Journal: Advanced Science

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

doi: 10.1002/advs.78304

Figure Lengend Snippet: Endothelial TRX2 restrains osteoclastogenesis and osteoporotic bone loss through FOXO3a‐mediated TRAIL signaling. A) Venn diagram analysis identifying candidate downstream targets. Overlap between aging‐up‐regulated genes and FOXO3a target genes identified Tnfsf10, which encodes TNF‐related apoptosis‐inducing ligand (TRAIL), as a candidate mediator. B) Bar graph showing the log 2 fold change of selected FOXO3a target genes in the GSE230665 dataset. C) Box plot showing normalized TRAIL expression in normal controls (NC) and patients with osteoporosis (OP) from the GSE230665 dataset. D) Western blot analysis of FOXO3a, TRAIL, and TRX2 protein levels in HEK293T cells transfected with pL‐vector, pL‐FOXO3a wild type (WT), pL‐FOXO3a ΔNLS mutant, or pL‐TRX2. β‐actin served as the loading control. E) Secreted TRAIL levels in the culture supernatants of the experimental groups in (D), as measured by enzyme‐linked immunosorbent assay (ELISA) (n = 4), with absorbance read at 450 nm. F) Representative TRAP staining images of osteoclasts derived from bone marrow‐derived macrophages (BMMs) isolated from Trx2 WT and Trx2 ECKO mice (n = 5). Scale bars, 400 µm. G, H) In vitro assessment of endothelial‐osteoclast crosstalk. (G) Left: schematic of the Transwell coculture system. Right: representative images of TRAP‐stained BMMs cocultured with control endothelial cells (shControl) or TRX2‐depleted endothelial cells (shTRX2) in the presence of immunoglobulin G (IgG) control or a TRAIL‐neutralizing monoclonal antibody (TRAIL‐mAb). Scale bars, 400 µm. (H) Quantification of the TRAP‐positive area fraction in (G) (n = 5). I‐K) In vivo rescue of TRX2 deficiency‐induced bone loss by TRAIL blockade. Representative 3D micro‐CT reconstructions of distal femurs from Trx2 WT and Trx2 ECKO mice treated with IgG or TRAIL‐mAb (I) (n = 4). Scale bars, 200 µm. Quantitative micro‐CT analysis of trabecular bone volume/tissue volume (BV/TV) (J) and trabecular number (Tb.N) (K). L, M) Histological evaluation of osteoclast formation in femoral sections. Representative images of TRAP‐stained femoral sections (L) and corresponding histomorphometric quantification of osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (Oc.N/BS) (M) (n = 4). Scale bars, 50 µm. Source data for the unedited raw immunoblots shown in Figure . Data in (C) are presented as means ± SEM, and statistical significance was determined by a two‐tailed unpaired Student's t ‐test. All other quantitative data are presented as means ± SEM, with statistical significance determined by one‐way ANOVA with Tukey's multiple‐comparisons test. * p < 0.05, ** p < 0.01, **** 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: Analysis, Expressing, Western Blot, Transfection, Plasmid Preparation, Mutagenesis, Control, Enzyme-linked Immunosorbent Assay, Staining, Derivative Assay, Isolation, In Vitro Evaluation, In Vivo, Micro-CT, Tissue, Two Tailed Test

Journal: Advanced Science

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

doi: 10.1002/advs.78304

Figure Lengend Snippet: TRAIL blockade restores angiogenesis–osteogenesis coupling and attenuates OVX‐induced osteoporotic bone loss. A) Representative 3D micro‐CT reconstructions of trabecular bone (top) and cortical bone (bottom) in distal femurs from mice in the Sham +IgG, ovariectomy (OVX) + IgG, and OVX + TRAIL Ab groups (n = 5). Scale bars, 200 µm (top) and 400 µm (bottom). B) Quantitative micro‐CT analysis of trabecular bone volume/tissue volume (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), cortical thickness (Ct.Th), and bone mineral density (BMD) in the indicated groups. C) Representative H&E–stained distal femoral sections showing trabecular architecture and bone marrow adiposity. Scale bars, 200 µm. D, E) Representative immunofluorescence images of CD31 + vessels (red) and RUNX2 + osteoblast‐lineage cells (green) in the femoral metaphysis (D), with corresponding quantification of the percentage of RUNX2 + cells (E). GP, growth plate; Tb, trabecular bone. Scale bars, 50 µm. F, G) Representative TRAP–stained distal femoral sections (F) and histomorphometric quantification of osteoclast surface per bone surface (Oc.S/BS) and osteoclast number per bone surface (Oc.N/BS) (G). Scale bars, 50 µm. H, I) Representative immunofluorescence images of CD31 + vessels (red) and ATP6V1B1/B2 + mature osteoclasts (green) in the femoral metaphysis (H), with corresponding quantification of the ATP6V1B1/B2 + area relative to the total area (I). GP, growth plate; Tb, trabecular bone. Scale bars, 50 µm. All quantitative data are presented as means ± SEM. Statistical significance was determined by one‐way analysis of variance (ANOVA) with Tukey's multiple‐comparisons test. * p < 0.05, ** p < 0.01, **** 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: Micro-CT, Analysis, Tissue, Staining, Immunofluorescence, H&E Stain

Journal: Advanced Science

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

doi: 10.1002/advs.78304

Figure Lengend Snippet: Mechanistic illustration of endothelial TRX2–FOXO3a–mediated TRAIL secretion and osteoclastogenesis in osteoporotic bone loss. In the healthy skeletal vascular niche, TRX2 binds FOXO3a through a redox‐sensitive Cys90–Cys31 disulfide interface, retaining FOXO3a at the mitochondrial outer membrane and preventing its nuclear translocation in BVECs. By contrast, in the osteoporotic state, TRX2 loss or oxidative stress disrupts this disulfide‐mediated interaction, allowing FOXO3a nuclear translocation and activation of the TRAIL promoter. Increased endothelial TRAIL secretion remodels the extracellular vascular–skeletal niche by promoting osteoclastogenesis, enhancing osteoclastic bone resorption, and suppressing osteoblast‐associated osteogenesis, thereby driving osteoporosis progression.

