Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: 2D side-by-side comparison of the 2-DE Western blot images of HSP27, PRDX2, GRP78 and GRP75 in cholesteatoma tissues and retroauricular skin run with pI 4–7 and pI 3–10, respectively. Images A , B , C and D are for HSP27. Images E , F , G and H are for PRDX2. Images I , J , K and L are for GRP78. Images M , N , O and P are for GRP75.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Comparison, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Upregulation of Phosphorylated HSP27, PRDX2, GRP75, GRP78 and GRP94 in Acquired Middle Ear Cholesteatoma Growth

doi: 10.3390/ijms140714439

Figure Lengend Snippet: Validation of HSP27, GRP75, GRP78, GRP94 and PRDX2 by Western blotting analysis and RT-PCR. The tissues of cholesteatoma and retroauricular skin were collected from six individual patients. Cholesteatoma is presented by C, and retroauricular skin is presented by S. β-actin was used for normalization.

Article Snippet: Rabbit anti-human HSP27, GRP75, GRP78, GRP94 and PRDX2 antibodies were purchased from ProteinTech Group (Chicago, IL, USA).

Techniques: Biomarker Discovery, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Heatmap showing DEGs specific to ER stress. ( B ) qPCR validation of DEGs specific to ER stress revealed by RNA-seq. ( C ) Immunohistochemical GRP94 expression in peri-infarct myocardium. ( D ) Mice were subjected to MI surgery as described in Methods. GRP94 levels were detected by immunoblot (mean ± SD, n = 4). ( E ) GRP94 attenuates infarct sizes 1 day after MI. TTC staining on the left shows infarct size modulation by AAV2/9-GRP94 overexpression (Ad-GRP94) or siRNA knockdown (Si-GRP94) at 24 hours after MI. Right panel shows the percentage of infarct sizes. n = 4. ( F and G ) GRP94 markedly improves cardiac function after MI. Transthoracic echocardiographic analysis was performed at 1 week after surgery. n = 6. LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. * P < 0.05; ** P < 0.01 by unpaired, 2-tailed Student’s t test ( D ) or 1-way ANOVA with Tukey-Kramer post hoc analysis ( E – G ).

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Biomarker Discovery, RNA Sequencing, Immunohistochemical staining, Expressing, Western Blot, Staining, Over Expression, Knockdown

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Cardiomyocytes were exposed to the indicated dose of H 2 O 2 . After 24 hours, cells were harvested for the analysis of GRP94 levels by immunoblot. n = 4. ( B ) Cardiomyocytes were cultivated under hypoxia for the indicated time. After 24 hours, cells were harvested for the analysis of GRP94 levels by immunoblot ( n = 4). Data analyzed versus the untreated group. ( C ) GRP94 attenuates cells’ sensitivity to hypoxia-induced apoptosis. Cardiomyocytes were infected with adenoviruses overexpressing GRP94 (Ad-GRP94) or Glb1 (Ad-Glb1) at MOIs of 50, or transfected with 60 nM GRP94 siRNA (Si-GRP94) or siRNA scramble (Si-NC). After 36 hours, cells were cultivated for another 12 hours under hypoxic conditions. The TUNEL assay was utilized to detect apoptosis. Quantitative analysis of apoptosis is shown in right panel. CON, control. Scale bar: 50 μm. ( D ) GRP94 attenuates mitochondrial fission caused by hypoxia stress. Cardiomyocytes were treated as in C . Representative photos show mitochondrial fission (left). The cells were stained with MitoTracker, and then the ratios of cells with lamentous network, intermediate, or completely fragmented mitochondrial structures were calculated. At least 50 cells were counted from different optical fields of each sample ( n = 4). CON, control. Scale bars: 20 μm. ( E and F ) GRP94 attenuates the cleavage of Opa1 into (S)-Opa1 caused by hypoxia stress. Cardiomyocytes were treated as in C . The levels of MFN1, MFN2, Opa1, DRP1, p-DRP1, and FIS1 were analyzed by immunoblot. n = 4. ( G ) GRP94 attenuated AMI-induced mitochondrial over-fragmentation. Adult male C57BL/6 mice (8–10 weeks old) were injected with AAV2/9 carrying GRP94 , Glb1 , GRP94 siRNA, or siRNA scramble and subjected to permanent left anterior descending coronary artery ligation. The left panels are representative photos of TEM images of cardiac slices. Scale bars: 1 μm (left images) and 100 nm (enlarged images). * P < 0.05; ** P < 0.01 by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A – C , E , and F ) or 2-way ANOVA followed by Bonferroni’s multiple-comparison test ( D ).

