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