Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 drives tumor-educated M2-like polarization of macrophages and promotes tumor-cell aggressiveness. (A) Workflow for generating TC-hMDMs and TC-BMDMs, circSMAD4 knockdown, and downstream functional assays. (B) RT–qPCR analysis of M1-associated markers (MHC-II [HLA-DRA in TC-hMDMs; H2-Ab1 in TC-BMDMs], NOS2, and CD86) and M2-associated markers (CD163, CD206, and ARG1) in TC-hMDMs and TC-BMDMs. (C) Representative flow-cytometry histograms for HLA-DR, iNOS, CD86, CD163, CD206, and ARG1 in TC-hMDMs. Gating strategy and marker thresholds were defined based on FMO controls (see ). (D) Flow-cytometry quantification of marker-positive cells in TC-hMDMs and TC-BMDMs. (E) ELISA of IL-10, TGF-β, and iNOS in culture supernatants. (F) CCK-8 assays of A549 and LLC cells. (G) Colony-formation assays of A549 and LLC cells with quantification. (H) Bioluminescence-based growth readouts of patient-derived LUAD organoids (PDO #1 and PDO #2) after co-culture with TC-hMDMs. (I) Immunoblot analysis of EMT-related proteins (E-cadherin, N-cadherin, Vimentin) in A549 and LLC cells. (J) Transwell migration and invasion assays of A549 and LLC cells with quantification. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: Knockdown, Functional Assay, Quantitative RT-PCR, Flow Cytometry, Marker, Enzyme-linked Immunosorbent Assay, CCK-8 Assay, Derivative Assay, Co-Culture Assay, Western Blot, Migration

Journal: Non-coding RNA Research

Article Title: CircSMAD4 shapes matrix-remodeling TAMs in lung adenocarcinoma

doi: 10.1016/j.ncrna.2026.03.003

Figure Lengend Snippet: circSMAD4 depletion in macrophages restrains LUAD growth and metastasis in vivo. (A) Schematic of orthotopic lung implantation and experimental metastasis models using LLC cells mixed with BMDMs expressing shNC or sh-circSMAD4. (B) Representative images of orthotopic lung tumors. (C) Tumor weight of orthotopic implants. (D) Overall survival of mice bearing orthotopic tumors. (E) Immunofluorescence showing F4/80 and circSMAD4 signals in tumor tissues. Scale bar, 50 μm. (F, G) Representative Ki-67 IHC staining and quantification in orthotopic tumors. Scale bar, 50 μm. (H) Representative bioluminescence images of lung tumor burden in the metastasis model. (I) Tumor weight in the metastasis model. (J) Overall survival of mice in the metastasis model. (K–M) Representative IHC staining and quantification of E-cadherin and vimentin in tumors. Scale bar, 50 μm. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001; ∗∗∗∗P < 0.0001; ns, not significant.

Article Snippet: Sections were incubated with primary antibodies against Ki-67 (Servicebio, Cat# GB111499 ), E-cadherin (Proteintech, Cat# 20874-1-AP), and Vimentin (Proteintech, Cat# 10366-1-AP).

Techniques: In Vivo, Expressing, Immunofluorescence, Immunohistochemistry

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 interacts with HEV-ORF2 through its substrate-binding domain. ( A ) SDS-PAGE analysis for recombinant GRP75. Recombinant GRP75 was expressed from the E. coli system and refolded into PBS, then subjected to SDS-PAGE analysis. ( B ) Far-western blot assay for detecting the interaction between K239 and GRP75. Different doses of recombinant K239 protein were subjected to SDS-PAGE and transferred to a PVDF membrane. Next, the membrane was incubated with recombinant GRP75 protein expressed in E. coli , followed by detection using an anti-GRP75 antibody and corresponding secondary antibody to assess the binding between K239 and recombinant GRP75. Recombinant SUMO protein was included as an irrelevant control. ( C ) Co-immunoprecipitation (CoIP) analysis of the GRP75 and HEV-ORF2 interaction in HEV-infected HepG2/C3A cells. HepG2/C3A cells stably infected with HEV-3 KernowC1-p6 were lysed using NP-40 buffer and subjected to immunoprecipitation (IP) with an ORF2-specific monoclonal antibody (Mab) 2G8 to detect GRP75 within the ORF2 complex using a GRP75-specific antibody. Normal mouse IgG (mIgG) served as an antibody isotype control for IP. ( D ) CoIP assay for GRP75 and HEV-ORF2 interaction in HEV RNA-transfected S10-3 cells. S10-3 cells were transfected with HEV-3 KernowC1-p6 RNA and cultured for 7 days. Cells were then harvested for Co-IP analysis, following the same protocol as described above. Uninfected control cells were included for comparison. ( E ) Schematic illustration of GRP75 function domains. ( F ) The interaction of GRP75 with ORF2 depends on the substrate-binding domain (SBD) of GRP75. HEK-293T cells were transfected with plasmids encoding MYC-tagged full-length GRP75 (GRP75-MYC), nucleotide-binding domain (NBD) (NBD-MYC), or substrate binding domain (SBD-MYC), along with a plasmid encoding HEV-ORF2, for 48 h. Next, cells were lysed with NP-40 buffer, and proteins were immunoprecipitated using the ORF2-specific Mab-2G8, followed by western blotting using a MYC tag-specific monoclonal antibody to elucidate domain-specific interactions. The proteins from whole cell lysate (WCL) were probed to confirm the expression of the transfected plasmids.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Binding Assay, SDS Page, Recombinant, Far Western Blot, Membrane, Incubation, Control, Immunoprecipitation, Infection, Stable Transfection, Co-Immunoprecipitation Assay, Transfection, Cell Culture, Comparison, Plasmid Preparation, Western Blot, Expressing