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Western Blot, Infection, Transfection, TUNEL Assay, Control, Staining, Injection, Ligation, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Hypoxia-induced GRP94 mRNA upregulation is suppressed by Yy1 knockdown. Mouse cardiomyocytes transfected with 40 nM siRNAs targeting Cebpb , c-Jun , Cebpa , Yy1 , or scramble control (Si-NC) were exposed to hypoxia for 12 hours. GRP94 mRNA levels were quantified by qRT-PCR ( n = 4). ( B ) Yy1 knockdown inhibits hypoxia-induced GRP94 protein expression. Cardiomyocytes transfected with Si-Yy1 or Si-NC were subjected to hypoxia. GRP94 protein was analyzed by immunoblot with total protein as loading control ( n = 4). ( C ) Yy1 binds to the GRP94 promoter under hypoxia. ChIP assays were performed using Yy1 or β-actin (negative control) antibodies in cardiomyocytes exposed to hypoxia for the indicated durations. ( D and E ) Yy1 regulates GRP94 protein in a hypoxia-dependent manner. ( D ) GRP94 levels were unaffected by Yy1 overexpression (Ad-Yy1, MOI = 40) under normoxia. ( E ) Ad-Yy1 enhanced hypoxia-induced GRP94 expression ( n = 4). ( F – H ) Yy1 activates GRP94 promoter via direct binding. ( F ) Luciferase reporter assays in MCM cells transfected with GRP94 promoter (pGL-GRP94) or empty vector (pGL-4.17), followed by Ad- Yy1 /Ad- Glb1 (MOI = 40) and hypoxia ( n = 4). ( G ) The –500 to +1 bp promoter region mediates Yy1 responsiveness. ( H ) Mutation of Yy1-binding site (pGL-GRP94-YS-mut) abolishes Yy1-driven activation. ( I ) ER stress response element (ERMS) mutation (pGL-GRP94-ERMS-mut) attenuates Yy1-dependent promoter activity ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group (or vs. Si-NC in A ) by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A , B , and D – G ) or 2-way ANOVA with Bonferroni’s multiple-comparison test ( H and I ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Knockdown, Transfection, Control, Quantitative RT-PCR, Expressing, Western Blot, Negative Control, Over Expression, Binding Assay, Luciferase, Plasmid Preparation, Mutagenesis, Activation Assay, Activity Assay, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Heatmap shows differentially expressed lncRNAs in the mouse model after MI. n = 3. ( B ) Hypoxia enhances Yy1-dependent enrichment of Lnc- AI662270 . Cardiomyocytes infected with Ad-Glb1 or Ad-Yy1 (MOI = 40) for 24 hours were exposed to hypoxia (12 hours). RNA immunoprecipitation (RIP) using anti-Yy1 or IgG antibody, followed by qRT-PCR ( n = 3). ( C ) Hypoxia redirects Yy1 binding from Lnc-Oip5os1 to Lnc- AI662270 . RNA pull-down with biotinylated probes (Bio- AI662270 , Bio-Oip5os1, or Bio-NC) in cardiomyocytes. Yy1 binding was analyzed by immunoblot (actin as loading control). ( D ) Subcellular localization of Lnc- AI662270 (cytoplasmic) and Lnc-Oip5os1 (nuclear) under hypoxia. Fluorescence in situ hybridization (FISH) in cardiomyocytes after 12-hour hypoxia. Scale bar: 20 μm. ( E ) Lnc- AI662270 regulates hypoxia-induced GRP94 mRNA. Cardiomyocytes infected with Ad- AI662270 or Ad-Glb1 (MOI = 40), or transfected with 40 nM ASO- AI662270 /ASO-NC for 36 hours, were exposed to hypoxia (12 hours). GRP94 mRNA levels analyzed by qRT-PCR ( n = 4). ( F and G ) AI662270 or Oip5os1 does not alter nuclear Yy1 abundance. ( F ) Nuclear Yy1 levels in cells infected with Ad-Oip5os1 or Ad-Glb1 (MOI = 40). ( G ) Nuclear Yy1 in cells transfected with ASO- AI662270 /ASO-NC. Cells were exposed to hypoxia (12 hours) prior to nuclear fractionation ( n = 4). ( H and I ) Lnc- AI662270 modulates Yy1 occupancy on the GRP94 promoter. ( H ) ASO- AI662270 reduces Yy1 binding to the GRP94 promoter. ( I ) Ad- AI662270 enhances Yy1-promoter interaction. ChIP-qPCR with anti-Yy1 or IgG after 36 hr treatment ( n = 4). ( J and K ) Hypoxia-dependent GRP94 regulation by Lnc- AI662270 . ( J ) ASO- AI662270 attenuates hypoxia-induced GRP94 protein. ( K ) Ad- AI662270 elevates GRP94 under hypoxia. Immunoblots quantified from 48 hours after transfection ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( B , E , J , and K ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Infection, RNA Immunoprecipitation, Quantitative RT-PCR, Binding Assay, Western Blot, Control, Fluorescence, In Situ Hybridization, Transfection, Fractionation, ChIP-qPCR

