Corticotomy is a clinical procedure to accelerate orthodontic tooth movement characterized by the regional acceleratory phenomenon (RAP). Despite its therapeutic effects, the surgical risk and unclear mechanism hamper the clinical application. Numerous evidences support macrophages as the key immune cells during bone remodeling. Our study discovered that the monocyte-derived macrophages primarily exhibited a pro-inflammatory phenotype that dominated bone remodeling in corticotomy by CX3CR1^CreERT2; R26^GFP lineage tracing system. Fluorescence staining, flow cytometry analysis, and western blot determined the significantly enhanced expression of binding immunoglobulin protein (BiP) and emphasized the activation of sensor activating transcription factor 6 (ATF6) in macrophages. Then, we verified that macrophage specific ATF6 deletion (ATF6^f/f; CX3CR1^CreERT2 mice) decreased the proportion of pro-inflammatory macrophages and therefore blocked the acceleration effect of corticotomy. In contrast, macrophage ATF6 overexpression exaggerated the acceleration of orthodontic tooth movement. In vitro experiments also proved that higher proportion of pro-inflammatory macrophages was positively correlated with higher expression of ATF6. At the mechanism level, RNA-seq and CUT&Tag analysis demonstrated that ATF6 modulated the macrophage-orchestrated inflammation through interacting with Tnfα promotor and augmenting its transcription. Additionally, molecular docking simulation and dual-luciferase reporter system indicated the possible binding sites outside of the traditional endoplasmic reticulum-stress response element (ERSE). Taken together, ATF6 may aggravate orthodontic bone remodeling by promoting Tnfα transcription in macrophages, suggesting that ATF6 may represent a promising therapeutic target for non-invasive accelerated orthodontics.
Animals ; Mice ; Macrophages/metabolism* ; Tumor Necrosis Factor-alpha/genetics* ; Tooth Movement Techniques/methods* ; Activating Transcription Factor 6/metabolism* ; Bone Remodeling ; Flow Cytometry ; Blotting, Western

Objective To investigate prokaryotic expression of the antigen sequence (amino acids 59-145) of mouse leukemia-related protein 16 (LRP16) protein and preparation of rabbit anti-mouse LRP16 polyclonal antibody. Methods The prokaryotic expression plasmid pLS962-LRP16 was constructed by the molecular cloning method and transferred into E.coli Rosetta to express LRP16 protein induced by IPTG. The recombinant protein was purified using Ni-NTA affinity columns followed by gel filtration chromatography. New Zealand white rabbits were immunized with the purified antigen to generate polyclonal antiserum, with antibody titer quantified by ELISA. Antigen-specific IgG was affinity-purified using Sepharose-coupled LRP16 and validated through Western blot and immunofluorescence assays. Results SDS-PAGE analysis confirmed insoluble expression of the LRP16 fusion protein as inclusion bodies. ELISA demonstrated exceptional antiserum titer (1:256 000). Western blot and immunofluorescence verified that the polyclonal antibody could specifically recognize endogenous LRP16 in murine tissues. Conclusion The prokaryotic expression of the LRP16 gene is successfully achieved, and the rabbit anti-mouse LRP16 polyclonal antibody exhibiting high specificity is prepared. This lays the foundation for further studies on the function of the LRP16 gene.
Animals ; Rabbits ; Mice ; Antibodies/immunology* ; Escherichia coli/metabolism* ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Antibody Specificity

Plant bioreactor is a new production platform for expression of recombinant protein, which is one of the cores of molecular farming. In this study, the anti DYKDDDDK (FLAG) antibody was recombinantly expressed in tobacco (Nicotiana benthamiana) and purified. FLAG antibody with high affinity was obtained after immunizing mice for several times and its sequence was determined. Based on this, virus vectors expressing heavy chain (HC) and light chain (LC) inoculated into Nicotiana benthamiana leaves by using Agrobacterium-mediated delivery. Accumulation of the HC and LC was analyzed by SDS/PAGE followed by Western blotting probed with specific antibodies from 2 to 9 days postinfiltration (dpi). Accumulation of the FLAG antibody displayed at 3 dpi, and reached a maximum at 5 dpi. It was estimated that 66 mg of antibody per kilogram of fresh leaves could be obtained. After separation and purification, the antibody was concentrated to 1 mg/mL. The 1:10 000 diluted antibody can probe with 1 ng/mL FLAG fused antigen well, indicating the high affinity of the FLAG antibody produced in plants. In conclusion, the plant bioreactor is able to produce high affinity FLAG antibodies, with the characteristics of simplicity, low cost and highly added value, which contains enormous potential for the rapid and abundant biosynthesis of antibodies.
Animals ; Mice ; Antibodies ; Nicotiana/genetics* ; Agrobacterium/genetics* ; Bioreactors ; Blotting, Western

