Journal: Advanced Science

Article Title: Thioredoxin‐2 Alleviates Osteoporosis by Restraining FOXO3a Nuclear Translocation

doi: 10.1002/advs.78304

Figure Lengend Snippet: TRX2 interacts with FOXO3a through a redox‐sensitive Cys90–Cys31 interface and restrains FOXO3a nuclear translocation. A) Liquid chromatography–tandem mass spectrometry (LC‐MS/MS) analysis identifying FOXO3a as a TRX2‐binding partner. The representative MS/MS spectrum shows the peptide sequences of FOXO3a associated with TRX2. B) Structured illumination microscopy (SIM) images and 3D reconstructions showing colocalization of TRX2 (red) and FOXO3a (green) in HEK293T cells. Mitochondria and nuclei were labeled with MitoTracker (magenta) and Hoechst (blue), respectively. Scale bars, 2 µm. C) Proteinase K protection assay in isolated mitochondria treated with proteinase K in the presence or absence of Triton X‐100. VDAC1 and HSP60 served as mitochondrial outer membrane and matrix markers, respectively. D) Representative fluorescence images of HUVECs transfected with control siRNA (siCon) or TRX2‐targeting siRNA (siTRX2) and subjected to Duolink proximity ligation assay (PLA) using antibodies against TRX2 and FOXO3a. Green puncta represent PLA signals indicating close proximity between TRX2 and FOXO3a. Mitochondria were labeled with MitoTracker (magenta), and nuclei were counterstained with Hoechst (blue) (n = 3). Scale bars, 5 µm. E) Representative immunofluorescence images of distal femoral sections from 12‐week‐old Trx2 WT and Trx2 ECKO mice stained for CD31 (red), FOXO3a (green), and Hoechst (blue) (n = 3). Dashed boxes indicate the regions shown at higher magnification, with the corresponding single‐channel images presented on the right. Scale bars: 50 µm (overview images) and 20 µm (magnified and single‐channel images). F) Co‐immunoprecipitation (Co‐IP) assay in HEK293T cells transfected with FOXO3a‐Flag and different TRX2‐HA constructs, including wild‐type (WT), C90S, C93S, and C90/93S mutants, showing the requirement of TRX2 active‐site cysteines for FOXO3a binding. G) Molecular docking illustrating the predicted interface of the TRX2‐FOXO3a complex. The enlarged view shows the spatial proximity of TRX2 Cys90/Cys93 and FOXO3a Cys31. H) Co‐IP assay showing the interaction between endogenous TRX2 and FOXO3a‐Flag, including WT and cysteine‐mutant FOXO3a constructs. I) Co‐IP assay showing that the TRX2–FOXO3a interaction is sensitive to oxidative stress induced by hydrogen peroxide (H 2 O 2 ). J, K) Western blot analysis of nucleocytoplasmic fractionation (J) and representative immunofluorescence images (K) showing the effect of siCon or siTRX2 treatment on FOXO3a subcellular distribution. Lamin B1 and GAPDH served as nuclear and cytosolic markers, respectively. Scale bars: 5 µm. L) Quantification of the nuclear fraction of FOXO3a corresponding to the immunofluorescence images in (K) (n = 5, with three technical replicates). M) Proposed model summarizing the mechanism by which TRX2 regulates FOXO3a subcellular localization. Source data for the unedited raw immunoblots shown in Figure . All quantitative data are presented as means ± SEM. Statistical significance was determined by a two‐tailed unpaired Student's t ‐test. **** p < 0.0001.

Article Snippet: Primary antibodies included: TRX2 (1:5000, Invitrogen, PA5‐81102), TRX2 (1:5000, Abcam, ab185544), TRAIL (1:2000, Proteintech, 66756‐1‐Ig), TRAIL (1:2000, Thermo Fisher Scientific, 1J5S5), FOXO3a (1:3000, Proteintech, 10849‐1‐AP), VDAC1 (1:5000, Proteintech, 10866‐1‐AP), HSP60 (1:5000, Proteintech,15282‐1‐AP), Histone H3 (1:5000, Proteintech, 68345‐1‐Ig), Lamin B1 (1:5000, Selleck, F0523), Flag (1:5000, Beyotime, AF2855), HA (1:5000, Proteintech, HRP‐66006), GAPDH (1:20000, Servicebio, ZB15004), and β‐Actin (1:20000, Servicebio, ZB15001).

Techniques: Translocation Assay, Liquid Chromatography, Mass Spectrometry, Liquid Chromatography with Mass Spectroscopy, Analysis, Binding Assay, Tandem Mass Spectroscopy, Microscopy, Labeling, Isolation, Membrane, Fluorescence, Transfection, Control, Proximity Ligation Assay, Immunofluorescence, Staining, Co-Immunoprecipitation Assay, Construct, Docking Assay, Mutagenesis, Western Blot, Fractionation, Two Tailed Test