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A ) Hypoxia treatment induces an increase in AI662270 levels. Cardiomyocytes were exposed to hypoxia for the indicated durations. AI662270 mRNA analyzed by qRT-PCR ( n = 4). ( B ) Tunicamycin (TN, 15 mg/L) induces AI662270 expression. Time-course analysis after TN treatment ( n = 4). ( C ) ER stress inhibitor TUDCA (120 mg/L) blocks hypoxia-induced AI662270 upregulation. Cells were pretreated with TUDCA for 2 hours before 12-hour hypoxia ( n = 4). ( D ) AI662270 knockdown specifically attenuates TN-induced GRP94. Cardiomyocytes were transfected with 40 nM ASO- AI662270 /ASO-NC for 24 hours, and then treated with TN (15 mg/L, 12 hours). ER stress markers (IRE1α, GRP78, PDI, GRP94, CHOP) analyzed by immunoblotting ( n = 4). ( E ) AI662270 silencing exacerbates hypoxia-induced Opa1 cleavage. Cells transfected with ASO- AI662270 /ASO-NC were exposed to hypoxia (12 hours). Opa1 protein was analyzed by immunoblot. ( F ) AI662270 deficiency promotes mitochondrial fragmentation. Mitochondrial morphology was assessed by MitoTracker staining (≥100 cells/group). Categories: filamentous (>80% tubular), intermediate (30%–80%), fragmented (<30%). Scale bar: 20 μm ( n = 4). ( G ) AI662270 knockdown enhances hypoxia-induced apoptosis. TUNEL assay in cells treated as in E . Scale bar: 50 μm ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( A – E and G ) or 2-way ANOVA with Bonferroni’s multiple-comparison test ( F ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Quantitative RT-PCR, Expressing, Knockdown, Transfection, Western Blot, Staining, TUNEL Assay, Comparison

