Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: Composite hydrogel promotes the polarization of BV2 cells to M2 types and alleviates PC12 cell pyroptosis in vitro inflammatory environment. (A) Representative western blots showing protein expression of iNOS and Arg-1 in each group, β-actin was utilized as a loading control. (B) Quantitative analysis of relative expression of iNOS and Arg-1. (C) Representative immunofluorescence images of CD68 positive and iNOS positive BV2 cells (scale bar: 20 μm). (D) Representative immunofluorescence images of CD68 positive and Arg-1 positive BV2 cells (scale bar: 20 μm). (E, F) Quantitative analysis of relative fluorescence intensity of iNOS and Arg-1. (G) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein associated with pyroptosis, β-actin was utilized as a loading control. (H) PI staining of PC12 cells in each group (scale bar, 100 μm). (I) Quantitative analysis of PI staining of PC12 cells (n = 3). (J) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Primary antibodies against NLRP3 (cat. no. 15101) were obtained from Cell Signaling Technology (Beverly, Massachusetts, USA).

Techniques: In Vitro, Western Blot, Expressing, Control, Immunofluorescence, Fluorescence, Staining, Comparison

Journal: Bioactive Materials

Article Title: 3D-MSCs apoptotic bodies-integrated conductive hydrogel mitigates spinal cord injury via immunoregulation and alleviating neuronal pyroptosis

doi: 10.1016/j.bioactmat.2026.01.043

Figure Lengend Snippet: The composite hydrogel inhibits post-SCI pyroptosis in neurons. (A) Immunofluorescence images of residual neurons existing in the anterior horn of the spinal cord (scale bar, 500 μm and 200 μm). (B) Immunofluorescence image of Caspase-1 expression of neurons 3 days after SCI (scale bar, 20 μm). (C) Quantitative analysis of relative fluorescence intensity of Caspase-1 protein expression in neurons within the specified groups (n = 3). (D) Immunofluorescence image of GSDMD-N expression of neurons 3 days after SCI (scale bar, 20 μm). (E) Quantitative analysis of relative fluorescence intensity of GSDMD-N protein expression in neurons within the specified groups (n = 3). (F) Representative western blots showing the expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N, and IL-18 protein 3 days after SCI, GAPDH was utilized as a loading control. (G) Quantitative analysis of relative expression of NLRP3, Caspase-1, IL-1β, ASC, GSDMD-N and IL-18 (n = 3). The data are presented as the means ± SEMs (n = 3); ∗p < 0.05, indicates significant differences; ns, is not significant. Statistical analysis was performed using two-way ANOVA followed by Tukey's multiple comparison test.

Article Snippet: Primary antibodies against NLRP3 (cat. no. 15101) were obtained from Cell Signaling Technology (Beverly, Massachusetts, USA).

Techniques: Immunofluorescence, Expressing, Fluorescence, Western Blot, Control, Comparison

