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2021 ◽  
Vol 18 (1) ◽  
Lilan Li ◽  
Yu Li ◽  
Bingqiang He ◽  
Hui Li ◽  
Huiyuan Ji ◽  

Abstract Background Two activation states of reactive astrocytes termed A1 and A2 subtypes emerge at the lesion sites following spinal cord injury (SCI). A1 astrocytes are known to be neurotoxic that participate in neuropathogenesis, whereas A2 astrocytes have been assigned the neuroprotective activity. Heat shock transcription factor 1 (HSF1) plays roles in protecting cells from stress-induced apoptosis and in controlling inflammatory activation. It is unknown whether HSF1 is involved in suppressing the conversion of A1 astrocytes following SCI. Methods A contusion model of the rat spinal cord was established, and the correlations between HSF1 expression and onset of A1 and A2 astrocytes were assayed by Western blot and immunohistochemistry. 17-AAG, the agonist of HSF1, was employed to treat the primary cultured astrocytes following a challenge by an A1-astrocyte-conditioned medium (ACM) containing 3 ng/ml of IL-1α, 30 ng/ml of TNF-α, and 400 ng/ml of C1q for induction of the A1 subtype. The effects of 17-AAG on the phenotype conversion of astrocytes, as well as underlying signal pathways, were examined by Western blot or immunohistochemistry. Results The protein levels of HSF1 were significantly increased at 4 days and 7 days following rat SCI, showing colocalization with astrocytes. Meanwhile, C3-positive A1 astrocytes were observed to accumulate at lesion sites with a peak at 1 day and 4 days. Distinctively, the S100A10-positive A2 subtype reached its peak at 4 days and 7 days. Incubation of the primary astrocytes with ACM markedly induced the conversion of the A1 phenotype, whereas an addition of 17-AAG significantly suppressed such inducible effects without conversion of the A2 subtype. Activation of HSF1 remarkably inhibited the activities of MAPKs and NFκB, which was responsible for the regulation of C3 expression. Administration of 17-AAG at the lesion sites of rats was able to reduce the accumulation of A1 astrocytes. Conclusion Collectively, these data reveal a novel mechanism of astrocyte phenotype conversion following SCI, and HSF1 plays key roles in suppressing excessive increase of neurotoxic A1 astrocytes.

2021 ◽  
Sheng Han ◽  
Ziyi Wang ◽  
Wenjie Yu ◽  
Chenyang Jia ◽  
Chenyu Jiao ◽  

Abstract BackgroudsTrem2 is a core member of the Triggering recptor expressed on myeloid cell family, and its role in response to pathological damagewas first discovered in Alzheimer’s disease. Recently, the role of Trem2 played in tumor progression has been highlighted. However, the specific role and related mechanisms of trem2 in HCC (hepatocellular carcinoma) are still ambiguous.MethodsThe 371 HCC data sets in the TCGA cohort combined with the TIMER2.0 online database were used to verify the potential role and prognostic value of trem2 in HCC.TIMER2.0 online database,immunohistology, immunofluorescence, Western Blot and other methods are applied to identify the correlation between Trem2 and HCC patient samples.Animal models of bile duct ligation, intraperitoneal injection of CCL4 and feeding of CDAHFD60 were used to verify the potential mechanism of trem2 to regulate HCC.ResultsTrem2 is significantly overexpressed in HCC and is an independent risk factor predicts the poor prognosis of HCC patients. Trem2 mainly regulates the progression of HCC in response to the mutations of TP53 and CTNNB1, which also suggests that Trem2 plays an important role in tumor pathological metabolism. Since HCC patients often accompanied by different degrees of liver fibrosis, we suspect that Trem2 may promotes the progression of HCC by regulating liver fibrosis.ConclusionOur research highlights the status of trem2 in the tandem or independent events of HCC's pathological metabolism, immune checkpoint coordination, and liver fibrosis. We predict that targeting Trem2 combined with immune checkpoint inhibitors may be an effective treatment for HCC with rich immune and metabolic microenvironment.