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A and B ) AI662270 regulates GRP94 and L-Opa1 in vivo. C57BL/6 mice (8–10 weeks old) injected with AAV2/9- AI662270 (1 × 10 11 ), Ad-Glb1, ASO- AI662270 (40 nM), or ASO-NC underwent permanent LAD ligation. ( A ) GRP94 and Opa1-L protein levels at 24 hours after MI. ( B ) Quantification of L-Opa1/S-Opa1 ratio ( n = 4). ( C and D ) AI662270 suppresses MI-induced mitochondrial fragmentation. ( C ) TEM images of cardiomyocyte mitochondria. ( D ) Quantification of fragmented mitochondria ( n = 3). Scale bar: 1 μm. ( E ansd F ) AI662270 reduces infarct size. ( E ) TTC-stained heart sections (red, viable; white, infarct). ( F ) Infarct area quantification at 24 hours after MI ( n = 4). ( G and H ) AI662270 preserves cardiac function. Echocardiography at 1 week after MI. ( G ) LVEF and ( H ) LVFS ( n = 4). LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening. * P < 0.05; ** P < 0.01 versus the indicated group by 1-way ANOVA with Tukey-Kramer post hoc analysis ( B , D , and F – H ). All data are shown as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: In Vivo, Injection, Ligation, Staining

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: ( A and B ) Hypoxia and ER stress remodel GRP94 interactomes. Cardiomyocytes exposed to ( A ) hypoxia (12 hours) or ( B ) tunicamycin (TN, 15 mg/L, 12 hours). GRP94 coimmunoprecipitated proteins identified by LC-MS/MS. ( C ) Anxa2 knockdown (si-Anxa2, 40 nM) blocks GRP94-mediated L-Opa1 stabilization. Cells infected with Ad- GRP94 or Ad- Glb1 (MOI = 50) and transfected with si-Anxa2/si-NC for 36 hours, followed by hypoxia (12 hours). Opa1 isoforms analyzed by immunoblotting ( n = 4). ( D ) Endogenous GRP94-Anxa2 interaction under hypoxia. Co-IP with anti-GRP94 or IgG in hypoxic cardiomyocytes (12 hours). ( E ) GRP94 overexpression enhances Anxa2 and p-Akt1 levels. Ad-GRP94–infected cells (MOI = 50, 48 hours). Anxa2, p-Akt1 (Ser473), and L-Opa1 analyzed ( n = 4). ( F ) Anxa2 deletion abrogates GRP94-induced p-Akt1 and L-Opa1. Ad-GRP94 (MOI = 50) + si-Anxa2/si-NC (40 nM, 48 hours). Protein levels quantified ( n = 4). ( G ) Akt1 inhibitor LY294002 (50 μM) reverses GRP94 effects. Ad-GRP94–infected cells treated with LY294002 for 48 hours ( n = 4). Anxa2, p-Akt1, and Opa1 protein was analyzed by immunoblot ( n = 4). ( H ) Anxa2 knockdown negates GRP94-mediated mitochondrial protection. Mitochondrial morphology was assessed by MitoTracker (≥50 cells/group). Categories: filamentous (>80% tubular), intermediate (30%–80%), fragmented (<30%). Scale bar: 20 μm ( n = 4). * P < 0.05; ** P < 0.01 versus the indicated group by 2-way ANOVA with Bonferroni’s multiple-comparison test. ( I ) Anxa2 deletion abolishes the GRP94 antiapoptotic effect. TUNEL assay in cells treated as in F . Scale bar: 50 μm ( n = 4). All data are expressed as mean ± SD.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Liquid Chromatography with Mass Spectroscopy, Knockdown, Infection, Transfection, Western Blot, Co-Immunoprecipitation Assay, Over Expression, Comparison, TUNEL Assay

Journal: JCI Insight

Article Title: AI662270 /GRP94 axis couples the unfolded protein response to mitochondrial dynamics during acute myocardial infarction

doi: 10.1172/jci.insight.188904

Figure Lengend Snippet: The red arrow represents the effect of the AI662270 /GRP94 signaling axis activation induced by early hypoxic stress.

