pLKO.3G

[Fat 1 inhibits cell proliferation through the ERK signaling pathway in esophageal squamous cell carcinoma]

Elucidation of the mechanism of action of lipid-1 in proliferating esophageal squamous cell carcinoma (ESCC). METHODS: KYSE450 cells were transfected with the plasmid Plko.1-puro-GFP-shRNA-Fat1 and real-time polymerase chain reaction (PCR) was used to verify the efficiency of Fat1 removal. The effects of lipid 1 and the extracellular protein kinase (ERK) pathway inhibitor U0126 on ESCC cell proliferation were revealed by methylthiazolyl tetrazolium (MTT). Colony forming assay was used to detect colony forming ability. The cell cycle was detected using live cell imaging. Western blot was used to monitor the level of the target protein. The murine xenograft assay was applied to detect the effect of Fat1 knockdown on KYSE450 cell tumor growth. Immunohistochemistry was used to detect relevant protein expressions in tumor sections. RESULTS: The removal efficiency of Fat1 was (77.1 ± 6.9%) in the Fat1sh1 group and (77.7 ± 7)% in the Fat1sh2 group. Compared with the control group, cell proliferation and p-ERK1/2 expression were significantly increased in Fat1 sh1 and Fat1 sh2 group (P < 0.05). After U0126 treatment, the effect of Fat1 knockdown on KYSE450 cell proliferation disappeared and the expression of p-ERK1/2 in KYSE450 cells decreased to a level similar to that of the control group. The number of clones in the control group was (72 ± 8), less than (155 ± 28) and (193 ± 9) in the Fat1sh1 and Fat1sh2 groups, respectively (P < 0.05). In the KYSE450 cell, the cleavage time was reduced from 1622 ± 32 min in the control group to 1408 ± 29 min in the Fat1 sh1 group, the difference was statistically significant (P < 0.05). Compared with the control group, the tumor size in the Fat1 knockout group increased significantly. Tumor weight of control group and Fat1 knockout group was (0.224 ± 0.028) g and (1.532 ± 0.196) g respectively after 4 weeks post-inoculation, and the difference was statistically significant (P < 0.05) . ). Conclusion: Lipid-1 inhibits cell proliferation through ERK signaling in ESCC.

Diabetic nephropathy in mice is exacerbated by the absence of IRE1 podocytes and is associated with decreased renal expression of ADH1.

Inositol-requiring enzyme 1 (IRE1) plays an important role in relieving endoplasmic reticulum (ER) stress associated with kidney injury that may also be a factor in diabetic nephropathy (DN). Alcohol dehydrogenase type I (ADH1) activity is prominent in the kidney, and ADH1 activity has also been reported to exert protective effects against ER stress that is not caused by alcohol consumption. However, the role of IRE1 in DN and the relationship between IRE1 and ADH1 remains unclear.
IRE1α knockdown mice (Ire1f/f) were generated from the C57BL/6J background and hybridized with Ire1αf/f mice to produce mice that were cell-specific IRE1α knockdown. Male db/db mice (C57BLKS/J-leprdb/leprdb mice) were used as a DN model. Male mice were made diabetic by injection of streptozotocin. pLKO.1-based vectors encoding the short hairpin RNA (shRNA) of the IRE1α gene were transfected into HEK293T cells to knock out IRE1α in murine cells. ELISA, Masson staining and electron microscopy were performed to analyze the development of DN. ADH1 expression was evaluated by qPCR and western blot.
We found that IRE activity was increased in the glomeruli in DN rat models. In contrast, ADH1 expression was decreased in these models and mice with a cell-specific disorder of IRE1 (PKO mice). Streptococcus-treated diabetic PKO mice showed accelerated proteinuria, enhanced glomerular fibrosis, and podocyte apoptosis. Furthermore, in cultured podocytes, IRE1 knockdown downregulated ADH1 mRNA expression and induced ER stress, consistent with the result from PKO mice, while its deleterious effects were reversed by increased ADH1 expression.
IRE1 activation in podocytes limits DN progression. The dependence of renal expression of ADH1 on the IRE1 podocyte also indicates that ADH1 activity may play an important role downstream of IRE1 in protection against DN.