2021 ◽  
Ken Christensen

Transferring your proteins from your SDS-PAGE gel to a nitrocellulose membrane for western blot is quick and straightforward.

BioTechniques ◽  
2021 ◽  
Kurpad Nagaraj Shashidhar ◽  
Venkata Lakshmaiah ◽  
Chandrappa Muninarayana ◽  
Krishna Sumanth Nallagangula

Background: SERPINA4/kallistatin is a multifunctional protein expressed from the liver; its concentration in blood circulation reflects the degree of liver dysfunction and may serve as a diagnostic/prognostic biomarker for chronic liver diseases (CLD). Materials & methods: Antibodies specific for SERPINA4/kallistatin were used for the development of an enzyme-linked immunosorbent assay (ELISA). For correlative studies, blood samples from patients with cirrhotic liver and healthy patients were collected from R.L. Jalappa Hospital & Research Centre, Kolar. Results: Interference of other SERPINs was ruled out using western blot analysis. Quantitative ELISA was developed using monospecific antibodies as capture antibodies. Conclusion: The accuracy of the developed ELISA was determined by inter- and intra-assay precision. Linearity was defined using a spiked sample with serial dilutions. Reduced levels of SERPINA4/kallistatin were observed in patients with CLD compared with healthy controls.

2021 ◽  
Vol 8 ◽  
Lulu Fu ◽  
Heming Shi ◽  
Wenfang Dai ◽  
Hanhan Yao ◽  
Yongbo Bao ◽  

The relationship between carotenoid and shellfish shell color has gained increasing attention. β, β-carotene-9′,10′-oxygenase 2 (BCDO2) is a key enzyme in animal carotenoid metabolism, and its accumulation affects the change in body color, as demonstrated in mammals, birds, and fish. However, it is unclear whether BCDO2 is involved in the formation of the red shell color of clam. To explore the molecular structure and biological function of BCDO2 gene in the process of carotenoids accumulation, in this study, the BCDO2 from hard clam Meretrix meretrix (designated as Mm-BCDO2) was cloned and characterized, and the single-nucleotide polymorphisms (SNPs) associated with shell color were detected. The results of qRT-PCR indicated that Mm-BCDO2 gene was expressed in all six tested tissues, and the expression of mantle was significantly higher than other tissues (P < 0.05). The association analysis identified 20 SNPs in the exons of Mm-BCDO2, among which three loci (i.e., c.984A > C, c.1148C > T, and c.1187A > T) were remarkably related (P < 0.05) to the shell color of clam. The western blot analysis revealed that the expression level of Mm-BCDO2 in the mantle of red shell clams was stronger than that of white shell clams (P < 0.05). Further, the immunofluorescence analysis indicated that the single-layer columnar cells at the edge of the mantle were the major sites for the Mm-BCDO2 secretion. This study explored the potential impacts of BCDO2 gene on the shell color of M. meretrix, which provided a theoretical basis for a better understanding of the important role of BCDO2 in carotenoid metabolism.

2021 ◽  
Junhao Zhou ◽  
Hu Tian ◽  
Xi Zhi ◽  
Zhuoyu Xiao ◽  
Taoyi Chen ◽  

Abstract Background In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. Methods Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. Results ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/β-catenin pathway. Conclusions This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

2021 ◽  
Ilenia Giordani ◽  
Carlo M. Scornajenghi ◽  
Francesco Marampon ◽  
Antonella Stoppacciaro ◽  
Silvia Di Agostino ◽  