Article Snippet: Total GRP94, Anxa2, and rabbit IgG control antibodies were conjugated overnight with Protein A/G Magnetic Beads (MedChemExpress), which were first blocked by incubating in 2% fatty acid–free BSA (Beyotime) on a rotator.

Techniques: Activation Assay

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: A Large-scale analysis of the key binding regions of antigens with GP96 using AlphaFold3 ( n = 22). B The possible structural pattern diagram of the combination of RBD and GP96 modeled by AlphaFold3. C The schematic representation of RBD-GP96-Fusion (monomer). RBDs (prototype strain) are truncated at the C-terminal residue G545 and connected to GP96 protein N-terminal residue D22 to construct the fusion protein (SP, signal peptide). The structure of the fusion protein was predicted based on AlphaFold3 (yellow RBM motif, hACE2 binding domain). D , E Molecular dynamics of GP96-dimer and RBD-GP96-dimer predicted by AlphaFold2-multimer were performed using AMBER. D The RMSD was analyzed. The red line represents RBD-GP96-dimer and the black line represents GP96-dimer. E The last frame of the molecular dynamics operation of the dimer conformations is captured for display. F Analytical gel filtration of the RBD-GP96-Fusion protein was performed with Superdex 200 Increase 10/300 GL. The resulting 280-nm absorbance curve is shown. The reduced SDS-PAGE migration profile of the pooled sample is shown. The data are representative of two independent experiments with similar results. G Ultracentrifugation sedimentation profiles of RBD-GP96-Fusion. H Transmission electron microscopy of RBD-GP96-Fusion and GP96 protein. The red frames represent typical transmission electron microscopy (TEM) structures. Below is a 3D cartoon sketch of the RBD-GP96-Fusion (surface, cone). I Analysis of GP96 and RBD-GP96-Fusion dimerization models.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Binding Assay, Residue, Construct, Filtration, SDS Page, Migration, Sedimentation, Transmission Assay, Electron Microscopy

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: A Representative BIAcore diagrams of RBD and RBD-GP96-Fusion bound to hACE2 protein (baculovirus expression). The K D value was calculated using the software BIAevaluation Version 4.1 (GE Healthcare). The values shown are mean ± SD of two independent experiments. B The complex structure of hACE2 (PDB: 1R42) is docked onto RBD-GP96-Fusion dimer by ZDOCK, showing the complete exposure of dual RBMs. Two RBD protomers are shown as surface and colored in hot pink and cyan, respectively. Two hACE2 proteins are shown as cartoon and colored in yellow. C The difference in thermal aggregation degree between RBD, GP96 and RBD-GP96-Fusion proteins was analyzed using the PR.NT.48 instrument. D The SDS-PAGE migration profiles of RBD, GP96 and RBD-GP96-Fusion hydrolyzed by trypsin at different pH were analyzed to determine the difference in anti-enzymatic ability. E Representative in vivo fluorescein image of 6- to 8-week-old female BALB/c mice on days 0, 7, and 30 after dorsal vaccination with RBD (with aluminum hydroxide adjuvant), GP96 and RBD-GP96-Fusion vaccines. In ( C , E ), n = 3 samples per group were analyzed. The data are representative of two independent experiments with similar results. Geometric mean ± geometric SD are shown. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, * p ≤ 0.05, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Expressing, Software, SDS Page, Migration, In Vivo, Adjuvant, Vaccines, Two Tailed Test