Serum insulin growth factor-binding protein 2 as a biomarker for thyroid cancer-associated pancreatic adenocarcinoma and muscle atrophy

Malnutrition and muscle wasting are common features frequently seen in pancreatic ductal adenocarcinoma (PDAC) patients with cachexia. They are associated with a lower survival rate and quality of life. Nutritional therapy is an important part of multimodal cancer care in PDAC. However, due to the complexity of nutritional assessment, only 30-60% of patients with nutritional risks currently receive nutritional therapy. It is important to identify biomarkers that can be used to improve the management of malnutrition associated with PDAC. Insulin-like growth factor-binding protein 2 (IGFBP2) has emerged as a potential serum biomarker in a variety of tumors. However, its association with malnutrition and muscle wasting in PDAC is unclear.
We evaluated tumor IGFBP2 expression and serum IGFBP2 level in 98 PDAC patients by immunohistochemistry and enzyme-linked immunosorbent assay and analyzed their relationship. In addition, we explored the relationship between IGFBP2 for tumor, serum, and nutritional status (patient-generated global self-assessment and skeletal muscle index). Stable transfection Pan02 IGFBP2 cell lines, Pan02 PLV-IGFBP2 cells and PLKO-IGFBP2 cells were injected subcutaneously into the flank of C57BL/6 mice. Serum IGFBP2 levels, food intake, and body weight of these mice were measured. The degree of muscle atrophy is characterized by hematoxylin, eosin, oil red O, and Masson’s trichrome staining. The mRNA and protein expression of several essential muscle-associated signaling proteins, such as atrogin-1 and RING finger muscle 1, were measured.
Among all 98 patients, we found that tumor IGFBP2 expression correlated with plasma IGFBP2 levels (rs = 0.562, P < 0.001), and were significantly increased among patients with a patient-generated global self-assessment ≥9 and correlated with overall survival. Furthermore, serum IGFBP2 level was negatively correlated with skeletal muscle index (rs = -0.600, P < 0.001) and Hounsfield units (rs = -0.532, P < 0.001). In mice injected with Pan02 PLV-IGFBP2 cells, the level of circulating IGFBP2 increased while body weight and food intake decreased compared to the Pan02 PLV-Control group. These mice also showed significant worsening of muscle fiber atrophy, fat deposition, increased collagen tissue, and increased expression of mRNA, ER-1, and cyclic muscle-1 in calf muscle. In contrast, these symptoms were alleviated in the PLKO-IGFBP2 group.
In the current study, there is a significant association between serum IGFBP2 levels, malnutrition, and muscular dystrophy in PDAC. Our results suggest that serum IGFBP2 level may be a promising biomarker and targets for intervention for severe malnutrition associated with PDAC and muscle atrophy.

pLKO.3G

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pLKO.1-UFM1 shRNA-7 Plasmid

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SEMA-3G/ Rat SEMA- 3G ELISA Kit

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pLKO.1- pLKO.1- TRC

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Anti-CHO:HRP Conjugate, 3G

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Semaphorin 3G (SEMA3G) Antibody

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Semaphorin 3G (SEMA3G) Antibody

20-abx339745 Abbexa
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Semaphorin 3G (SEMA3G) Antibody

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Semaphorin 3G (SEMA3G) Antibody

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Semaphorin 3G (SEMA3G) Antibody

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pLKO.1-PMP22shRNA4

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pLKO.1-PMP22shRNA2

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pLKO.1- DDit3shRNA1

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Relationship between abnormal expression of central binding factor in human cartilage and osteoarthritis

 

This study aimed to investigate the effect of abnormal BCF expression on chondrocyte proliferation, differentiation, and death, and to elucidate the relationship between BCF-related markers, osteoarthritis, and degenerative joint disease.
Cartilage tissue was collected from healthy subjects and osteoarthritis patients for histology and expression of core binding factor-β, MMP-13, IL-1β, COMP, and YKL-40. Human articular chondrocytes were cultured in vitro and a viral vector was generated to regulate cellular expression of the essential cell attachment factor. Cell proliferation and apoptosis were observed, and the expression of osteoporosis-associated inflammatory factor and the formation of chondrogenic metabolites were examined.
Human osteoporotic lesions caused alteration of cartilage structure and cell arrangement, and increased emptying of cartilage lacunae. The number of normal cells was significantly reduced, the extracellular cartilage matrix was clearly damaged, and the expression of type II collagen was significantly reduced. Nucleus-binding factor-β was highly expressed in osteoarthritic cartilage (P < 0.001), and expression of MMP-13, IL-1β, COMP, and YKL-40 was higher than in normal cartilage (P < 0.001). 0.001). Cell proliferation was reduced in the basal-binding factor high expression group and overall apoptosis rate increased (p < 0.05), while the opposite was found in the basal-binding factor inhibition group (p < 0.05). 0,

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