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is recruited to lipid droplets (LDs) in a GTPase-dependent manner in Hep3B cells . Super-resolution confocal micrographs of Hep3B human hepatoma cells expressing ( A ) WT Rab5 (GFP-Rab5 WT), ( B ) constitutively active mutant (GFP-Rab5 Q79L), and ( C ) dominant-negative mutant (GFP-Rab5 S34N). LDs were stained with Oil Red O (ORO, red ) to visualize neutral lipid stores. GFP-Rab5 fluorescence (green ) marks Rab5 localization. Inlays depict Rab5 localization around LD borders, indicated further by yellow arrows . D , quantification of Rab5–LD colocalization by the ratio of LD:cytoplasm GFP-Rab5 intensity. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using one-way ANOVA with Tukey’s post hoc test. ∗∗ p < 0.01 and ∗∗∗ p < 0.001.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Expressing, Mutagenesis, Dominant Negative Mutation, Staining, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Enhanced recruitment of Rab5 to lipid droplets (LDs) upon lipophagy stimulation . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in regular medium (CT) or 4 h HBSS starvation in Hep3B cells. B , quantification of the fold change of GTP-bound/total Rab5 as well as EEA1 from n = 4 independent experiments. C , quantification of total Rab5–β-actin confirming that starvation does not change overall Rab5 expression. D , Western blot analysis of LDs isolated from Hep3B cells by density gradient centrifugation showing abundant levels of Rab5 in isolated LDs following 4 h of HBSS starvation compared with the regular medium (CT). E , quantification of Rab5–Plin2 in the biochemically isolated LDs. F , fluorescence micrograph showing immunofluorescence staining of Hep3B cells expressing Myc-tagged Rab5 ( green ). Cells were treated with oleic acid for 16 h and then either maintained in regular medium (control) or subjected to 4 h of HBSS starvation before being fixed and stained with Oil Red O (ORO; red ) to label LDs. Note the marked increase in Rab5 staining around LDs in HBSS-starved cells versus control ( yellow arrowheads ). Graphs represent mean ± SD from n = 3 to 4 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; EEA1, early endosome antigen 1; HBSS, Hank’s balanced salt solution; Plin2, perilipin-2.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Western Blot, Expressing, Isolation, Gradient Centrifugation, Fluorescence, Immunofluorescence, Staining, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Inhibition of Rab5 GTPase activity reduces lipid droplet (LD) catabolism via its GTPase activity . A , Western blot analysis of Rab5 and EEA1 GTP-pulldown in Hep3B cells treated with DMSO (CT) or NAP (100 μM, 48 h). B , quantification of GTP/total Rab5 as well as EEA1 from n = 3 independent experiments. C , confocal micrograph showing ORO-stained LDs ( red ) and DAPI-stained nuclei from Hep3B cells treated with DMSO (control) and NAP (100 μM). D , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. E , confocal micrograph showing ORO-stained LDs (red ) and DAPI-stained nucleus from Hep3B cells treated with DMSO (control) and NAP (100 μM) with oleic acid (OA, 150 μM). F , bar graphs depict quantification of LD number/cell, total LD area/cell, and LD size. G , cartoon depicting NAP alteration of Rab5 GTP binding. Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001. CT, control; DAPI, 4′,6-diamidino-2-phenylindole; DMSO, dimethyl sulfoxide; EEA1, early endosome antigen 1; NAP, neoandrographolide; ORO, Oil Red O.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Inhibition, Activity Assay, Western Blot, Staining, Control, Binding Assay, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Inhibition of Rab5 decreases HCC energy homeostasis . A , Seahorse metabolic analysis showing NAP (100 μM, 48 h) inhibition of Rab5 decreases oxygen consumption rate (OCR) compared with control in complete media. n = 3 independent experiments. B , quantification of basal and spare respiratory capacity in complete media following treatment with NAP. C , Seahorse metabolic analysis showing DMSO versus NAP treatment in Hep3B cells under 4 h HBSS starvation. D , quantification of basal and spare respiratory capacity from C . Graphs represent mean ± SD from n = 3 independent experiments. Statistical significance was determined using an unpaired two-tailed t test. ∗ p < 0.05, ∗∗ p < 0.01. DMSO, dimethyl sulfoxide; HBSS, Hank’s balanced salt solution; HCC, hepatocellular carcinoma; NAP, neoandrographolide.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Inhibition, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Rab5 is upregulated in HCC and associated with cancer hallmarks . A , volcano plot from TNMplot showing expression of known lipid droplet (LD)–associated proteins, including Rab5A/B/C, in HCC tumors versus adjacent normal tissues from n = 53 patients. B , representative immunohistochemistry images from a liver tissue microarray showing Rab5 staining in normal liver (n = 4) versus HCC specimens (n = 7). C , quantification of Rab5 immunohistochemistry scores from the tissue microarray (mean ± SD, ∗∗∗ p < 0.0002; Welch's t test). D , cancer hallmark gene set enrichment analysis based on the study by Menyhart et al . , showing Rab5-associated genes enriched in proliferation, invasion, and evading growth suppressors. E , Kaplan–Meier survival analysis from the TCGA–LIHC dataset stratified by Rab5 expression. High Rab5 expression correlates with poorer overall survival (log-rank test, p = 0.06). HCC, hepatocellular carcinoma; LIHC, Liver Hepatocellular Carcinoma Cohort; TCGA, The Cancer Genome Atlas.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Expressing, Immunohistochemistry, Microarray, Staining