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: Mice ( n = 6 per group) were intramuscularly immunized with SARS-CoV-2 RBD vaccines (prototype or XBB.1.5 strain). A Schematic of RBD-GP96-Fusion induced humoral immunity. B–G Serum analyses at indicated time points. B RBD-specific IgG kinetics at day 7 post-immunization and 6 months after the 3 rd dose. C , D S1-specific and cross-reactive RBD IgG against variants at day 7 post-3 rd dose. E Serum ADCC activity. F , G Neutralization titers (NT50) against SARS-CoV-2 variants at day 7 post-3 rd dose. H Immunization and challenge timeline. I Lung viral RNA by reverse transcription quantitative PCR (RT-qPCR) at day 3 post-infection. J H&E staining of lung tissues (scale bars: 400/100 µm). K , L Bone marrow RBD-specific and XBB.1.5-RBD-specific IgG + ASCs by ELISPOT at 6 months. M , N scRNA-seq of Cy5 + cells in draining LNs: t-SNE visualization of B cell clusters ( M ) and absolute numbers/percentages of subsets ( N ). O – R Inguinal LN analysis at day 7 post-1 st dose. O Flow cytometry of GC B cells and RBD-specific GC B cells. P , Q Frequencies of GC B cells and RBD-specific GC B cells. R Frequency of RBD-specific CCR6 + MBC precursors in LZ. S IgG1 + and IgG2a/2b + MBCs frequencies at 6 months. Data represent two independent experiments. Geometric mean ± SD shown; individual points represent mice. L.O.D. indicated (dotted line). Statistical analysis by one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Vaccines, Activity Assay, Neutralization, Reverse Transcription, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Infection, Staining, Enzyme-linked Immunospot, Flow Cytometry, Two Tailed Test

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: Mice were immunized i.m. with SARS-CoV-2 RBD vaccines. Inguinal LNs were collected 7 days post-1 st immunization. A Galaxy plots of monocyte subsets. B Absolute numbers and percentages of each cell type ( n = 3). C Frequency of FDCs and RBD + /RBD-GP96-Fusion + FDC cells ( n = 6). D Flow cytometry analysis of LRP1 and TLRs expression on FDCs (isotype: PE Rat IgG2a, κ). E–G Frequency of RBD + /RBD-GP96-Fusion + FDCs after blocking with indicated mAbs. H , I RBD-specific Ab titers at day 14 post-immunization with FcR/LRP1 blocking. J–L IP and Western blot of LRP1-GP96-antigen-BCR complex in FDCs from immunized ( J , L ) or non-immunized ( K ) mice. ( J ) FDC cell sorting was subjected to IP with RBD mAb and Western blotting using RBD and LRP1 mAbs. K FDC cell sorting was incubated with the recombinant RBD protein, RBD protein with GP96 and RBD-GP96-Fusion protein. The incubated FDCs were subjected to IP with RBD mAb and Western blotting by LRP1 mAb. L Lymph node cells were directly subjected to IP by Protein A/G agarose and Western blotting with LRP1, RBD and GP96 mAbs, respectively. M–O GP96-stimulated FDCs enhance GC B cell interactions in LRP1-dependent manner. M FDCs from inguinal LNs were treated with GP96, RBD-GP96-Fusion, and with or without α-LRP1 or isotype control antibody. Hoechst-labeled GC B cells were then added at a 1:1 ratio. Time-lapse images were captured every 5 minutes for 1–2 h to analyze cell interactions and motility. (N) B cell tracking and interaction time quantification ( n = 20). O Percentage of B cells with long (> 10 min) vs short (0–10 min) interactions ( n = 20). P Frequency of Cy5 + FDC or Cy5 + GCB cells. Q ZDOCK model of RBD-GP96-Fusion bound to CD91-α receptor. R Schematic of antigen acquisition by FDCs. Data are from n = 6 mice/group unless specified, representative of two experiments. Geometric mean ± SD shown; individual points represent mice. Statistics: one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, ***p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Vaccines, Flow Cytometry, Expressing, Blocking Assay, Western Blot, FACS, Incubation, Recombinant, Control, Labeling, Cell Tracking Assay, Two Tailed Test