Abstract Background: Human Dachshund homologue 1 (DACH1) is involved in carcinogenesis with opposite roles reported in different tumor types. Four alternatively spliced transcripts encoding different DACH1 isoforms were described but their specific role in human cancers is still unknown. Prostate cancer (PCa) is a heterogeneous disease with a very wide variability, so there is yet a relevant need to find new diagnostic and therapeutic biomarkers to make a safe clinical evaluation. It is well known that the differential expression of protein isoforms can induce distinct transcriptional programs with opposing effects on tumor progression and therapy. Thus, in this study we aimed to correlate the functional role of DACH1 with its splicing variants expression in PCa.Methods: The expression and functional role of DACH1 splicing variants in PCa were investigated using tumor (PC3) and normal (RWPE-1) cell lines, patient biopsies and TCGA dataset. Flow-cytometry, western blots and RT-qPCR were used for in vitro molecular characterization; invasion, adhesion, clonogenic assays and cell cycle analysis for functional characterization. Immunohystochemistry and western blot were performed on human PCa biopsies.Results: RT-qPCR and Western Blot revealed that DACH1-positive PC3 cells predominantly expressed DACH1 variant 4 (DACH1-v4), whereas RWPE-1 cells mostly expressed DACH1 variant 3. Stable DACH1-v4 overexpression enhanced the transformed phenotype of PC3 cells by inducing proliferation, colony formation, invasion ability, epithelial to mesenchymal transition. Given its intrinsic radioresistance, PCa frequently recurs after radiotherapy. Of note, DACH1-v4-overexpressing PC3 cells displayed higher radioresistant behavior. Overexpression of DACH1-v4 also transformed RWPE-1 cells to oncogenic phenotype, suggesting a pro-oncogenic role for this specific isoform. PCa biopsies analysis showed DACH1 nuclear staining enhanced throughout the increase of the tumor grade. Remarkably, tumor glands were found to express a long DACH1 variant, while normal prostate tissue expressed the short DACH1 isoform, in line with data from TCGA-PRAD analysis and our data in RWPE-1 cells. Conclusions: Our findings highlight the oncogenic role of DACH1-v4 in PCa and suggest that the longer DACH1 variants could be associated to pro-tumor function, while the shortest DACH1 variant would perform tumor suppression. The expression of specific DACH1 isoforms could represent a novel diagnostic/prognostic marker in PCa.

2021 ◽  
Vol 11 (1) ◽  
Xin-hui Wang ◽  
Rui Lang ◽  
Qin Zeng ◽  
Ying Liang ◽  
Nan Chen ◽  

AbstractJianpi Qushi Heluo Formula (JQHF) is an empirical traditional Chinese medicine prescription for treating Membranous Nephropathy (MN) clinically in China. The therapeutic effect of JQHF has been reported in our previous studies. However, the exact mechanism is still unknown. In this study, by establishing an experimental rat model of MN induced by Sheep anti-rat Fx1A serum, we evaluated the effects of JQHF and Tetrandrine (TET), and Benazepril was used as a positive control. As an autophagy agonist, TET is one of the most active components in JQHF. After 4 weeks, significant kidney damage was observed in the rats in the Model group; comparatively, JQHF markedly decreased 24 h urinary protein, Total Cholesterol (TC), and increased serum total Albumin (ALB). Histology showed that JQHF caused significant improvements in glomerular hyperplasia, renal tubular damage, IgG immune complex deposition, and the ultrastructure of mitochondria in MN rats. Flow cytometry analysis showed that treatment with JQHF reduced the level of reactive oxygen species and apoptosis rate, and upregulated mitochondrial membrane potential. Western blot analysis demonstrated that JQHF could protect against mitochondrial dysfunction and apoptosis by upregulating the expression of PINK1, Mitochondrial Parkin, and LC3-II/I, downregulating the expression of Cytoplasmic Parkin, P62, Cytochrome c, and Caspase-3 in the kidneys of MN rats. From images of co-immunofluorescence, it is observed significantly increase in the co-localization of PINK1 and Parkin, as well as LC3 and mitochondria. Similarly, TET treatment significantly upregulated the mitochondrial autophagy and reduced apoptosis in rats after 4 weeks compared with the model group. Comparatively, the ability of JQHF to alleviate renal damage was significantly higher than those of Benazepril and TET. It was demonstrated that JQHF could delay pathology damage to the kidney and hold back from the progression of MN by inhibiting apoptosis and upregulating the mitochondrial autophagy by PINK1/Parkin pathways.