Journal: The Journal of Biological Chemistry

Article Title: Rab5 nucleotide binding promotes oxidative metabolism to fuel hepatocellular carcinoma cell proliferation

doi: 10.1016/j.jbc.2026.111321

Figure Lengend Snippet: Working model depicting Rab5-mediated lipid droplet (LD) catabolism in HCC . Under nutrient deprivation, Rab5 GTP binding is increased and promotes a higher frequency of interaction with LDs, driving tethering and fusion with lysosomes via microlipophagy. Inside the lysosome, lysosomal acid lipase (LAL) degrades LD, releasing free fatty acids (FFAs), which are then shuttled to mitochondria for β-oxidation to fuel HCC cell proliferation and survival. HCC, hepatocellular carcinoma.

Article Snippet: Formalin-fixed, paraffin-embedded sections were stained with an anti-Rab5 primary antibody (Cell Signaling Technology, 3547) followed by standard immunohistochemistry detection procedures.

Techniques: Binding Assay

Journal: Journal of the Endocrine Society

Article Title: Insulin receptor trafficking and interactions in muscle cells

doi: 10.1210/jendso/bvag020

Figure Lengend Snippet: Colocalization and interaction between INSR and ANXA2 under different insulin concentrations. (A, B) Representative STED microscopy images of C2C12 myoblasts expressing INSR-A-SNAP (surface labeled) that were fixed after stimulation with 0, 0.2, or 20 nM insulin for (A) 15 or (B) 30 minutes and stained for ANXA2 (scale bar = 5 µm). (C, D) Colocalization between INSR-A-SNAP and ANXA2 at (C) 15 or (D) 30 minutes of insulin-stimulated were quantified by Object Pearson Coefficient ( n = 4-13 images, 1-3 cells per image. # P < .05, 1-ANOVA of all groups. Box represents median and 25th to 75th percentiles.).

Article Snippet: Similarly, for AXNA2 experiment, cells labeled with SNAP-Surface Alexa Fluor 488 substrate were incubated with insulin for 15 or 30 minutes, and labeled with Annexin A2 primary antibody (1:150, Rabbit mAb, Cat. # 8235S, Cell Signaling, RRID: AB_11129437).

Techniques: Microscopy, Expressing, Labeling, Staining

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: A panel of MR1 mutants differentially translocates to the cell surface in response to stabilizing ligand . A , MR1-GFP expression was induced in clonal cell lines derived from the polyclonal parent cell line described in with 2 μg/ml of doxycycline (dox, dark colors) or not ( light gray ) and either treated with 100 μM of 6-FP (filled histograms) or an equal volume of solvent control 0.01 M NaOH (empty histograms) overnight. Data are representative of three independent experiments. The same unstained BEAS-2B WT control is included in each graph for reference. Calibration beads were included in each experiment. B , MR1-GFP expression was measured over time after removal of 2 μg/ml dox. Data are pooled from four independent experiments resulting in three or four data points for each time point with each cell line normalized to its starting level in each experiment and shown as mean with standard deviation (SD). Best fit parameters for straight lines were determined by least-squares regression. The fit of one curve fit to all the data sets was compared with the fit of individual curves fit to each data set using the extra sum-of-squares F test. For full test results see . C , whole cell lysates from cell lines induced with 2 μg/ml dox overnight were analyzed for expression of MR1-GFP and loading control Vinculin (VINC) by WB. Primary antibodies were from different species and detected in parallel with species-specific secondary antibodies conjugated to distinct IRDyes, and both channels exported as grayscale. Molecular weight markers are indicated in kDa on the left . Data are representative of three independent experiments. See for remaining blots. D , the region immediately around the sgRNA target site was analyzed by next-generation sequencing and mutations resulting in intact reading frames are summarized. See for complete sequence analysis. aa, amino acid; GeoMFI, geometric mean fluorescence intensity; M, marker; MR1, MHC class I-related protein 1; RF, reading frame; sgRNA, single-guide RNA; WT, wild type.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Expressing, Derivative Assay, Solvent, Control, Standard Deviation, Molecular Weight, Next-Generation Sequencing, Sequencing, Fluorescence, Marker