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: BALB/c or HLA-A2 transgenic mice were immunized with indicated vaccines. A t-SNE plot of T cell subpopulations. B Absolute numbers and percentages of each cell type ( n = 3). C Cell-cell contact analysis between CD8 + effector and DC cells by CellPhoneDB. D Schematic of cellular immune responses induced by RBD-GP96-Fusion. E , F Spleen and G–K BALF collected at day 14 post-3 rd immunization; L Spleen at 6 months post-3 rd immunization. E , F ICS assay of splenic CD8 + T cells producing IFN-γ upon epitope stimulation. G–I An ICS assay was performed to assess the ability of BALFs to secrete IFN-γ following stimulation with cocktail or dominant CD8 + epitope S 535-543 . G Representative flow plots of BALF CD8 + IFN-γ + cells. H Frequencies of epitope-specific CD8 + IFN-γ + cells in BALF. I CD11a and CXCR6 expression on BALF CD8 + IFN-γ + cells. J , K Frequencies of Trm ( J ) and α4β7 + ( K ) cells in BALF CD4 + /CD8 + T cells. L AIM + (CD137 + OX40 + ) CD4 + and AIM + (CD137 + CD69 + ) CD8 + T cells in splenocytes after RBD peptide stimulation. M MFI of RBD-TRITC on BMDCs after incubation with indicated proteins. N The kinetics of MFI as ( M ) over time. O Confocal microscopy of BMDCs showing nucleus (DAPI), LRP1 (FITC), and RBD (TRITC). P Percentage of ROI with LRP1-RBD colocalization. Q Schematic of RBD-GP96-Fusion mediated antigen cross-presentation via CD91/LRP1. Data from n = 6 mice/group (E-L) or n = 6 fields ( M – P ), representative of two experiments. Geometric mean ± SD shown. Statistics by one-way ANOVA with Bonferroni correction or unpaired two-tailed t-test. ns p > 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Transgenic Assay, Vaccines, Expressing, Incubation, Confocal Microscopy, Two Tailed Test

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: Groups of 6- to 8-week-old female BALB/c or HLA-A2 transgenic mice ( n = 6) were i.m. immunized with HPV-18 L1 vaccines adjuvanted with AS04C, AS04D, MF59, or GP96, and L1-GP96-Fusion vaccine. Naïve mice were also included. A A schematic diagram of HPV-18 L1-GP96-Fusion (monomer). The structure of the L1-GP96-Fusion dimer was predicted based on AlphaFold3. B Reduced SDS-PAGE migration profiles of the pooled samples are shown. C Transmission electron microscopy of HPV-18 L1 and L1-GP96-Fusion protein. The red frames represent typical TEM structures. D Immunization schedule of HPV-18 L1 or L1-GP96-Fusion vaccines. E , F Serum was collected at various time points for analysis by ELISA. Kinetics of L1-specific IgG titers are shown (7 days after immunization). G–I Frequency of GC B cells ( G ), L1-specific GC B cells ( H ) or L1-specific MBC precursors ( I ) at 12 days post 3 rd immunization by flow cytometry. J Frequencies of CD8 + IFN-γ + cells following stimulation with cocktail in the spleen at 12 days post-3 rd immunization are shown by flow cytometry. K–L The frequency of AIM + (CD137 + OX40 + ) cells ( K ) among CD4 + T cells and the frequency of AIM + (CD137 + CD69 + ) cells (L) among CD8 + T cells were analyzed (12 days post 3 rd immunization) by flow cytometry. M Representative analysis of various vaccines immune group-mediated killing of MS751 cell line (infected with HPV-18) by flow cytometry. N The frequency of killing percentage at different cell ratios was analyzed as ( M ). O Spearman correlation of L1-specific CD8 + IFN-γ + cells frequency and killing percentage. In ( E – O ), n = 6 mice per group were analyzed. The data are representative of two independent experiments with similar results. The geometric mean ± geometric SD is shown, and each data point represents an individual mouse or field. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Transgenic Assay, Vaccines, SDS Page, Migration, Transmission Assay, Electron Microscopy, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Infection, Two Tailed Test