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 inhibits HEV infection by targeting ORF2. ( A ) The qPCR validation of GRP75 knockdown using siRNA. S10-3 cells either GRP75-specific siRNA-4, 5, and 6 or control siRNA (siNCtrl). After 48 hours, the cells were harvested using TRIzol reagent. Next, qPCR was performed to quantify the mRNA level of GRP75. All data are presented as mean ± SD and were analyzed using Student’s t -test. ****, P < 0.0001. All results were derived from three independent biological replicates. ( B ) Western blotting validation of GRP75 knockdown using siRNA. S10-3 cells either GRP75-specific siRNA-4, 5, and 6 or control siRNA (siNCtrl). After 48 h, the cells were harvested for SDS-PAGE, followed by western blotting to assess GRP75 protein levels. Tubulin served as a loading control. ( C ) Knockdown of GRP75 increased HEV-ORF2 expression in HEV-infected cells. HepG2/C3A cells stably infected by HEV were transfected with either GRP75-specific siRNA-6 or control siRNA (siNCtrl). After 48 h, the cells were harvested for SDS-PAGE, followed by western blotting to assess GRP75, HEV-ORF1, and ORF2 protein levels. ( D ) Knockdown of GRP75 promotes HEV-RNA levels in HEV-replicating cells. S10-3 cells stably transfected by HEV were further transfected with either GRP75-specific siRNA or control siRNA, followed by RNA extraction using TRIzol reagent. The qPCR was performed to quantify both positive-strand (+) and negative-strand (−) HEV RNA levels. Untreated HEV-replicating S10-3 cells (MOCK control) served as a control. All data are presented as mean ± SD and were analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ****, P < 0.0001; ns, not significant. All results were derived from three independent biological replicates. ( E ) Knockdown of GRP75 promotes the release of HEV virion and secreted ORF2 (sORF2). S10-3 cells stably transfected by HEV were further transfected with either GRP75-specific siRNA or control siRNA. Then, cell culture supernatant was harvested for qPCR to evaluate the HEV-RNA copy numbers (left panel), for virus titration in HepG2/C3A cells (middle panel), or for sandwich ELISA to evaluate secreted ORF2 (sORF2). All data are presented as mean ± SD and were analyzed using Student’s t -test. ***, P < 0.001; ****, P < 0.0001. All results were derived from three independent biological replicates. ( F ) Overexpression of GRP75 inhibits HEV-ORF2 protein level in HEV-replicating cells. S10-3 cells stably transfected with HEV RNA were further transfected with either a plasmid encoding GRP75-MYC or an EV. After 48 h, the cells were harvested for western blotting to assess GRP75, ORF1, and HEV-ORF2 protein levels. Tubulin served as a loading control. ( G ) GRP75 inhibits HEV-RNA levels in HEV-replicating cells. S10-3 cells stably infected by HEV were further transfected with either a plasmid encoding GRP75-MYC or EV. After 48 h, total RNA was extracted, and qPCR was performed to quantify total HEV RNA levels. GAPDH expression from the same cDNA was used as an internal control. All data are presented as mean ± SD and were analyzed using Student’s t -test. **, P < 0.01; ****, P < 0.0001; ns, not significant. All results were based on three independent biological replicates. ( H ) GRP75 inhibits releasing of HEV virion and secreted ORF2 (sORF2). S10-3 cells stably infected by HEV were further transfected with either a plasmid encoding GRP75-MYC or empty vector (EV). After 48 h, the cell culture supernatant was harvested for qPCR to evaluate the HEV-RNA copies numbers (left panel), for virus titration in HepG2/C3A cells (middle panel), or for sandwich ELISA to evaluate secreted ORF2 (sORF2). All data are presented as mean ± SD and were analyzed using Student’s t -test. **, P < 0.01; ***, P < 0.001. All results were derived from three independent biological replicates. ( I ) GRP75 did not affect HEV-ORF1 level in HEV-zsGreen replicon system. S10-3 cells transfected with HEV-zsGreen replicon RNA were further transfected with either GRP75 encoding plasmid (left panel) or GRP75-specific siRNA-6 (right panel). After 48 h, the cells were harvested for SDS-PAGE, followed by western blotting to assess GRP75 and HEV-ORF1 and ORF2 protein levels. Cells transfected with empty vector (EV) or control siRNA (siNCtrl) were included as controls, respectively. ( J ) GRP75 did not affect HEV-RNA level in HEV-zsGreen replicon system. S10-3 cells transfected with HEV-zsGreen replicon RNA were further transfected with either GRP75 encoding plasmid (left panel) or GRP75-specific siRNA-6 (right panel). Cells transfected with empty vector (EV) or control siRNA (siNCtrl) were included as controls, respectively. After 48 hours, total RNA was extracted, and qPCR was performed to quantify total HEV RNA levels. GAPDH expression from the same cDNA was used as an internal control. All data are presented as mean ± SD and were analyzed using Student’s t -test. ns, not significant. ( K ) GRP75 inhibits HEV-ORF2 expression in a dose-dependent manner. HEK-293T cells were transfected with the HEV-ORF2-encoding plasmid along with increasing amounts of a GRP75-encoding plasmid. After 48 h, the cells were harvested for western blotting to assess GRP75 and HEV-ORF2 expression levels. GAPDH served as a loading control to normalize total protein input. ( L ) Inhibition of HEV-ORF2 protein level requires the ATPase activity of GRP75. S10-3 cells stably replicating HEV RNA were transfected with plasmids encoding MYC-tagged wild-type GRP75 (WT GRP75), an ATPase-deficient mutant (GRP75 K73M ), or EV for 48 h. Western blotting was performed to examine ORF2 protein expression levels. Tubulin served as a loading control.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Infection, Biomarker Discovery, Knockdown, Control, Derivative Assay, Western Blot, SDS Page, Expressing, Stable Transfection, Transfection, RNA Extraction, Cell Culture, Virus, Titration, Sandwich ELISA, Over Expression, Plasmid Preparation, Inhibition, Activity Assay, Mutagenesis

