Journal: Redox Biology

Article Title: Histone H3K9 lactylation activates the TXNIP/NLRP3 pathway to drive macrophage inflammation after spinal cord injury

doi: 10.1016/j.redox.2026.104078

Figure Lengend Snippet: Molecular Mechanism of Macrophage H3K9la-Mediated Activation of TXNIP Transcription. (A) Representative immunofluorescence images of spinal cord sections from sham mice and SCI mice at 7 and 14dpi, stained for F4/80 (green), TXNIP (red) and DAPI (blue). Scale bar, 100 μm. White boxes denote regions shown at higher magnification. (B) Higher magnification of the 14dpi section in (A), Scale bar, 10 μm. (C) Line-scan analysis of relative fluorescence intensity for TXNIP and F4/80 along the same pixel coordinates indicated in (A). (D) Spatial transcriptomic feature plot illustrating TXNIP expression in spinal cord sections from sham and 14 dpi mice. The color bar denotes relative expression levels. (E) Temporal expression trajectory of TXNIP in spinal cord macrophages, as inferred from the single-cell RNA-seq dataset GSE162610 . (F) Representative immunofluorescence images of spinal cord sections from sham mice or from mice at 7 and 14dpi, stained for F4/80 (green), NLRP3 (red) and DAPI (blue). Scale bar, 100 μm; white boxes indicate regions shown at higher magnification. (G) Line-scan analysis of relative fluorescence intensity for NLRP3 and F4/80 along identical pixel coordinates indicated in (F). (H) WB analysis of Co-IP samples, demonstrating the presence of TXNIP and its interaction partner NLRP3. (I) Representative WB of NLRP3, pro‐Caspase-1, cleaved Caspase-1, TXNIP, and β-actin. (J) Quantification of NLRP3, cleaved Caspase-1 (normalized to total Caspase-1), and TXNIP band intensities relative to β-actin in (I) (n = 3; one-way ANOVA). (K) Representative WB of TNF-α, iNOS, IL-1β, and β-actin in BMDMs treated with lactate and/or 2-DG. (L) Quantification of TNF-α, iNOS, and IL-1β band intensities normalized to β-actin in (K) (n = 3; one-way ANOVA. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.) (M) Intracellular ROS levels in BMDMs assessed by DCFH-DA staining and flow cytometry. (N) Oxygen consumption rate (OCR) profiles measuring mitochondrial respiration in BMDMs (n = 5; mean ± SD). O, oligomycin; F, FCCP; R/A, rotenone/antimycin A. (O) Quantification of OCR parameters from panel N, including basal respiration, ATP‐linked respiration, maximal respiration, and spare respiratory capacity (n = 5; one-way ANOVA. ns, not significant; ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001).

Article Snippet: Samples were blocked in 5% BSA in PBS for 30 min at room temperature, then incubated overnight at 4 °C with primary antibodies diluted in PBS: anti- F4/80 (ab6640, Abcam, 1:400), anti-IBA1 (011-27991, WAKO, 1:400), anti- Pankla (PTM-1401, PTM Bio, 1:50), anti- NLRP3 (MA5-32255, Invitrogen, 1:200), anti- TXNIP (MA5-32771, Invitrogen, 1:100), anti-β3-Tubulin (D71G9, CST, 1:400), anti-Neurofilament-L (C28E10, CST, 1:400), anti- GAP43 (D9C8, CST, 1:400) and anti- H3K9la (PTM-1421RM, PTM Bio, 1:50).