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1 mutants have a defect in presenting exogenous ligands but present ligands derived from intracellular infection and endosomal processing . MR1-GFP expression was induced with adjusted dox concentrations (see and text) overnight, and cells were used as APCs in IFNγ ELISpots. A , B , D , cells were preincubated with indicated dilutions of M . smegmatis ( M . smeg ) supernatant ( A ), deazalumazine ( B ), or 5-A-RU prodrug ( D ) for at least 1 hour before addition of MAIT cell clone D481-C7. C , APCs in C were infected with auxotroph Mtb (Aux Mtb) overnight and diluted as indicated. MR1-GFP expression at the time of the ELISpot was measured by flow cytometry and is shown to the right of each plot. Each dot represents a single technical replicate from one independent experiment. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration, and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test and statistically significant differences are indicated. 5-A-RU, 5-amino-6-D-ribitylaminouracil; aa, amino acid; APC, antigen presenting cell; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Derivative Assay, Infection, Expressing, Enzyme-linked Immunospot, Flow Cytometry, Concentration Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Mutated MR1 does not accumulate in vesicular compartments at baseline . A , expression of MR1-GFP was induced in clonal cell lines with twice the adjusted dox concentrations (1 μg/ml, 2 μg/ml, and 4 μg/ml, respectively—see and text), and cells were incubated with 100 μM of 6-FP or an equal volume of the solvent control 0.01 M NaOH overnight. Images are representative of three independent experiments. Scale bar indicates 10 μm. B–D , clonal cell lines D4 and D6 were induced to express MR1-GFP with 1 μg/ml and 2 μg/ml of dox, respectively, and treated with CellLight BacMam 2.0 for late endosomes. B , representative images. Scale bar indicates 20 μm. C , enumeration of MR1-GFP + vesicles per cell. D , colocalization of MR1-GFP + vesicles and Rab7a + vesicles. Data in C and D represent 24 cells from 24 images for each cell line, pooled from three independent experiments. Experimental groups in C were compared by two-tailed, unpaired t test. 6-FP, 6-formylpterin; MR1, MHC class I-related protein 1.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Expressing, Incubation, Solvent, Control, Two Tailed Test