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 inhibits HEV infection in primary porcine hepatocyte and HepaRG cells. ( A ) IFA examination of HEV replication in primary porcine hepatocyte and HepaRG cells. Porcine hepatocyte and HepaRG cells were transfected with HEV-RNA for 7 days; then, the cells were fixed and stained for ORF2 protein using ORF2-specific Mab-2G8, followed by observation under fluorescence microscopy. Scale bar = 100 μm. ( B ) Knockdown of GRP75 promotes HEV infection in primary porcine hepatocyte and HepaRG cells. Porcine hepatocyte (left panel) and HepaRG cells (right panel) were transfected with HEV-RNA for 7 days; then, the cells were further transfected with siRNA targeting porcine GRP75 (left panel) and human GRP75 (right panel). After 48 h post-siRNA transfection, the cells were harvested for western blotting to assess GRP75 and HEV-ORF2 expression levels. Tubulin was served as a loading control to normalize total protein input. ( C ) Knockdown of GRP75 promotes virion release of HEV from infected cells. HEV-RNA transfected porcine hepatocyte (left panel) and HepaRG cells (right panel) were further transfected with corresponding GRP75 siRNA for 48 h. Then, the cell culture supernatant was harvested for qPCR to quantify HEV-RNA copies. ( D ) Knockdown of GRP75 increases infectious viral titer of HEV in cell culture supernatant. HEV-RNA transfected porcine hepatocyte (left panel) and HepaRG cells (right panel) were further transfected with corresponding GRP75 siRNA for 48 h. Then, the cell culture supernatant was harvested for the titration of infectious HEV virion in HepG2/C3A cells. ( E ) Knockdown of GRP75 promotes secretion of ORF2. HEV-RNA transfected porcine hepatocyte (left panel) and HepaRG cells (right panel) were further transfected with corresponding GRP75 siRNA for 48 h. Then, the cell culture supernatant was harvested for sandwich ELISA to evaluate secreted ORF2 (sORF2). ( F ) Overexpression of GRP75 inhibits HEV infection in primary porcine hepatocyte and HepaRG cells. Porcine hepatocyte (left panel) and HepaRG cells (right panel) were transfected with HEV-RNA for 7 days; then, the cells were further transfected with plasmids encoding MYC tagged porcine GRP75 (left panel) and human GRP75 (right panel). After 48 h post-plasmid transfection, the cells were harvested for western blotting to assess GRP75-MYC and HEV-ORF2 expression levels. Tubulin was served as a loading control to normalize total protein input. ( G ) Overexpression of GRP75 inhibits virion release of HEV from infected cells. HEV-RNA transfected porcine hepatocyte (left panel), and HepaRG cells (right panel) were further transfected with plasmids encoding corresponding GRP75s. Then, the cell culture supernatant was harvested for titration in HepG2/C3A cells for infectious viral particles. ( H ) Overexpression of GRP75 inhibits infectious viral titer of HEV in cell culture supernatant. HEV-RNA-transfected porcine hepatocyte (left panel) and HepaRG cells (right panel) were further transfected with plasmids encoding corresponding GRP75s. Then, the cell culture supernatant was harvested for titration in HepG2/C3A cells for infectious viral particles. ( I ) Overexpression of GRP75 inhibits secretion of ORF2. HEV-RNA-transfected porcine hepatocyte (left panel) and HepaRG cells (right panel) were further transfected with plasmids encoding corresponding GRP75s. Then, the cell culture supernatant was harvested for sandwich ELISA to evaluate secreted ORF2 (sORF2). All data above are presented as mean ± SD and were analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. All results were derived from three independent biological replicates.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Infection, Transfection, Staining, Fluorescence, Microscopy, Knockdown, Western Blot, Expressing, Control, Cell Culture, Titration, Sandwich ELISA, Over Expression, Plasmid Preparation, Derivative Assay

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 promotes ORF2 degradation via a lysosome-dependent pathway. ( A ) GRP75 reduces the half-life of ORF2. HEV-replicating S10-3 cells were transfected with either empty vector (EV, left panel) or a GRP75-encoding plasmid (right panel) before treatment with cycloheximide (CHX, 10 μg) to inhibit global protein translation. Cells were harvested at 0, 2, 4, 8, 12, 16, 20, and 24 h post-CHX treatment for western blot analysis to determine the half-life of ORF2. GAPDH served as a loading control to normalize total protein input. ( B ) Degradation of HEV-ORF2 is not dependent on the ubiquitin-proteasome pathway. HEK-293T cells were transfected with either an EV or a GRP75-encoding plasmid, along with an ORF2-encoding plasmid, and incubated for 36 h. Next, the proteasome inhibitor MG132 (10 μM) was added, followed by incubation for an additional 16 h before cell harvest. Western blotting was performed to assess HEV-ORF2 protein levels, with GAPDH serving as a loading control. Cells treated with DMSO alone served as a solvent control. ( C ) Rescue of ORF2 protein levels depends on lysosome inhibitors. HEK-293T cells were transfected with either EV or a GRP75-encoding plasmid, along with an ORF2-encoding plasmid, and incubated for 36 hours. Cells were then treated with 3-MA (5 mM), CQ (50 μM), Baf-A1 (200 nM), and NH 4 Cl (15 mM) for 16 h before collection for western blot analysis. ORF2 expression levels were assessed to evaluate potential rescue effects, with GAPDH used as a loading control. Cells treated with DMSO alone were included as a solvent control. ( D ) Lysosome inhibitors rescue ORF2 protein in HEV-replication cells after GRP75 overexpression. S10-3 cells stably transfected with HEV RNA were further transfected with either a plasmid encoding GRP75-MYC or empty vector (EV) for 48 h. Cells were then treated with Baf-A1 (200nM) for another 16 h before collection for western blot analysis. ORF2 expression levels were assessed to evaluate potential rescue effects, with GAPDH used as a loading control. Cells treated with DMSO alone were included as a solvent control.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Transfection, Plasmid Preparation, Western Blot, Control, Ubiquitin Proteomics, Incubation, Solvent, Expressing, Over Expression, Stable Transfection

