Fig 2: CHOP directly interacts with FOXO3a in response to ER stress.(A) Nuclear and cytoplasmic fractions from untreated SH-SY5Y cells and those treated with tunicamycin (1µM) for 24h were examined for levels of total and phosphorylated FOXO3a and CHOP. GAPDH was used as a loading control for whole cell lysates or cytoplasmic fractions, while HDAC was used as a loading control for nuclear fractions (B) Cell lysates of SH-SY5Y cells treated with tunicamycin or controls were subjected to immunoprecipitation using FOXO3a polyclonal antibody or control IgG. CHOP protein was detected from immunoprecipitates by western blotting in comparison to input controls.

Fig 3: CHOP knockdown prevents dephosphorylation of AKT and FOXO3a.(A) CHOP knockdown prevents dephosphorylation of AKT (Ser 473) in comparison to control cells treated with tunicamycin. (B) CHOP knockdown prevents dephosphorylation of FOXO3a (Thr 32) in comparison to control cells treated with tunicamycin.

Fig 4: Proposed scheme for the mechanism of AKT activation by mTORC2 in early endosomes during growth factor stimulation. PDGF-dependent PI3K activation triggers AKT phosphorylation through PDK1 and mTORC2. PtdIns(3,4,5)P3 molecules produced by class I PI3K at the plasma membrane provide a binding platform for PDK1 and AKT through each protein’s PH domain. PtdIns(3,4)P2 molecules produced predominantly by SHIP1/2 5-phosphatase from PtdIns(3,4,5)P3 play a role in connecting mTORC2 to AKT by providing a binding site for mSIN, a component of the complex, and AKT. This local activation of AKT is required for a subset of substrates represented by GSK3ß and FoxO1/3a.

Fig 5: Hepatic miRNA-205 expression in mice and humans. A-B, miR-205-5p expression in liver of WT vs. L-Foxo1,3a,4 (n = 5 per group) and L-Foxo1 (n = 7 and 9, respectively) measured by qPCR. C, miR-205-5p expression in primary hepatocytes from control WT or L-Foxo1 mice after 7-hr treatment with vehicle, cAMP/dex, cAMP/dex/insulin (n = 4 per group). D-G, miR-205-5p expression in liver of WT vs. iL-Sin3a/b (D) (n = 6 and 7, respectively), chow vs. diet-induced obese (DIO) mice (E), n = 5 per group), WT vs. ob/ob mice (F) (n = 5 per group), WT vs. hyperinsulinemic db/db (inshigh) (G, n = 7/4, respectively). Data are means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001.