Techniques: Activation Assay, Immunofluorescence, Staining, Fluorescence, Expressing, Single Cell, RNA Sequencing, Co-Immunoprecipitation Assay, Flow Cytometry

Journal: Redox Biology

Article Title: Histone H3K9 lactylation activates the TXNIP/NLRP3 pathway to drive macrophage inflammation after spinal cord injury

doi: 10.1016/j.redox.2026.104078

Figure Lengend Snippet: H3K9la-pe Inhibits TXNIP Downstream Gene Expression and Promotes Axon Regeneration After SCI. (A) Representative in vivo imaging of SCI mice at 12, 24, and 48 h following intraperitoneal injection of H3K9la-pe, demonstrating targeted accumulation at the lesion site. Postmortem images of dissected organs were also captured, showing fluorescence intensity in mice injected with saline, DiR, or DiR-H3K9la-pe. (B) Schematic diagram illustrating the anatomical locations of major dissected organs. (C) Quantitative analysis of fluorescence signal intensity at the injury site at 12, 24, and 48 h post-injection in the saline, DiR, and DiR-H3K9la-pe groups (n = 3, one-way ANOVA). (D) Quantification of fluorescence efficiency (log-transformed) from dissected organs shown in (A) (n = 3, mean ± SD). (E) Quantitative analysis of fluorescence intensity specifically in spinal cord tissue in (A) (n = 3, mean ± SD, one-way ANOVA, ns: not significant, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.) (F) Representative WB images showing the expression levels of nuclear H3K9la, NLRP3, Caspase 1, Cleaved Caspase 1, TXNIP, β-actin, and H3 in the Sham, Control, and H3K9la-pe treatment groups. (G) Quantitative analysis of the relative protein levels in (F), including H3K9la normalized to H3, NLRP3 and TXNIP normalized to β-actin, and Cleaved Caspase 1 normalized to total Caspase 1 (n = 3, one-way ANOVA. ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001). (H) Quantitative analysis of the percentage of BDA- and NF-positive areas within the lesion site shown in (I-J) (n = 6, one-way ANOVA. ∗∗p < 0.01; ∗∗∗∗p < 0.0001.) (I) Representative immunofluorescence images of corticospinal tract tracing at 28dpi in Sham, Control, and H3K9la-pe-treated SCI mice (BDA: white, DAPI: blue; scale bar = 200 μm). (J) Representative immunofluorescence images of NF staining at day 28 post-injury in Sham, Control, and H3K9la-pe-treated SCI mice, including magnified views of the lesion area (NF: white, DAPI: blue; scale bar = 200 μm, magnified view = 50 μm).

Article Snippet: Samples were blocked in 5% BSA in PBS for 30 min at room temperature, then incubated overnight at 4 °C with primary antibodies diluted in PBS: anti- F4/80 (ab6640, Abcam, 1:400), anti-IBA1 (011-27991, WAKO, 1:400), anti- Pankla (PTM-1401, PTM Bio, 1:50), anti- NLRP3 (MA5-32255, Invitrogen, 1:200), anti- TXNIP (MA5-32771, Invitrogen, 1:100), anti-β3-Tubulin (D71G9, CST, 1:400), anti-Neurofilament-L (C28E10, CST, 1:400), anti- GAP43 (D9C8, CST, 1:400) and anti- H3K9la (PTM-1421RM, PTM Bio, 1:50).

Techniques: Gene Expression, In Vivo Imaging, Injection, Fluorescence, Saline, Transformation Assay, Expressing, Control, Immunofluorescence, Staining

Journal: bioRxiv

Article Title: Apollo-IRE1: A Genetically Encoded Sensor for Live Cell and Multiplexed Imaging of ER Stress