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75-mediated degradation of ORF2 depends on the KFERQ-like motif of ORF2. ( A ) Illustration of the KFERQ sequence standard as a motif for CMA. ( B ) Schematic illustration of the KFERQ-like motif in ORF2. The ORF2 sequences from all four HEV genotypes were compared and analyzed for the presence of potential CMA motifs using KFERQ finder V0.8. ( C ) Alphafold prediction for locations of KFERQ-like motif in HEV-ORF2 dimer from KernowC1-p6 strain. ( D ) Co-IP assay for ORF2-LAMP2A interaction in HEV-replicating S10-3 cells. S10-3 cells stably transfected with HEV-3 KernowC1-p6 RNA were lysed using NP-40 buffer, and ORF2-specific Mab-2G8 was used for IP. Western blotting was performed using a LAMP2A-specific monoclonal antibody to detect potential interactions. ( E ) Co-immunoprecipitation (Co-IP) assay for ORF2 interaction with LAMP2A in HEK-293T cells. HEK-293T cells were transfected with MYC-tagged LAMP2A (LAMP2A-MYC) and an HEV-ORF2-encoding plasmid for 48 h. Cells were lysed using NP-40 buffer, and immunoprecipitation (IP) was performed using MYC-specific and ORF2-specific monoclonal antibodies (Mab-2G8). The immunoprecipitated complexes were analyzed by western blot using MYC-specific monoclonal antibodies (Mab) for detecting LAMP2A and ORF2-specific polyclonal antibodies (pAb) for detecting ORF2, respectively. ( F ) Deletion of the KFERQ-like motif in ORF2 rescues ORF2 expression. HEK-293T cells were co-transfected with wild-type ORF2 (WT ORF2) or a KFERQ-like motif-deleted ORF2 mutant (ORF2ΔCMA). After 48 h, the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( G ) Deletion of the all CMA motif in ORF2 conferred resistance to GRP75-mediated degradation. HEK-293T cells were co-transfected with a GRP75-MYC-encoding plasmid or empty vector (EV), along with either wild-type ORF2 (WT ORF2) or a KFERQ-like motif-deleted ORF2 mutant (ORF2ΔCMA). After 48 h, the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( H ) KFERQ-like motif in aa421-425 contributed more role for the degradation of ORF2. HEK-293T cells were transfected with a plasmid encoding a complete KFERQ-like motif-deleted ORF2 mutant (ORF2ΔCMA), or an ORF2 mutant bearing the deletion of a different KFERQ-like motif (ORF2ΔCMA 45-49A , ORF2ΔCMA 421-425A , and ORF2ΔCMA 648-653A ). After 48 h, the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( I ) Screening for the key aa residue of the second KFERQ-like motif in aa421-425. HEK-293T cells were transfected with plasmids encoding deletion of KFERQ-like motif in aa421-425 in ORF2 mutant (ORF2ΔCMA 421-425A ), or ORF2 mutant bearing point mutation in KFERQ-like motif in aa421-425 (ORF2 Q421A , ORF2, D422A ORF2 K423A , G424A and ORF2 I425A ). After 48 h, the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( J ) Q421A mutation of ORF2 confers resistance to GRP75-mediated degradation through CMA. HEK-293T cells were co-transfected with a GRP75-MYC-encoding plasmid, along with plasmids encoding deletion of KFERQ-like motif in aa421-425 in ORF2 mutant (ORF2ΔCMA 421-425A ), or ORF2 mutants bearing single point mutation in aa421-425 (ORF2 Q421A , ORF2 D422A ORF2 K423A ORF2 G424A , and ORF2 I425A ). After 48 h, the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( K ) HEV bearing ORF2 Q421A confers resistance to GRP75-mediated degradation through CMA in HEV-replicating S10-3 cells. S10-3 cells transfected RNA of wild type HEV-3 KernowC1-p6 (HEV-WT) or HEV-3 KernowC1-p6 bearing Q421A mutation in ORF2 (HEV-Q421A) for 7days. Then, the cells were transfected with GRP75-MYC-encoding plasmid or empty vector (EV) for 48 h before the cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Sequencing, Co-Immunoprecipitation Assay, Stable Transfection, Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation, Bioprocessing, Expressing, Mutagenesis, Control, Residue