Journal: Nature Communications

Article Title: Alternative antigen retention by a gp96-fusion approach induces long-lasting and broad immunity in mice

doi: 10.1038/s41467-025-67092-x

Figure Lengend Snippet: Groups of 6- to 8-week-old female HLA-A2 transgenic mice ( n = 6) were i.m. immunized with HPV-18 E7 vaccines adjuvanted with MF59, GP96, and E7-GP96-Fusion vaccine. Naïve mice were also included. A–D Spleens were collected 14 days after 3 rd immunization and stimulated with cocktail for detection. A Representative flow cytometry contour plots of CD8 + IFN-γ + /Granzyme B + /Perforin + cells. B The frequency of ( A ) was analyzed. C Representative flow cytometry contour plots of CD4 + IFN-γ + /Granzyme B + /Perforin + cells. D The frequency of ( C ) was analyzed. E–J Analysis of the clearance effect of CD8 + T cell immune response induced by E7-GP96-Fusion in an HPV + tumor challenge model. E Schedule of animal experiments for T cell transfer strategies in HeLa-bearing mice. F Tumor growth curves of HeLa tumor-bearing mice receiving intravenous transfer of different formulations every 3 days, for four immunotherapy sessions. G Photographs and tumor weight H of the collected tumor tissues on day 30. I Representative flow cytometry contour plots of CD8 + T cells among tumor-infiltrating lymphocytes (TIL) on day 30. J Frequency, absolute count, and infiltration density of CD8 + T cells as shown in ( I ) was analyzed. In ( A – J ), n = 6 mice per group were analyzed. The data are representative of two independent experiments with similar results. The geometric mean ± geometric SD is shown, and each data point represents an individual mouse or field. One-way ANOVA with Bonferroni correction or unpaired two-tailed t tests were conducted according to the distribution of the data. ns p å 0.05, * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001.

Article Snippet: The primary antibodies for incubation are respectively RBD mAb (1:500, Abclonal, A23771), GRP94 (GP96) mAb (1:1000, CST, 20292 T), and LRP1 mAb (1:200, Abcam, ab92544).

Techniques: Transgenic Assay, Vaccines, Flow Cytometry, Two Tailed Test

Journal: Molecular Metabolism

Article Title: TALK-1 reduces delta-cell endoplasmic reticulum and cytoplasmic calcium levels limiting somatostatin secretion

doi: 10.1016/j.molmet.2018.01.016

Figure Lengend Snippet: TALK-1 channels are expressed in mouse and human δ-cells. (A) Mouse pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 mice). (B) Mouse pancreas section stained for TALK-1 (green), ER (GRP94, red), and SST (cyan). (C) Human pancreas section stained for TALK-1 (green) and somatostatin (red) (representative of N = 3 pancreata). (D) Human pancreas section stained for TALK-1 (green), ER (GRP94, red), and somatostatin (E) K2P currents recorded from WT and TALK-1 KO δ-cells ( N = 3 mice per genotype). (F) K2P currents recorded from human δ-cells expressing TALK-1 DN or control mCherry. ( N = 3 islet preparations); * P < 0.05, ** P < 0.005.

Article Snippet: Sections were stained using primary antibodies against somatostatin (Santa Cruz Biotechnology sc-7819: 1:250), TALK-1 (Novus Biologicals #NBP1-83071; 1:175) or TALK-1a (Antibody Verify AAS72353C; 1:250), glucagon (Proteintech #15954-I-AP: 1:500), and the endoplasmic reticulum marker GRP94 (Novus Biologicals #NB300-619; 1:100); secondary antibodies used were Alexa Fluor 488-conjugated donkey anti-rabbit (Jackson Immunoresearch #711-546-152; 1:300), DyLight 650-conjugated donkey anti-goat (Thermo Fisher #SA5-10089; 1:250), and Cy3-conjugated donkey anti-mouse (Jackson Immunoresearch #715-166-150; 1:500).

Techniques: Staining, Expressing, Control