=9–21; D–F correlations between placental P-gp protein levels and p-JNK, YB-1, and P300 expression levels in the female and male placenta; G, I correlations between fetal cord cortisol concentrations, neonatal birth weights, and YB-1 mRNA expression levels in female and male placenta, n=22–34. Mean ± S.E.M. *P<0.05, **P<0.01 vs. control