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75-mediated degradation of ORF2 through CMA is not HEV genotype-specific. ( A ) GRP75-mediated inhibition of HEV-ORF2 expression is not genotype-specific. HEK-293T cells were transfected with plasmids encoding HEV-ORF2 proteins from HEV-1, HEV-2, HEV-3, and HEV-4, along with a GRP75-encoding plasmid. After 48 h, cells were harvested for western blotting to assess GRP75 and HEV-ORF2 expression levels. GAPDH served as a loading control to normalize total protein input. ( B ) Deletion of KFERQ-like motif in ORF2 of HEV-1, 2, and 4 led to evaluation of ORF2 protein level. HEK-293T cells were co-transfected with wild-type ORF2 (WT ORF2) from HEV1, 2, and 4, or KFERQ-like motif-deleted ORF2 mutant (ORF2ΔCMA) from HEV1, 2, and 4. After 48 h, cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control. ( C ) Deletion of the all CMA motif in ORF2 of HEV-1, 2, and 4 conferred resistance to GRP75-mediated degradation. HEK-293T cells were co-transfected with a GRP75-MYC-encoding plasmid or empty vector (EV), along with either wild-type ORF2 (WT ORF2) or KFERQ-like motif-deleted ORF2 mutants (ORF2ΔCMA) from HEV1, 2, and 4. After 48 h, cells were harvested for western blot analysis to assess ORF2 protein levels. Tubulin served as a loading control.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Inhibition, Expressing, Transfection, Plasmid Preparation, Western Blot, Control, Mutagenesis

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: Degradation of ORF2 by GRP75 is dependent on the molecular chaperone HSC70. ( A ) Investigation of HSC70’s role in ORF2 degradation. HEV-replicating S10-3 cells were transfected with increasing doses of a MYC-tagged HSC70 (HSC70-MYC)-encoding plasmid for 48 hours. Cells were then harvested for western blot analysis using MYC-specific monoclonal antibodies (Mab) and ORF2-specific Mab-2G8. Tubulin served as a loading control to normalize protein input. ( B ) Knockdown of HSC70 does not affect HEV-ORF2 expression. HEV-replicating S10-3 cells were transfected with HSC70-specific siRNA (siRNA-6) for 36 h, followed by western blotting using antibodies against HSC70, GRP75, and ORF2. Tubulin served as a loading control. ( C ) Co-immunoprecipitation (Co-IP) assay for ORF2 and HSC70 interaction. HEV-replicating S10-3 cells were transfected with MYC-tagged HSC70 (HSC70-MYC) plasmid for 48 h before being lysed using NP-40 buffer. Co-IP was performed using ORF2-specific Mab-2G8, followed by western blot analysis with HSC70-specific antibody to determine potential interactions. ( D ) Co-IP assay for HSC70 and GRP75 interaction. HEK-293T cells were co-transfected with HA-tagged GRP75 (GRP75-HA) and MYC-tagged HSC70 (HSC70-MYC) plasmids for 48 h before lysis with NP-40 buffer. Immunoprecipitation was carried out using a HA-specific antibody, followed by western blot analysis using MYC-specific antibodies to confirm the interaction. ( E ) Co-IP analysis of the complex formed by HSC70, GRP75, and ORF2. HEK-293T cells were transfected with plasmids encoding HSC70 and GRP75, along with either wild-type HEV-ORF2 (WT ORF2) or an ORF2 mutant lacking the KFERQ motif (ORF2ΔCMA), for 48 h. Cells were then lysed using NP-40 buffer, and immunoprecipitation was performed using ORF2-specific Mab-2G8. Western blotting was conducted using antibodies against HSC70 and GRP75 to detect the corresponding targets in the immunoprecipitated complex.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Transfection, Plasmid Preparation, Western Blot, Bioprocessing, Control, Knockdown, Expressing, Co-Immunoprecipitation Assay, Lysis, Immunoprecipitation, Mutagenesis

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 blocks the translocation of ORF2 to mitochondria. ( A ) HEV-ORF2 co-localizes with mitochondria. HEV-replicating S10-3 cells were fixed and stained for ORF2 protein (green channel) and mitochondrial protein COX4 (red channel), followed by confocal microscopy. Scale bar = 10 μm. Co-localization across fluorescence channels was analyzed using ImageJ software. ( B ) Mitochondrial fractionation of HEV-replicating S10-3 cells. HEV-replicating S10-3 cells and uninfected S10-3 cells were subjected to mitochondrial extraction using a commercial kit. Extracted fractions were processed with Laemmli sample buffer for SDS-PAGE and western blotting. VDAC1 and tubulin were used as markers to verify successful fractionation of mitochondrial and cytoplasmic proteins, respectively. ( C ) The N-terminal 111 amino acids of ORF2 are responsible for mitochondrial translocation. HEK-293T cells were transfected with plasmids encoding full-length ORF2 (ORF2) or ORF2 truncation mutants, including ORF2ΔC54 (lacking the C-terminal 54 aa), ORF2ΔN111 (lacking the N-terminal 111 aa), p495 (lacking both the N-terminal 111 aa and C-terminal 54 aa), and FLAG-tagged ORF2N111 (N-terminal 111 aa only). After 48 h, cells were subjected to mitochondrial extraction, followed by SDS-PAGE and western blot analysis. VDAC1 and tubulin were used as markers for mitochondrial and cytoplasmic fractions, respectively. ( D ) HEV-ORF2-N111 colocalized with mitochondrial marker. Normal S10-3 cells transfected with plasmids expressed FLAG-tagged ORF2-N111 truncation for 48 h. Next, cells were fixed and stained for FLAG antibody protein (green channel) and mitochondrial protein VDAC1 (red channel), followed by confocal microscopy. Scale bar = 10 μm. Co-localization across fluorescence channels was analyzed using ImageJ software. ( E ) HEV-ORF2- localized in isolated mitochondrial fraction. Normal S10-3 cells transfected with plasmids expressed FLAG-tagged ORF2-N111 truncation for 48 h. Then, the cells were subjected to mitochondrial extraction. Extracted fractions were processed with Laemmli sample buffer for SDS-PAGE and western blotting. VDAC1 and tubulin were used as markers to verify successful fractionation of mitochondrial and cytoplasmic proteins, respectively. ( F ) Knockdown of GRP75 promotes ORF2 translocation to mitochondria. HEV-replicating S10-3 cells were transfected with GRP75-specific siRNA-6 or control siRNA, followed by mitochondrial extraction. Western blotting was performed to assess ORF2 protein levels in mitochondrial and cytoplasmic fractions, with VDAC1 and tubulin used as fractionation markers. Error bars represent quantification from three independent experiments. All data are presented as mean ± SD and were analyzed using Student’s t -test. **, P < 0.01. ( G ) Overexpression of GRP75 inhibits ORF2 translocation to mitochondria. HEV-replicating S10-3 cells were transfected with a plasmid encoding MYC-tagged GRP75 or empty vector (EV) before mitochondrial extraction. Western blotting was performed to assess ORF2 protein levels in different fractions, with VDAC1 and tubulin serving as markers of successful fractionation of mitochondria and cytoplasm. Error Bar represents quantification from three independent experiments. All data are presented as mean ± SD and were analyzed using Student’s t -test. *, P < 0.05.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Translocation Assay, Staining, Confocal Microscopy, Fluorescence, Software, Fractionation, Extraction, SDS Page, Western Blot, Transfection, Marker, Isolation, Knockdown, Control, Over Expression, Plasmid Preparation

