Journal: Oncotarget

Article Title: A DNA telomerase vaccine for canine cancer immunotherapy

doi: 10.18632/oncotarget.26927

Figure Lengend Snippet: ( A ) Schematic maps and alignments of wild type cTERT and pDUV5 proteins. VDD: deletion in catalytic site at amino acid positions 858-860. NoLS, nucleolar localization signal; Ubi, ubiquitin; Flu, influenza A HLA-A2 restricted epitope; V5, V5 tag. ( B ) Expression of pDUV5 protein monitored 24 h post-transfection in HEK293T cells. Protein was detected using an anti-V5 mouse monoclonal antibody. pcDNA3.1 empty vector backbone as negative control. JP: jetPRIME. NT: non-treated cells. β-actin protein detection was used as a loading control assessment. ( C ) Intracellular localization of wild type hTERT and pDUV5 proteins in transfected QT6 cells visualized 24 h post-transfection with a rabbit anti-hTERT antibody or a mouse anti-V5 antibody respectively and a goat anti-rabbit-Alexa Fluor 488 conjugate or a goat anti-mouse antibody-Alexa Fluor 488 conjugate (green fluorescence) respectively. pcDNA3.1 vector as negative control. The nuclei were stained with DAPI (blue). The cells were analyzed for both fluorescence wavelengths (merged) upon fluorescence microscopy. ( D ) Neutralization of pDUV5 telomerase catalytic activity. Total cell proteins were extracted from wild type hTERT and pDUV5 transfected CRFK cells and telomerase activity was assessed by Telomeric Repeat Amplification Protocol (TRAP) assay. Relative Telomerase Activity (RTA; sample/positive control ratio) of pDUV5 compared to wild type hTERT and non-treated (NT) CRFK cells are displayed (n = 3 for 2.1 μg of total protein samples) using absorbance measurements values (OD450/690 nm). Mann-Whitney non-parametric test against non-treated CRFK cells, ** p < 0.01 .

Article Snippet: HEK293T (Human embryonic kidney) cell line and CRFK (Crandell-Rees feline kidney) cell line (from the ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal calf serum (FCS), 1% sodium pyruvate, 1% penicillin/streptomycin pyruvate and 0.1% β-mercaptoethanol.

Techniques: Ubiquitin Proteomics, Expressing, Transfection, Plasmid Preparation, Negative Control, Control, Fluorescence, Staining, Microscopy, Neutralization, Activity Assay, Amplification, TRAP Assay, Positive Control, MANN-WHITNEY

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