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: Membrane, Translocation Assay, Activation Assay

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) Luciferase-based cytotoxicity assay to validate the impact of selected HITs on FluTC-mediated MO3.13-A2-Luc killing. MO3.13-A2-Luc cells were transfected with IRG-specific pool of 30 siRNA (siTOOLs) and co-cultured with FluTC as described in . Graph indicates cytotoxicity/viability ratio normalized to Scr control. (B-C) Luminex-based cytokine analysis of the FluTC – MO3.13-A2-Luc cell co-cultures. MO3.13-A2-Luc cells transfected either with Scr siRNA or IRG-specific siRNA pool and (B) co-cultured with FluTC for 24 h or (C) cultured in CM. (B) MCP-1 and (C) IL-8 levels were depicted. (B) Each line represents an independent experiment; values indicate the average of (B) triplicates or (C) duplicates. (A) Representative data of at least 2 independent experiments with triplicates per sample. (A-C) Graphs show mean +/- SD. P-values were calculated using (A) two-tailed student’s t-test (B) Ratio-paired t-test. * = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001. (D) Cytokine analysis to determine IFNγ, TNFα, FasL and TRAIL secretion by anti-CD3/CD28 activated FluTC. Values represent the mean ± SD of triplicates.

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Luciferase, Cytotoxicity Assay, Transfection, Cell Culture, Control, Luminex, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A) FACS analysis to determine the surface expression of IFNG-R1, TNF-R1, TNF-R2, FAS, TRAIL-R1, and TRAIL-R2 in MO3.13-A2-Luc cells. Grey histograms represent the isotype control. (B) Impact of IRG knockdown on TRAIL-R2 surface expression in MO3.13-A2-Luc cells. Left panel: Representative overlay of histograms to compare TRAIL-R2 expression on IRG +/- MO3.13-A2-Luc cells. Right panel: Compiled data of mean fluorescence intensity (MFI) for TRAIL-R2 expression. (C) Real-time cytotoxicity assay (IncuCyte® SX5 System) to analyze TRAIL-induced apoptosis over 48 hours in IRG +/- MO3.13-A2-Luc cells. Incucyte® Cytotox-Red Dye was added to the transfected MO3.13 oligodendrocytes together with TRAIL treatment as an indicator of apoptosis. The graph shows total red object integrated intensity per well (RCU x µm²/Image). (D) Western blot analysis of total/cleaved caspase-3/8/9, TRAF2, phosho(p)/total TAK1, MKK4, JNK, NF-κB, pLKB1 and Bcl2 in IRG +/- MO3.13-A2-Luc cells upon 4 h treatment with TRAIL. Experiment was run in two separate blots each including Scr samples. Each HIT was shown in comparison to Scr sample analyzed in the same blot (Blot1: Scr, siSTK11, siKCNH8 and siABCA2; Blot2: Scr, siSLC1A3 and siCHRNA1). Representative data of (A & D) two, (B-C) three independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01, *** = p < 0.005, **** = p < 0.001).

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Expressing, Control, Knockdown, Fluorescence, Cytotoxicity Assay, Transfection, Western Blot, Comparison, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: (A-B) Impact of SIK3 on TRAIL-mediated MO3.13-A2-Luc killing was determined by (A) Luciferase-based cytotoxicity assay as described in supp and (B) real-time cytotoxicity assay as described in . Representative data of (A) three (B) two independent experiments. Values represent the mean ± SD. P-value was calculated using paired two-tailed Student’s t-test (* = p < 0.05, ** = p < 0.01).

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Luciferase, Cytotoxicity Assay, Two Tailed Test

Journal: bioRxiv

Article Title: Multifaceted immune resistance landscapes in human oligodendrocytes protect against cytotoxic T cells and are dysregulated in MS brain cell subsets

doi: 10.64898/2026.04.21.719872

Figure Lengend Snippet: Summary of molecular pathways involved in the downstream signaling of STK11/LKB1, KCNH8, ABCA2, SLC1A3/EAAT1 and/or CHRNA1/AChR-mediated immune resistance. Activation of caspase-3/-8/-9, MKK4, JNK1, NF-κB and expression of TRAF2, TRAIL-R2, IL-8 was experimentally depicted in this study, whereas the potential involvement of the other molecular components was deducted from the previous studies as described and referred in the results and discussion sections above. Colors of the lines depicting the regulatory networks are matched with colors of the IRG icons. Activating and inhibitory effects are indicated by arrows and blocked lines respectively. The figure was generated using biorender.com (Created in BioRender. Beckhove, P. (2026) https://BioRender.com/2adzain )

Article Snippet: Supernatants of FluTC-MO3.13-A2-Luc co-cultures and anti-CD3/CD28 activated FluTC were further analyzed for the detection of IFNγ (Human IFN-γ ELISA Set, BD OptEIA, #555142), TNFα (Human TNF ELISA Set, BD OptEIA, #555212), Granzyme B (Human Granzyme B ELISA development kit, Mabtech, #3485-1H-20) and TRAIL (human TRAIL/TNFSF10 DuoSet ELISA, R&D Systems, #DY375-05) ( , ).

Techniques: Activation Assay, Expressing, Generated