Objective To evaluate the correlation between alterations in DNase1 and DNase1L3 enzyme activities and impairment of NET degradation in patients with sporadic SLE, and to investigate the underlying mechanism. Methods 46 sporadic SLE patients and 30 age- and sex-matched healthy individuals were recruited. Serum levels of DNase1, DNase1L3 and corresponding autoantibodies were detected by ELISA. DNase1 and DNase1L3 were isolated by immunoprecipitation; NETs and enzyme degradation activities were detected using a modified immunofluorescence. DNase1L3 secretion by PBMCs was analyzed by ELISPOT, Western blotting and reverse transcription PCR. Results Levels of H3-dsDNA and Ela-dsDNA complexes were significantly elevated in SLE patients. LDGs in SLE population was significantly higher than in the control group, and LDGs was positively correlated with H3-dsDNA and Ela-dsDNA NETs complexes. The ability of SLE patients to degrade NET in vitro was significantly lower than that of the control group. Degradation experiments of DNase1 and DNase1L3 in different proportions showed that the decrease in DNase1L3 activity was the primary contributor to the elevated NET residue level. The concentration of DNase1L3 autoantibodies in SLE patients was significantly elevated compared to the control group. In addition, the capacity of PBMCs to secrete DNase1L3 was significantly lower in the SLE patients compared to the control group. Conclusion Decreased secretion of DNase1L3 and the presence of relevant autoantibodies notably impede NET degradation in patients with SLE, offering new directions for the monitoring and treatment of SLE patients.
Humans ; Autoantibodies ; Blotting, Western ; Enzyme-Linked Immunosorbent Assay ; Extracellular Traps ; Lupus Erythematosus, Systemic

OBJECTIVE: This study investigated how the natural phytophenol and potent SIRT1 activator resveratrol (RSV) regulate necroptosis during Vibrio vulnificus (V. vulnificus)-induced sepsis and the potential mechanism. METHODS: The effect of RSV on V. vulnificus cytolysin (VVC)-induced necroptosis was analyzed in vitro using CCK-8 and Western blot assays. Enzyme-linked immunosorbent assays and quantitative real-time polymerase chain reaction, western blot, and immunohistochemistry and survival analyses were performed to elucidate the effect and mechanism of RSV on necroptosis in a V. vulnificus-induced sepsis mouse model. RESULTS: RSV relieved necroptosis induced by VVC in RAW264.7 and MLE12 cells. RSV also inhibited the inflammatory response, had a protective effect on histopathological changes, and reduced the expression level of the necroptosis indicator pMLKL in peritoneal macrophages, lung, spleen, and liver tissues of V. vulnificus-induced septic mice in vivo. Pretreatment with RSV downregulated the mRNA of the necroptosis indicator and protein expression in peritoneal macrophages and tissues of V. vulnificus-induced septic mice. RSV also improved the survival of V. vulnificus-induced septic mice. CONCLUSION Our findings collectively demonstrate that RSV prevented V. vulnificus-induced sepsis by attenuating necroptosis, highlighting its potency in the clinical management of V. vulnificus-induced sepsis.
Animals ; Mice ; Necroptosis ; Resveratrol/therapeutic use* ; Vibrio vulnificus ; Sepsis/drug therapy* ; Blotting, Western