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Mutated MR1 differentially interacts with calnexin, B2M, HLA-Ia, TPP1, OLFML2A, and SQSTM1/p62 . Expression of MR1-GFP was induced in clonal cell lines D4 and D6 with 10 μg/ml of dox overnight before incubation with 20 μg/ml dox and 100 μM 6-FP overnight. A , B , C , MR1-GFP was immunoprecipitated, and bound proteins were analyzed by mass spectrometry in DDA mode ( A ) or DIA mode ( B + C ). A , average size-adjusted intensity of proteins identified in both D4 and D6 normalized to MR1-GFP intensity in each sample. The MR1-GFP construct, B2M, and two isoforms of CALX (Uniprot IDs P27824 and P27824-2) are highlighted. Data in A are pooled from three independent experiments. Data in B and C are pooled from three independent experiments with three or four technical replicates each. Each dot in C represents one mass spectrometry injection for proteins with p adjusted ≤ 0.01 and |log2 fold-change| ≥ 1. For statistical analysis, see mass spectrometry Experimental Procedures section. For full lists of identified proteins and peptides see and . D , the lysates of the three experiments shown pooled in B and C were analyzed by WB. CANX and B2M were analyzed on one gel with the membrane cut horizontally before primary antibody incubation. Both the red and the green channel were exported in grayscale to allow visualization of the molecular weight marker. MR1-GFP and SQSTM1/p62 were analyzed on another gel with primary antibodies from different species. Species-specific secondary antibodies conjugated to distinct IRDyes were used, and each channel was exported individually in greyscale. aa, amino acid; B2M, β2-microglobulin; DDA, data-dependent acquisition; DIA, data independent acquisition; expt, experiment; HLA-Ia, human leukocyte antigen class Ia; LFQ, label-free quantification; M, marker; MR1, MHC class I-related protein 1; MW, molecular weight; OLFML2A, olfactomedin-like protein 2A; SQSTM1, sequestosome 1; TPP1, tripeptidyl-peptidase 1.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Expressing, Incubation, Immunoprecipitation, Mass Spectrometry, Construct, Injection, Membrane, Molecular Weight, Marker, Data-dependent acquisition, Data-independent acquisition, Quantitative Proteomics

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: SQSTM1/p62 is not required for MR1-mediated antigen presentation . A–D , clonal cell lines D4 and D6 ( A + B ) or BEAS-2B WT cells ( C + D ) were transfected with missense (mis, filled symbols) siRNA or siRNA targeting SQSTM1/p62 (KD, empty symbols) after inducing MR1-GFP expression with adjusted dox concentrations (see , A + B only) and used as APCs in IFNγ ELISpots with the indicated antigens. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right in A . Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups in A and B were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. Comparisons in C and D were performed with two-tailed, paired t tests. SQSTM1/p62 KD was confirmed by WB except for one of the independent experiments in B as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux, auxotroph; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units; SQSTM1, sequestosome 1.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Immunopeptidomics, Transfection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Two Tailed Test, Fluorescence, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1 in mutant cell line D6 binds B2M with lower apparent affinity. A , clonal cell lines D4 and D6 were transiently transfected to overexpress B2M (full symbols) or mock transfected (empty symbols) before use as APCs in an ELISpot. Data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. B2M overexpression was confirmed by WB as shown in . B , MR1-GFP was immunoprecipitated from clonal cell lines D4 and D6 induced to express MR1-GFP with 0.5 or 8 μg/ml dox, respectively, overnight followed by incubation with the same concentrations of dox plus 100 μM 6-FP overnight. Immunoprecipitated samples were incubated at 37 °C for the indicated time periods and MR1 bound to the beads, B2M bound to the beads, and B2M in the supernatant (unbound) were measured by WB. A representative blot is shown on the left and pooled data from three experiments, normalized to MR1 for each sample, and t = 0 for each cell line is shown on the right as mean with SD. Primary antibodies were detected in parallel with species-specific secondary antibodies conjugated to IRDye800, and both channels exported as grayscale to visualize the molecular weight markers. The 800 channel was exported individually for MR1-GFP. See for the remaining blots. C , clonal cell lines D4 and D6 were induced to express MR1-GFP with 4 and 8 μg/ml dox, respectively, overnight followed by incubation with the same concentrations of dox with 100 μM 6-FP overnight to bring MR1 to the cell surface before incubation with brefeldin A (BFA) for the indicated amounts of time. MR1 surface levels ( left ) and total MR1 expression ( right ) were measured by flow cytometry. Best fit parameters for one-phase exponential decay curves were determined by least-squares regression in B and C . The fit of one curve fit to both data sets was compared with the fit of individual curves fit to each data set using the extra sum-of-squares F test. For full test results, see . 5-A-RU, 5-amino-6-D-ribitylaminouracil; 6-FP, 6-formylpterin; A.U., arbitrary units; aa, amino acid; APC, antigen presenting cell; B2M, β2-microglobulin; IFNγ, interferon-γ; IP, immunoprecipitation; M, marker; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Mutagenesis, Transfection, Enzyme-linked Immunospot, Concentration Assay, Over Expression, Immunoprecipitation, Incubation, Molecular Weight, Expressing, Flow Cytometry, Marker