2021 ◽  
Zhiwen Wang ◽  
Juan Chen ◽  
Zhuanglin Zeng ◽  
Qing Zhang ◽  
Gaohui Du ◽  

Abstract Objective: Ox-LDL is the core factor in the development of atherosclerosis. However, there are few therapyaimed at eliminating Ox-LDL. Here in this study, we investigate whether the expression of the lectin-like oxidized low density lipoprotein receptor (LOX-1) in the liver could lead to the phagocytosis and degradationof circulating Ox-LDL and prevent the deposition of oxidized lipids in the vascular wall, thereby alleviatingthe progression of atherosclerosis.Methods: ApoE-/- mice were randomly divided into three groups, the control group,the AAV8-TBG-eGFP group and AAV8-TBG-LOX-1 group. In the viral group, mice received an injection of AAV8-TBG-LOX-1 (1.16×1011 virus genome (vg)/animal/100 μl). The mice in the control group and the AAV8-TBG-eGFP group received the same amount of sterile saline and AAV8-TBG-eGFP injections. The expression of LOX-1 in the liver was detected by immunofluorescent, Western blot and immunohistochemistry. The safety was assessed by H&E staining and blood biochemical analyses. The function of LOX-1 in the liver was detected by the co- localization of LOX-1 and Dil-Ox-LDL under laser scanning confocal microscope. The Ox-LDL in plasma was detected by ELISA. Changes in blood lipids were assessed through blood biochemical analysis. The pro- gression of atherosclerotic lesions was detected by oil red O(ORO) staining. And the expression of VCAM- 1 in endothelial cells and the extent of macrophages in plaques were detected by immunofluorescence staining. The protein expression in liver was assessed by qRT-PCR and Western blot. Results: The expression of LOX-1 was stable in liver within 4 weeks. Ectopically expressed LOX-1 in the liver phagocytosed and degraded Ox-LDL and reduced Ox-LDL in circulating plasma but did not have a significant effect on blood lipid levels. After the expression of LOX-1 in liver, Ox-LDL can be cleared by the liver, thereby reducing VCAM-1 expression in vascular endothelium and migration of macrophages in plaques, and eventually alleviating the progression of atherosclerosis. Hepatic LOX-1 expression may facilitate the metabolic clearance of Ox-LDL by upregulating the expression of ABCG5/G8. Conclusions: Ectopic liver-specific expression of LOX-1 alleviates the progression of atherosclerosis by clearing Ox-LDL in circulation.

2021 ◽  
Liang Hao ◽  
Jun Chen ◽  
Xiliang Shang ◽  
Wu Yang ◽  
Shiyi Chen

Abstract BackgroundOsteoarthritis (OA) adversely affects quality of life of elderly patients and is among hotspots and challenges of current research efforts. However, mechanism of occurrence and development of OA has not been fully elucidated. MethodsThrough qRT-PCR and Western blot assays, the current study established that levels of Rock2, a key protein in Rho signaling pathway, were significantly higher in OA cartilage. Furthermore, the current study explored effect of down-regulating Rock2 expression on growth and apoptosis of cartilage cells using CCK-8, Edu and Flow cytometry assays. Alkaline phosphatase (ALP) and Alizarin Red S (ARS) assays were then used to determine differentiation effects.ResultsFindings showed that expression of Rock2 is closely related to proliferation, apoptosis and differentiation of chondrocytes. Furthermore, the current study confirmed that Rock2 affects growth and differentiation of chondrocytes by activating β-catenin signaling pathway. Conclusionthe current study provided novel insights to targeted therapy of OA.

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