OBJECTIVE: To investigate the effect of ANP32A silencing on invasion and migration of colon cancer cells and the influence of the activity of AKT signaling pathway on this effect. METHODS: Colorectal cancer HCT116 and SW480 were transfected with a small interfering RNA targeting ANP32A via a lentiviral vector. At 24, 48 and 72 h after the transfection, the changes in cell proliferation and AKT activity in the cells were detected using MTT assay and Western blotting, respectively. HCT116 and SW480 cells were treated with the AKT agonist SC79 or its inhibitor MK2206 for 24, 48, 72 and 96 h, and the changes in cell migration and invasion ability were analyzed using Transwell chamber assay and cell proliferation was assessed using MTT assay. The effects of SC79 and MK2206 on migration and invasion abilities of HCT116 and SW480 cells with or without ANP32A silencing were examined using wound healing and Transwell chamber assays, and the changes in the expression of metadherin (MTDH), a factor associated with cells invasion and migration, was detected with Western blotting. RESULTS: Lentivirus-mediated ANP32A silencing significantly down-regulated the activity of AKT and inhibited the proliferation of both HCT116 and SW480 cells (P < 0.01). The application of AKT inhibitor MK2206 obviously inhibited the proliferation, invasion and migration of the colorectal cancer cells (P < 0.05), while the AKT agonist SC79 significantly promoted the invasion and migration of the cells (P < 0.01). In HCT116 and SW480 cells with ANP32A silencing, treatment with MK2206 strongly enhanced the inhibitory effects of ANP32A silencing on cell invasion and migration (P < 0.05) and the expression of MTDH, while SC79 partially reversed these inhibitory effects (P < 0.01). CONCLUSION ANP32A silencing inhibits invasion and migration of colorectal cancer cells possibly by inhibiting the activation of the AKT signaling pathway.
Humans ; Proto-Oncogene Proteins c-akt ; Cell Proliferation ; Blotting, Western ; Cell Movement ; Colonic Neoplasms ; Membrane Proteins ; RNA-Binding Proteins/genetics* ; Nuclear Proteins

OBJECTIVE: To investigate the changes in gray matter volume in depressive-like mice and explore the possible mechanism. METHODS: Twenty-four 6-week-old C57 mice were randomized equally into control group and model group, and the mice in the model group were subjected to chronic unpredictable mild stimulation (CUMS) for 35 days. Magnetic resonance imaging was performed to examine structural changes of the grey matter volume in depressive-like mice. The expression of brain-derived neurotrophic factor (BDNF) in the grey matter of the mice was detected using Western blotting and immunofluorescence staining. RESULTS: Compared with the control mice, the mice with CUMS showed significantly decreased central walking distance in the open field test (P < 0.05) and increased immobile time in forced swimming test (P < 0.05). Magnetic resonance imaging showed that the volume of the frontal cortex was significantly decreased in CUMS mice (P < 0.001, when the mass level was greater than or equal to 10 756, the FDRc was corrected with P=0.05). Western blotting showed that the expression of mature BDNF in the frontal cortex was significantly decreased in CUMS mice (P < 0.05), and its expression began to decrease after the exposure to CUMS as shown by immunofluorescence staining. The volume of different clusters obtained by voxel-based morphometry (VBM) analysis was correlated with the expression level of mature BDNF detected by Western blotting (P < 0.05). CONCLUSION The decrease of frontal cortex volume after CUMS is related with the reduction of mature BDNF expression in the frontal cortex.
Animals ; Mice ; Blotting, Western ; Brain-Derived Neurotrophic Factor ; Cerebral Cortex ; Depression/physiopathology* ; Frontal Lobe/pathology*

OBJECTIVE: To investigate the prognostic value of death-associated protein 5 (DAP5) in gastric cancer (GC) and its regulatory effect on aerobic glycolysis in GC cells. METHODS: We analyzed DAP5 expression levels in GC and adjacent tissues and its association with survival outcomes of GC patients using public databases. We collected paired samples of GC and adjacent tissues from 102 patients undergoing radical resection of GC in our hospital from June, 2012 to July, 2017, and analyzed the correlation of DAP5 expression level detected immunohistochemically with the clinicopathological parameters of the patients. Cox regression analysis, Kaplan-Meier analysis, and ROC curves were used to explore the independent risk factors and the predictive value of DAP5 expression for 5-year survival of the patients. In the cell experiments, we observed the changes in aerobic glycolysis in MGC-803 cells following lentivirus-mediated DAP5 knockdown or overexpression by measuring glucose uptake and cellular lactate level and using qRT-PCR and Western blotting. RESULTS: Analysis using the public databases showed that DAP5 was highly expressed in GC and correlated with tumor progression and poor survival outcomes of the patients (P < 0.05). In the clinical samples, DAP5 expression was significantly higher in GC than in the adjacent tissues (3.19±0.60 vs 1.00±0.12; t=36.863, P < 0.01), and a high expression of DAP5 was associated with a reduced 5-year survival rate of the patients (17.6% vs 72.5%; χ²=29.921, P < 0.05). A high DAP5 expression, T3-4, N2-3, and CEA≥5 ng/mL were identified as independent risk factors affecting 5-year survival outcomes of GC (P < 0.05), for which DAP5 expression showed a prediction sensitivity, specificity and accuracy of 73.2%, 80.4% and 79.0%, respectively. In MGC-803 cells, DAP5 knockdown significantly reduced glucose uptake, lactate level and the expressions of GLUT1, HK2 and LDHA, and DAP5 overexpression produced the opposite effects (P < 0.05). CONCLUSION A high expression of DAP5 in GC, which enhances cellular aerobic glycolysis to promote cancer progression, is correlated with a poor survival outcome and may serve as a biomarker for evaluating long-term prognosis of GC patients.
Humans ; Stomach Neoplasms ; Blotting, Western ; Databases, Factual ; Glucose ; Lactates