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: Knock down of B2M differentially affects exogenous and endosomal antigen presentation pathways . Clonal cell lines D4 and D6 were transfected with missense (mis, filled symbols ) siRNA or siRNA targeting B2M (KD, empty symbols ) before induction with adjusted dox concentrations (see ) and used as APCs in IFNγ ELISpots with the indicated antigens ( A ) or infected with auxotroph Mtb ( B ) and then used as APCs in IFNγ ELISpots. Dox concentrations during Aux Mtb infection were 0.4 and 0.8 μg/ml, respectively. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and shown to the right . Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test, and statistically significant differences are indicated. B2M KD was confirmed by WB as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux Mtb, Mycobacterium tuberculosis auxotroph; Aux, auxotroph; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Knockdown, Immunopeptidomics, Transfection, Infection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: The effect of B2M knock down depends on MR1 expression levels . A , B , BEAS-2 B WT cells ( A ) or BEAS-2B MR1 KO (KO) and BEAS-2B MR1 overexpressing (OE) cells ( B ) were transfected with missense (mis, filled symbols) siRNA or siRNA targeting B2M (KD, empty symbols) and used as APCs in IFNγ ELISpots with the indicated antigens. C , B2M was knocked down in the clonal cell line D4 as in A and B , and MR1-GFP expression was induced with the indicated concentrations of dox before use as APCs in IFNγ ELISpots with the indicated antigens. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right. Each dot represents a single technical replicate from one of three independent experiments. ELISpot data in all panels are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations ( A + B ), or the highest and lowest dox concentrations ( C ) are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test for more than two groups or a two-tailed, paired t test for two groups and statistically significant differences are indicated. Comparisons of all experimental groups for the flow cytometry analysis in C are shown in . B2M KD was confirmed by WB as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KD, knock down; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; SFU, spot forming units.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Knockdown, Expressing, Transfection, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Two Tailed Test, Fluorescence

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: B2M is required for endosomal antigen presentation in the presence of high levels of MR1 . Clonal cell lines D4 and D6 cells were electroporated with RNPs containing scrambled nontarget control sgRNAs (NT) or three pooled sgRNAs targeting B2M (KO). Single clones were induced to express MR1-GFP with adjusted dox concentrations (see ) and used as APCs in IFNγ ELISpots with the indicated antigens ( A ) or infected with auxotroph Mtb ( B ) and then used as APCs in IFNγ ELISpots. MR1-GFP expression was measured by flow cytometry at the time of the ELISpot and is shown to the right. Each dot represents a single technical replicate from one of three independent experiments. ELISpot data are pooled from three independent experiments, normalized to D4 at the highest antigen concentration and shown as mean with SD. IFNγ responses at the highest antigen concentrations are additionally shown as dot plots where each dot represents the mean of technical duplicates from one independent experiment. Experimental groups were compared by repeated-measures ANOVA with Tukey’s multiple comparisons test and statistically significant differences are indicated. B2M KO was confirmed by WB, PCR, and qRT-PCR as shown in . 5-A-RU, 5-amino-6-D-ribitylaminouracil; APC, antigen presenting cell; Aux, auxotroph; B2M, β2-microglobulin; GeoMFI, geometric mean fluorescence intensity; IFNγ, interferon-γ; KO, knock out; M . smeg , M . smegmatis ; MR1, MHC class I-related protein 1; RNP, ribonucleoprotein; SFU, spot forming units; sgRNA, single-guide RNA.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Immunopeptidomics, Control, Clone Assay, Infection, Expressing, Flow Cytometry, Enzyme-linked Immunospot, Concentration Assay, Quantitative RT-PCR, Fluorescence, Knock-Out

