Fig. 7

Verification of FoxO-related signaling pathways involved in cold acclimation of Songpu mirror carp. a Flow chart of these experiments. Larvae of Songpu mirror carp were pre-treated at 18 °C with or without inhibitors or activator for 24 h and then moved to severe low temperature at 8 °C for 24 h. Cold acclimation was monitored at 12 dpf by calculation of larval survival rates. b Cold acclimation of Songpu mirror carp was significantly affected by inhibition or activation of key signaling factors acting upstream of FoxO signaling. Larvae of Songpu mirror carp were treated with inhibitors (AS1842856 for FOXO at 5 μM, dorsomorphin 2HCl for AMPK at 10 μM or SP600125 for JNK at 20 μM) or an activator (SC79 for Akt at 4 μM) for 24 h. c The expression ratios of p-FOXO/GAPDH, p-AMPK/AMPK and p-Akt/GAPDH were calculated with the densitometries of Western blots measured using ImageJ software (NIH). Data of Western blots in Fig. S7 showed significant changes in phosphorylated protein levels after treatment with AS1842856 for FOXO at 5 μM, dorsomorphin 2HCl for AMPK at 10 μM or SC79 for Akt at 4 μM for 24 h. *, p < 0.05; **, p < 0.01; ***, p < 0.001. d A working model for the roles of FoxO-related signaling pathways in cold acclimation of carps