Objective To investigate antigen optimization of Shisa like protein 1 (SHISAL1) for preparing mouse anti-human SHISAL1 polyclonal antibody and to identify the specificity of the prepared antibody. Methods Bioinformatics was employed to predict the antigenic epitope region of SHISAL1 protein, and then a polypeptide composed of amino acid residues from the site of 28 to 97 of SHISAL1, termed SHISAL1-N, was selected as the antigen. The coding region of SHISAL1-N was cloned by molecular cloning technique, and then it was inserted into pET-28a to generate pET28a-SHISAL1-N recombinant plasmid. The two recombinant plasmids pET28a-SHISAL1-N and pET28a-SHISAL1 were transformed into BL21 (DE3) bacteria and induced to express by IPTG. The two proteins were purified and immunized to female Kunming mice, respectively. The specificities and sensitivities of the acquired antibodies were detected by Western blot analysis, immunoprecipitation and immunofluorescent cytochemical staining. Results pET28a-SHISAL1-N recombinant plasmid was successfully constructed, and the two fused proteins, SHISAL1 and SHISAL1-N, were induced to express. Moreover, two types of SHISAL1 mouse polyclonal antibodies, derived from SHISAL1-N and SHISAL1 antigens, were obtained. Western blot results showed that the antibody prepared from SHISAL1 antigen was less specific and sensitive compared with the antibody prepared from SHISAL1-N antigen which could specifically identify different endogenous SHISAL1 protein. Immunoprecipitation results showed that SHISAL1-N antibody could specifically pull down SHIISAL1 protein in hepatocellular carcinoma cells and immunofluorescence results demonstrated that SHISAL1-N antibody could specifically bind to SHISAL1 protein in the cytoplasm. Conclusion We have optimized the SHISAL1 antigen and prepared the mouse anti-human SHISAL1 polyclonal antibodies successfully, which can be used for Western blot analysis, immunoprecipitation and immunofluorescence cytochemical staining.
Animals ; Female ; Humans ; Mice ; Antibodies ; Antibody Specificity ; Blotting, Western ; Cloning, Molecular ; Epitopes/genetics*

Objective To prepare a rabbit anti-mouse coiled-coil domain containing 189 (Ccdc189) polyclonal antibody. Methods The pET-28a-Ccdc189 prokaryotic expression plasmid was constructed and transformed into E.coli BL21. IPTG was used to induce the expression of Ccdc189 prokaryotic protein. Adult male New Zealand rabbits were immunized with purified recombinant protein to obtain rabbit anti-mouse Ccdc189 polyclonal antibody. The specificity of the polyclonal antibody was identified by Western blot analysis, indirect ELISA and immunofluorescence histochemical staining. Results The pET-28a-Ccdc189 recombinant plasmid was successfully constructed and the expression of the Ccdc189 recombinant protein was induced. ELISA revealed that the titer of the polyclonal antibody was 1:1 000 000. Western blot and immunofluorescence staining demonstrated that the Ccdc189 polyclonal antibody could specifically identify the Ccdc189 prokaryotic protein and the Ccdc189 protein in adult wild-type mouse testis. Conclusion A polyclonal antibody with high specificity against mouse Ccdc189 was successfully created.
Rabbits ; Male ; Animals ; Mice ; Antibody Specificity ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; Blotting, Western ; Recombinant Proteins ; Escherichia coli/genetics*