Journal: The Journal of Biological Chemistry

Article Title: Mutations outside the MR1 antigen binding groove differentially inhibit presentation of exogenous antigens

doi: 10.1016/j.jbc.2026.111317

Figure Lengend Snippet: MR1-mediated presentation of exogenous antigen is limited by binding to B2M, whereas presentation of antigen derived from intracellular infection is limited by the availability of MR1 itself . Cellular mechanisms for MR1-dependent presentation of free antigen that originates in the extracellular environment ( left ) differ from those for pathogen-derived antigen generated within the endosomal compartment ( right ). A reduced ability to bind B2M due to either AB loop mutation (baseline in D6) or KD of B2M reduces ER-based presentation of exogenous ligand whereas endosomal presentation in the context of intracellular infection remains functional as long as MR1 protein levels are not limiting. Complete deficiency for B2M abrogates all MR1-mediated antigen presentation. B2M, β2-microglobulin; ER, endoplasmic reticulum; KD, knock down; KO, knock out; MR1, MHC class I-related protein 1; Mtb , Mycobacterium tuberculosis ; OE, overexpressing; WT, wild type.

Article Snippet: For staining with the polyclonal rabbit anti-MR1 antibody, BEAS-2B MR1 KO tet MR1-GFP clonal cell lines were incubated with 1 (D4), 2 (D6 + D8), or 4 (D16) μg/ml of dox and 100 μM of 6-FP or solvent control 0.01 M NaOH overnight before staining with 25 μl of 50 μg/ml polyclonal rabbit anti-MR1 primary antibody (Proteintech, #13260-1-AP; used 1:10) for at least 30 min at 4 °C.

Techniques: Binding Assay, Derivative Assay, Infection, Generated, Mutagenesis, Functional Assay, Immunopeptidomics, Knockdown, Knock-Out

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Morphological and functional changes of ovaries between Ctrl and LP groups. (a) The illumination pattern for SD rats. Time (hours) refers to clock time. (b) Serum melatonin levels of Ctrl (n = 4) and LP (n = 4) group of every ZT. (c) The expression phase of Bmal1 in ovaries. (d) Typical oestrous cycles from Ctrl (n = 12) and LP (n = 12) group. Proestrus (P), oestrus (E), metoestrus (M), dioestrus (D). (e) Composition of oestrous cycle (n = 12). (f) Ovaries from Ctrl (n = 3) and LP (n = 3) group. (Scale bar, 1 cm). (g) Ovary weight of Ctrl (n = 9) and LP (n = 7) group. (h) HE staining images of ovary section for rats, triangle means expanded cystic follicles, pentagram means corpus luteum. Scale bar, 1 mm. (i) Comparison of follicle numbers of Ctrl (n = 4) and LP (n = 6) group. (j) BrdU immunohistochemical staining (Scale bar, 500 μm). (k) The number of retrieved oocytes (n = 5) after ovulation induction. (l) Litter size of Ctrl (n = 24) and LP (n = 11) groups. Statistical differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated overnight at 4 °C with rotation using primary antibodies against BMAL1 (Cell Signalling Technology, 14020; 2 μg) or appropriate normal IgG (ABclonal, AS070; 2 μg), followed by incubation with secondary antibodies (Millipore, AP132; 1:100 dilution) for 1 h at room temperature.

Techniques: Functional Assay, Expressing, Staining, Comparison, Immunohistochemical staining

Journal: eBioMedicine

Article Title: Circadian rhythm disruption impairs ovarian follicular development via NAD + metabolic reprogramming

