Fig. 9.

Melatonin (MT) blocks environmental stress-activated GCN2/ATF4 singaling via suppressing ROS release in placental trophoblasts. (A–H) Pregnant mice in Cd and MT + Cd groups received intraperitoneal injection of Cd on GD8. In MT and MT + Cd groups, pregnant mice were treated with MT from GD7 to GD15. Pregnant mice in Ctrl group were treated with saline. n = 10–12 from 10 to 12 different pregnant mice. (A) GSH/GSSG ratio in mouse placentae. (B) Representative immunoblots of GCN2, p-eIF2α/eIF2α and ATF4 proteins in placentae. (C–E) Quantification for GCN2, p-eIF2α and ATF4. (F) Representative immunoblots of NOX2, NOX4 and HO-1 proteins in placentae. (G) Quantification for NOX2, NOX4 and HO-1. (H–Q) Human JEG-3 cells were pretreated with MT before Cd stimulation (n = 3 per group). (H) Representative immunoblots of GCN2, p-eIF2α/eIF2α and ATF4 proteins in the cells. (I-K) Quantification for GCN2, p-eIF2α and ATF4. (L) Representative fluorescent images of ROS detection. (M) Quantification for fluorescence recur to a Multimode Reader. (N) Representative immunoblots of NOX2, NOX4 and HO-1 proteins in placenta. (O–Q) Quantification for NOX2, NOX4 and HO-1. Data are expressed as the mean ± SEM. Multiple comparisons were performed using ANOVA. The post hoc test is executed adopting Bonferroni or Tamhane's T2 method following the result of homogeneity of variance test. *P < 0.05, **P < 0.01 versus PBS/NS group, ^#P < 0.05, ^##P < 0.01 versus Cd group.