Journal: Journal of Virology

Article Title: GRP75 blocks hepatitis E virus infection by targeting HEV-ORF2 for degradation through chaperone-mediated autophagy and promoting IRF3 activation

doi: 10.1128/jvi.01344-25

Figure Lengend Snippet: GRP75 antagonizes ORF2’s ability to block IFN induction by enhancing the MAVS-TBK1 interaction. ( A ) GRP75 enhances poly(I:C)-induced IFN-β expression. HEK-293T cells were transfected with a plasmid encoding MYC-tagged GRP75 or empty vector (EV) for 24 h. Cells were then further transfected with different doses of high-molecular-weight (HMW) poly(I:C) for 24 h, whereas a non-transfected group served as a control. After treatment, cells were harvested using TRIzol reagent, and qPCR was performed to measure IFN-β mRNA levels. GAPDH transcription from the same cDNA was used as an internal control. All data are presented as mean ± SD and analyzed using Student’s t -test. *, P < 0.05; **, P < 0.01; ns, not significant. All results are based on at least three independent biological replicates. ( B ) GRP75 antagonizes HEV-ORF2’s inhibition of IFN-β induction in HEK-293T cells. HEK-293T cells were co-transfected with a plasmid encoding MYC-tagged GRP75 or empty vector (EV), along with ORF2 encoding plasmid for 24 h. Then, cells were further transfected with 1 μg HMW poly(I:C) for 24 h to induce expression of IFN-β. Next, cells were harvested using TRIzol reagent, and qPCR was performed to measure IFN-β mRNA levels. GAPDH transcription from the same cDNA was used as an internal control. All data are presented as mean ± SD and analyzed using Student’s t -test. ***, P < 0.001; ns, not significant. ( C ) GRP75 antagonizes HEV-ORF2’s inhibition of RIG-I activated IFN-β. HEK-293T cells were co-transfected with a plasmid encoding RIG-I N terminal (RIG-I N), MYC-tagged GRP75, and ORF2, along with IFN-β promoter reporter plasmid and pRL-TK control plasmid for 24 h. Then, cells were lyzed for Dual-luciferase reporter assay to evaluate activation of IFN-β promoter. Firefly luciferase in cells transfected with IFN-β promoter reporter and pRL-TK control plasmid was set as 1-fold. All data are presented as mean ± SD and analyzed using Student’s t -test for indicated groups. ****, P < 0.0001. ( D ) GRP75 antagonizes HEV-ORF2’s inhibition of IFN-β induction in S10-3 cells. S10-3 cells were co-transfected with a plasmid encoding MYC-tagged GRP75 or empty vector (EV), along with ORF2 encoding plasmid for 24 h. Then, cells were further transfected with 1 μg HMW poly(I:C) for 24 h to induce the expression of IFN-β. Next, cells were harvested using TRIzol reagent, and qPCR was performed to measure IFN-β mRNA levels. GAPDH transcription from the same cDNA was used as an internal control. All data are presented as mean ± SD and analyzed using Student’s t -test. *, P < 0.05. ( E ) GRP75 antagonizes HEV-ORF2’s inhibition of IFN-λ1 induction similar to type I IFN. S10-3 cells were co-transfected with plasmids similar to above, then cells were further transfected 1 μg HMW poly(I:C) for 24 h to induce expression of IFN-λ1. Next, cells were harvested using TRIzol reagent, and qPCR was performed to measure IFN-β mRNA levels. GAPDH transcription from the same cDNA was used as an internal control. All data are presented as mean ± SD and analyzed using Student’s t -test for indicated groups. ****, P < 0.0001. ( F ) GRP75 enhances the interaction between TBK1 and MAVS. HEK-293T cells were co-transfected with plasmids encoding FLAG-tagged TBK1 and MYC-tagged MAVS, along with either an HA-tagged GRP75 plasmid or EV. After 48 h, cells were transfected with 1 μg poly(I:C) before lysed using NP-40 buffer, and IP was performed using a TBK1-specific antibody. Western blotting was conducted using a MAVS-specific antibody to assess the levels of MAVS-TBK1 complexes pulled down by IP. ( G ) GRP75 enhances downstream IRF3 activation. HEK-293 cells stably expressing Venus-tagged IRF3 (HEK-293-Venus-IRF3) were transfected with a plasmid encoding MYC-tagged GRP75 or empty vector (EV) for 24 h. Cells were then further transfected with 1 μg poly(I:C) for 0, 2, 4, or 6 h. After treatment, cells were harvested using Laemmli sample buffer, and western blot analysis was performed using antibodies specific for phosphorylated IRF3 (pIRF3-S396) and total IRF3. Tubulin served as a loading control.