doi: 10.1016/j.ebiom.2026.106200

Figure Lengend Snippet: Disruption of NAMPT oscillated expression through NAMPT after long photoperiod exposure. (a) Ovaries were obtained at different ZT from Ctrl and LP group for WB, β-Tubulin was used as control. (b) The relative expression of BMAL1 at different ZT based on ZT0 to show the dynamic changes of Ctrl and LP group (n = 3). (c) The relative expression of NAMPT at different ZT based on ZT0. (d) WB of ovaries from Ctrl and LP group at each ZT (n = 3). (e) BMAL1 expression of LP group relative to Ctrl at each ZT (n = 3). (f) NAMPT expression of LP group relative to Ctrl at each ZT (n = 3). (g) Linear correlation of NAMPT and BMAL1 expression in Ctrl and LP group, which were showed for calculated expression based on its fold change to raw expression of ZT0. (h) Genomic views of BMAL1 CUT&Tag-seq assay enrichment at the promoters of the Nampt . (i) The non-canonical E-box motifs in the promoter and first intron of the Nampt gene. Chromatin immunoprecipitation (ChIP) qPCR was used to show that BMAL1 physically and specifically associate to the E-boxes on the Nampt promoter, since the comparative analysis with 1% input as a reference revealed a significant decrease in fold change at the LP group site. (j) BMAL1-NAMPT-SIRT3-Deacetylation SOD2 axis. Statistically significant differences between the Ctrl and LP groups were determined using Tukey's test; ∗ p < 0.05, ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: Samples were incubated overnight at 4 °C with rotation using primary antibodies against BMAL1 (Cell Signalling Technology, 14020; 2 μg) or appropriate normal IgG (ABclonal, AS070; 2 μg), followed by incubation with secondary antibodies (Millipore, AP132; 1:100 dilution) for 1 h at room temperature.

Techniques: Disruption, Expressing, Control, Chromatin Immunoprecipitation, ChIP-qPCR

Journal: Journal of Advanced Research

Article Title: The GOLM1-ACLY pathway regulates macrophage-secreted EFEMP1 via H3K27ac modifications to drive tumor progression

doi: 10.1016/j.jare.2025.07.003

Figure Lengend Snippet: Nucleic P-ACLY within Golm1 -knockout macrophage regulates Efemp1 expression through H3K27 acetylation (A) Total histone H3 acetylation levels in Thp1 nc and Thp1 −/− cells determined by Histone H3 acetylation detection Kit. n = 9 replicated wells. (B-C) WB detecting (B) and quantitative analysis (C) of the protein levels of various acetylated histone H3. n = 3 independent experiments. (D) PCA plot of CUT&Tag-seq data of experiments with Thp1 nc and Thp1 −/− cells. n = 3 samples/group. (E) Pie chart showing the distribution of H3K27ac peaks across annotated genomic regions in Thp1 nc and Thp1 −/− cells. (F) Heatmap showing the genomic occupancy of H3K27ac from −3 kb flanking TSS to + 3 kb flanking TES in Thp1 nc or Thp1 −/− cells. The x-axis represents the position of reads relative to the TSS, and the y-axis represents the read density. (G) CUT&Tag-seq volcano plot of different gene peaks between Thp1 nc and Thp1 −/− cells. (H) RNA-seq volcano plot of DEGs between BMDMs wt and BMDMs mko cells. n = 3 mice/group. (I) Overlap of differentially expressed genes identified by RNA-seq and CUT&Tag-seq. Secreted proteins with statistically significant differences are listed below. (J) IGV snapshot of Thp1 nc and Thp1 −/− cells showing the H3K27ac CUT&Tag signals at the Efemp1 genomic loci. (K) H3K27ac occupancy in the genome of Efemp1 analysed by ChIP-qPCR. n = 4 duplicates/group. (L) Relative expression of Efemp1 genes in Thp1 nc and Thp1 −/− cells determined by qPCR. n = 6 duplicates/group. The data are presented as the means ± SEMs. The results presented in (A, C, K, and L) were analyzed via two-tailed Student's t test. ns, not statistically significant. **p < 0.01; ***p < 0.001.

Article Snippet: The Bead-bound cells were resuspended and sequentially incubated with anti-H3K27ac primary antibody (8173S, Cell Signaling Technology, USA) and secondary antibody (TransGen Biotech, China).

Techniques: Knock-Out, Expressing, RNA Sequencing, ChIP-qPCR, Two Tailed Test