Figure 8(A) Molecular docking indicating the interaction between GHTT and active motifs (AMs) of Pdia4. AM 1, 2 and 3 represent the first, second and third CGHC domains. The sulfur, nitrogen and oxygen atoms are shown in yellow, blue, and red, respectively. The hydrogen bond and hydrophobic interaction between GHTT (light pink) and the amino acid residues (gray) of Pdia4 model are shown by the green and yellow dashed lines, respectively. (B) Insulin-based turbidity assays were used. Insulin was incubated with recombinant Pdia4 in the presence of PBS and inhibitors at 25°C. After 30 min, the reaction was stopped and measured the absorbance at 595 nm. The relative activity (%) of Pdia4 was obtained by the formula, 100% × (OD595 of PBS − OD595 of GHTT)/(OD595 of PBS). Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (C) Min6 cells were incubated with medium containing low glucose (LG, 0.5 mM) and high glucose (HG, 25 mM) in the absence or presence of 28 μM GHTT (HG+GHTT) and then stained with MitoSOX or CellROX for an additional 30 min. Signal from CellROX (right) and MitoSOX (left) was quantified and re-plotted into histograms. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (D) 293T cells, which were transfected with the construct encoding Flag-Pdia4 and that expressing Myc/Flag-tagged p22phox or Ndufs3, were treated with GHTT (28 μM) for 30 min. The cells were lysed and incubated with anti-Pdia4 antibodies plus protein G beads. Their total lysates (TL) and immunoprecipitates (IP) underwent immunoblotting analysis with anti-Flag antibody. (E) Eight-week-old diabetic Leprdb/db mice were orally fed with a daily dose of PBS (CTR), sitagliptin (STG, 90 mg/kg) and GHTT (1, 5 and 25 mg/kg) from 8 to 24 weeks. Their FBG and PBG were measured. Data information: Data from 3 experiments and more are presented as the mean ± SD. Log rank test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. The number of mice (n) is indicated in parentheses. (F) The sections of pancreata of the mice (E) were stained with anti-insulin (αIns) antibody and dihydroethidium (DHE) (left). Islet area (μm2) and relative fluorescence intensity (RFI) were quantified (right). Scale bar: 100 μm. The dash circles indicate islet regions. Data information: Data from 3 experiments are presented as the mean ± SD. one-way ANOVA test were used for statistical analysis of differences between groups, and P (*) < 0.05; P (**) < 0.01 and P (***) < 0.001 are considered statistically significant. (G) A schema illustrating the regulation of Ndufs3 and p22phox by Pdia4 via a CGHC-dependent intermolecular interaction. Pdia4 interacts with Ndufs3 and p22phox, leading to an increased activity of ETC CI and Nox. Consequently, the exuberant ROS induces β-cell pathology and diabetes. Conversely, Pdia4 deficiency and inactivation of Pdia4 by GHTT can abolish the interaction of Pdia4 and p22phox or Ndufs3, leading to reduction of ROS production and β-cell failure and mitigation of diabetes.