Article Snippet: The MYC tag Mab (ProteinTech, Wuhan, Hubei, China), anti-GRP75 Mab (Santa Cruz Biotechnology, Santa Cruz, CA, USA), the anti-VDAC1 Mab (ProteinTech), anti-COX4 Mab (ProteinTech), anti-HSC70 Mab (Santa Cruz Biotechnology), anti-LAMP2A polyclonal antibodies (pAb, Thermo Fisher Scientific, 51-2200) and anti-TBK1 Mab (Santa Cruz Biotechnology), anti-MAVS rabbit polyclonal antibodies pAb, (Cell Signaling Technology, Danvers, MA, USA), anti-GRP75 rabbit pAb (ProteinTech), and anti-Phos-IRF3-S396 rabbit Mab (Cell Signaling Technology) were obtained from commercial suppliers.

Techniques: Blocking Assay, Expressing, Transfection, Plasmid Preparation, High Molecular Weight, Control, Inhibition, Luciferase, Reporter Assay, Activation Assay, Western Blot, Stable Transfection

Journal: Nature Communications

Article Title: Expression of the vault RNA protects cells from undergoing apoptosis

doi: 10.1038/ncomms8030

Figure Lengend Snippet: ( a ) qPCR identifies 10 apoptosis marker genes. Only mRNAs with expression levels that differ more than 2-fold between BL41 and BL41 cells expressing vtRNA1-1 (after a 4.5 h staurosporine treatment) were taken into consideration. Upregulation is reflected by a positive and downregulation by a negative fold-change value. The data shown derive from averaging two biological replicates. ( b ) Protein levels of Bcl-xL, ARC and the cleaved caspases Casp-9 and Casp-3 in BL41 cells expressing vtRNA1-1 or vtRNA1-2 in the absence (−) or presence (+) of staurosporine (Stau) were assessed by western blot analyses. Cleavage of Casp-8 was monitored after Fas-L treatment. Proteins GAPDH or L9 served as loading controls. See also . ( c ) Western blot analyses (upper panel) were used to monitor the kinetics and levels of phosphorylated IκB expression in BL41 cells, or in BL41 cells expressing vtRNA1-1 or vtRNA1-2, respectively, as a function of TNFα incubation (in minutes). Hsp90 served as loading control. The same cell lines were used to test for Bcl-xL mRNA expression by qPCR (lower panel). The data represent the mean and standard deviation of two independent experiments. Locations of molecular weight markers (kDa) are shown on the left ( b ) or right ( c ), respectively. ( d ) A putative model showing how vtRNA1-1 confers apoptosis resistance by modulating the intrinsic as well as the extrinsic pathways. When vtRNA1-1 levels are high (green arrow), such as in the presence of EBV infection and LMP1 signalling (orange), ARC and Bcl-xL become upregulated (green arrows). These proteins subsequently contribute to inhibiting the intrinsic as well as the extrinsic apoptotic pathway resulting in reduced levels of cleaved caspases 9 (intrinsic), 8 (extrinsic) and 3 (both pathways). In the latter case Bcl-xL neutralizes caspase-8 processed Bid at the outer mitochondrial membrane that links the extrinsic pathway to the mitochondrial pathway required for full effector caspase activation thus leading to resistance to Fas Ligand-induced cell death.

Article Snippet: The following antibodies and dilutions for western blotting were used: mouse anti-MVP 1:500 (Santa Cruz); mouse anti-HA tag (16B12, Covance) 1:500; rabbit anti-FLAG 1:5,000 (F7425, Sigma), Histidine Tag (6xHis) mouse anti-histidine Tag monoclonal Antibody (clone 3D5, Life Technologies); rat anti-EBNA1 1:50 (ref. ); rat anti-EBNA2 (1:20, ref. ); mouse anti-EBV 1:1,000 (Dianova),rabbit anti–PDCD4 1:1,000 (D29C6, Cell Signaling); rabbit anti-cleaved caspase-3 1:1,000 (5A1E, Cell Signaling); rabbit anti-cleaved caspase-9 1:1,000 (D2D4, Cell Signaling); mouse anti-cleaved caspase-8 1:1,000 (11G10, Cell Signaling); goat anti-mouse IgG HRP Conjugate 1:10,000 (Invitrogen); goat anti–rabbit IgG HRP Conjugate 1:15,000 (Pierce); anti-ribosomal protein L9 (Santa Cruz); rabbit anti-Bcl-xL 1:1,000 (54H6, Cell Signaling), rabbit anti-ARC 1:1,000 (sc-11435, Santa Cruz); rabbit anti-GAPDH 1:1,000 (14C10, Cell Signaling), rabbit anti-NFκB p65 (Santa Cruz, sc-372) 1: 500, rabbit anti Phospho-IkBα (Ser32) 1:250 (14D4, Cell Signaling), mouse anti-HSP 90 1:1,000 (AC-16, Santa Cruz).