doi: 10.64898/2026.03.20.712661

Figure Lengend Snippet: (a) Schematic representation of the Apollo-IRE1 homoFRET sensor and unfolded protein response (UPR). Top: Accumulation of misfolded proteins in the ER lumen triggers IRE1 activation, leading to three major downstream signaling outcomes: BiP upregulation, XBP1 mRNA splicing, and TXNIP activation. A cartoon showing the crystal structures of the IRE1 lumenal domain, kinase/RNase domain, and mVenus fluorescent protein connected by the transmembrane domain, which is represented only schematically as an amorphous section that connects all three crystal structures. The mVenus fluorescent protein is positioned at the cytoplasmic edge of the transmembrane domain. Bottom: The principle of homoFRET-based anisotropy sensing: monomeric sensor excited with polarized light emits polarized light (high anisotropy). Upon IRE1 dimerization, fluorescent proteins come within FRET distance (<10 nm), resulting in homoFRET and depolarization of emission (low anisotropy). (b) Representative fluorescence intensity (top row) and calculated anisotropy images (bottom row) of INS1E cells expressing Apollo-IRE1 under control conditions (Cont) or following treatment with thapsigargin (Tg; 1 µM, 6 h) or DTT (5 mM, 3 h). Anisotropy is displayed on a pseudocolor scale ranging from 0.25 to 0.38. Scale bar, 20 µm. (c) Quantification of Apollo-IRE1 anisotropy under control, Tg, and DTT treatment conditions. Data are presented as mean ± S.E.M., n = 3 independent experiments. * P < 0.05, ** P < 0.01; statistical significance assessed by ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (95% CI).

Article Snippet: Primary antibody solution was prepared using 0.2% BSA (4 μL), 1.5% NGS, and 1:100 dilution of a recombinant monoclonal TXNIP primary antibody (ThermoFisher, Cat# MA5-32771) in AD buffer.

Techniques: Activation Assay, Fluorescence, Expressing, Control

Journal: bioRxiv

Article Title: Apollo-IRE1: A Genetically Encoded Sensor for Live Cell and Multiplexed Imaging of ER Stress

doi: 10.64898/2026.03.20.712661

Figure Lengend Snippet: (a) Comparison of Apollo-IRE1 anisotropy measured in live cells versus after paraformaldehyde fixation following DTT treatment (5 mM, 3 h). The change in anisotropy (Δ Anisotropy) relative to untreated controls is preserved after fixation, demonstrating compatibility with immunofluorescence workflows. ns, not significant; unpaired t-test (95% CI). (b) Quantification of TXNIP subcellular localization expressed as cytoplasmic-to-nuclear intensity ratio in INS1E cells under control conditions, following high glucose treatment (30 mM, 24 h), or DTT treatment (5 mM, 3 h). DTT treatment induces significant TXNIP nuclear-to-cytoplasmic translocation. ns, not significant; ** P < 0.01, *** P < 0.001; ordinary one-way ANOVA followed by Tukey’s multiple comparisons test (95% CI). (c) Representative multiplexed images of INS1E cells expressing Apollo-IRE1 fluorescence intensity (top row), Apollo-IRE1 anisotropy (middle row), and immunostained TXNIP intensity (bottom row) under control, high glucose (30 mM, 24 h), and DTT (5 mM, 3 h) conditions. Anisotropy is displayed on a pseudocolor scale (0.25–0.38). TXNIP fluorescence intensity is displayed on a pseudocolor scale (1000–4000 intensity units). Scale bar, 20 µm. (d) Ridgeline density plots showing the distribution of Apollo-IRE1 anisotropy (solid line, filled) and TXNIP cytoplasmic-to-nuclear intensity ratio (dashed line) across treatment conditions: control, high glucose (30 mM), and DTT time course (3, 4, and 5 h). Progressive DTT treatment shifts Apollo-IRE1 anisotropy slightly toward lower values (indicating increased oligomerization) while TXNIP cytoplasmic-to-nuclear ratio increases more dramatically. (e) Correlation between Apollo-IRE1 anisotropy and TXNIP cytoplasmic-to-nuclear intensity ratio across all treatment conditions. Each data point represents the mean of n = 3 biological replicates. Error bars represent S.E.M..

Article Snippet: Primary antibody solution was prepared using 0.2% BSA (4 μL), 1.5% NGS, and 1:100 dilution of a recombinant monoclonal TXNIP primary antibody (ThermoFisher, Cat# MA5-32771) in AD buffer.

Techniques: Comparison, Immunofluorescence, Control, Translocation Assay, Expressing, Fluorescence