Techniques: Marker, Expressing, Western Blot, Incubation, Standard Deviation, Molecular Weight, Infection, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Hyperglycemia increased the gingival senescent burden and induced serum SASP in diabetic mice. A, all protocols were performed strictly according to the procedure. C57 mice were rendered diabetic by STZ injections and sacrificed every 2 weeks. B, fasting glucose levels were determined every 2 weeks from weeks 5 to 17. The p value between control mice (N group) and diabetic mice (D group) is shown. **, p < 0.01. C, Western blotting analysis showing p16 and p21 specific immunoreactivity in the gingival tissue of the N group and the D group. The optical density (O.D.) values of p16 and p21 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. Panel C here and panels A and E in Fig. 3 use the same band of β-actin because of the identical protein sample in same Western blotting experiment. D, immunohistochemistry using antibody against p16 and p21 was analyzed in the gingival tissue of N group and D group. Scale bar, 50 μm. The percentage of positive cells is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus N group. E, the SASP factors in the serum of N group and D group were determined every 2 weeks by a Luminex assay customization tool and shown in the heat map. F, gingival tissues of N group and D group were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16 antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 50 μm.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Control, Western Blot, Immunohistochemistry, Luminex, Staining, Immunofluorescence

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: High glucose induced cellular senescence and SASP in macrophage derived from RAW 264.7 cell. A, the activity of SA–β-gal was determined in macrophage exposed to 5–30 mm glucose for 6 or 24 h. Scale bar, 100 μm. The rate of SA–β-gal–positive (blue-stained) cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus 30 mm glucose for 6 h. ##, p < 0.01 versus 30 mm glucose for 24 h. B, the expression levels of p16 and p21 in macrophage were analyzed by Western blotting. The O.D. values of p16 and p21 levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus 30 mm glucose for 6 h. #, p < 0.05; ##, p < 0.01 versus 30 mm glucose for 24 h. Panel B here and panels C and F in Fig. 3 use the same band of β-actin because of the identical protein sample in same Western blotting experiment. C, the SASP factors in the supernatant of macrophage were determined by a Luminex assay customization tool and shown in the heat map. D, cell proliferation was detected using EdU detection kits to analyze the incorporation of EdU during DNA synthesis. Scale bar, 100 μm. The percentage of proliferating cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus 30 mm glucose for 6 h. ##, p < 0.01 versus 30 mm glucose for 24 h.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Derivative Assay, Activity Assay, Staining, Expressing, Western Blot, Luminex, DNA Synthesis

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: High glucose was incapable of inducing cellular senescence and SASP in in IRF8−/− or NLRC4−/− macrophage exposed to 30 mm glucose for 24 h. A, the activity of SA–β-gal was determined in control, NLRC4−/−, and IRF8−/− macrophage exposed to 5 and 30 mm glucose for 24 h. Scale bar, 100 μm. The rate of SA–β-gal–positive cells is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). B, p16 and p21 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose). C, the change of SASP-associated factors is displayed in the heat map. D, Western blotting analysis showing IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 specific immunoreactivity. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus control CRISPR/Cas9 plasmid (30 mm glucose).

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Control, CRISPR, Plasmid Preparation, Immunofluorescence, Staining, Fluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin ameliorated the burden of senescent cells in gingival tissue and the SASP in serum of diabetic mice. A, all protocols were performed strictly according to the procedure. The diabetic mice were treated with the metformin (300 mg/kg body weight, everyday) from weeks 9 to 17. B, fasting blood glucose levels were determined at sacrifice (week 17) among control mice (N group), diabetic mice (D group), and diabetic mice treated with metformin (DM group). The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group. C, representative of immunohistochemical staining of p16, p21, IRF8, and NLRC4 on the gingival sections from the N, D, and DM groups. Scale bar, 50 μm. The percentage of positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus the D group. D, the SASP factors in the serum of the N, D, and DM groups were determined at sacrifice (week 17) by a Luminex assay customization tool and shown in the heat map. E, the gingival tissues of the N, D, and DM group mice were stained for immunofluorescence using an F4/80 antibody targeting macrophages (red) and a p16 antibody (green). The nuclei were stained with DAPI (blue). Scale bar, 50 μm. F, IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in the gingival tissue of the N, D, and DM groups were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus D group.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Control, Immunohistochemical staining, Staining, Luminex, Immunofluorescence, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Hyperglycemia-induced inflamm-aging accelerates gingival senescence via NLRC4 phosphorylation

doi: 10.1074/jbc.RA119.010648

Figure Lengend Snippet: Metformin attenuated high glucose–induced cellular senescence and SASP in macrophage exposed to high glucose (30 mm) for 24 h. A, the activity of SA–β-gal were determined in macrophage treated with low glucose (5 mm) (N), high glucose (30 mm) (HG), and high glucose (30 mm) + metformin (10 mm) (HGM). Scale bar, 100 μm. The rate of SA–β-gal–positive cells was calculated and is represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG. B and E, p16, p21, IRF8, and NLRC4 levels were measured by immunofluorescence staining. Scale bar, 50 μm. The fluorescence intensity is represented in bar histograms. The data are means ± S.D. (n = 3). **, p < 0.01 versus HG. C, the change of SASP factors are displayed in the heat map. D, the expression levels of IRF8, p-NLRC4, NLRC4, Caspase-1, cleaved Caspase-1, NF-κB, p16, and p21 in N, HG, and HGM were analyzed by Western blotting. The O.D. values of these proteins' levels relative to β-actin are represented in bar histograms. The data are means ± S.D. (n = 3). *, p < 0.05; **, p < 0.01 versus HG.

Article Snippet: The membranes were incubated overnight at 4 °C with primary antibodies against β-actin (1:1000; ta-09; ZSGB-BIO), p-NLRC4 (1:500; NM5491; ECMbiosciences), NLRC4 (1:1000; ab99860; Abcam), IRF8 (1:1000; sc-365042; Santa Cruz), Caspase-1 (1:1000; ET1608–69; Huabio), NF-κB p65 (1:1000; sc-514451; Santa Cruz), p16 (1:1000; sc-166760; Santa Cruz), and p21 (1:1000; sc-166630; Santa Cruz) followed by either secondary anti-rabbit (1:5000; 1706515; Bio-Rad) or anti-mouse (1:5000; ZB-2305; ZSGB-BIO) antibody for 1 h at room temperature.

Techniques: Activity Assay, Immunofluorescence, Staining, Fluorescence, Expressing, Western Blot