Gormsenschmitt4518

GST pull-down assay revealed that GST-IGF1R β-PKD could interact with MEMO in vitro.

GST-IGF1R β-ED and GST-IGF1R β-PKD were successfully cloned and purified. In addition, GST-IGF1R β-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.

GST-IGF1R β-ED and GST-IGF1R β-PKD were successfully cloned and purified. In addition, GST-IGF1R β-PKD could interact with MEMO in vitro, which demonstrated the good activity of the purified proteins.

To explore the feasibility of applying molecular chaperones to the soluble expression of e23sFv/His fusion proteins.

The molecular chaperone plasmid pGro7 or pKJE7 was transformed into BL21 (DE3) competent cells together with the prokaryotic expression vector harboring His-tagged e23sFv. The soluble expression of e23sFv/His proteins was induced at 16 °C. The yield and antigen-binding activity of the soluble products were compared with those of the insoluble products conventionally purified from inclusion bodies.

Both the overall yield and the purification ratio of soluble e23sFv/His proteins were relatively lower. The binding affinity of the soluble products to immobilized HER2 was not superior to that of the insoluble products from inclusion bodies.

The molecular chaperone plasmids pGro7 and pKJE7 partially facilitate the soluble expression of e23sFv/His proteins, but both the yield and the purification ratio are still limited.

The molecular chaperone plasmids pGro7 and pKJE7 partially facilitate the soluble expression of e23sFv/His proteins, but both the yield and the purification ratio are still limited.

To design a biphasic pulsed electrical stimulation (BPES) system for cardiac tissue engineering and investigate the effect of BPES on the cardiomyocyte-like differentiation of adipose-derived stem cells (ADSCs) in vitro.

ADSCs were isolated and cultivated from Sprague Dawley rats. Surface protein expression was detected by flow cytometry to identify the cell phenotype. The multi-differentiation potential of ADSCs was verified by adipogenic and osteogenic inducers. The cells of the third passage were randomly divided into 2 groups BPES group was subjected to BPES 24 hours after the cells were plated in 6-well culture plates, and BPES was set as 2 ms square pulses delivered at 2 Hz, 2 V voltage amplitude, and lasted 2 hours per day, for 3, 4 or 7 days. The culture medium was replaced every 3 days. Control group was cultured with the same condition but without BPES. The cell morphology was observed under an inverted phase-contrast microscope. Cell proliferation was evaluated by the MTT assay. The expression of cardiac-specific homeobox Nkx-2.5 and connexin 43(CX43) was determined by immunofluorescence cytochemistry.

The flow cytometry proved that the isolated cells were ADSCs. Oil red O staining showed the fat droplets in the cytoplasm of ADSCs after adipogenic induction. alkaline phosphatase and Von Kossa staining showed calcium nodes in the ADSCs after osteogenic induction. No obvious changes were found in the proliferation of ADSCs. The levels of Nkx-2.5 and CX43 proteins were significantly higher in the BPES group than in the control group.

BPES system we designed for tissue engineering could promote the differentiation of ADSCs into cardiomyocyte-like cells in vitro.

BPES system we designed for tissue engineering could promote the differentiation of ADSCs into cardiomyocyte-like cells in vitro.

To observe the effect of metastasis-associated gene 1 (MTA1) on the growth and metastasis of laryngeal squamous cell carcinoma in nude mice using RNA interference technology (RNAi).

Lentiviral vector of short hairpin RNA (shRNA) targeting MTA1 was constructed and packaged to transfect Hep-2 cells. Hep-2 cells transfected with scramble shRNA and MTA1 shRNA were injected into the paw pad of nude mice (n=5 per group). Nine weeks after modeling of the lymphatic metastasis of laryngeal carcinoma, the mice were sacrificed, and tumor tissues and inguinal lymph node were harvested to be subjected to HE staining, reverse transcription PCR and Western blotting.

The gene screening showed the lentiviral vector of MTA1 shRNA was constructed successfully, and those tumor cells were transplanted and grew well in all mice. The size of tumor in the mice of MTA1 shRNA tansfected group was obviously smaller compared to the scramble shRNA transfected group at the same time point. No inguinal lymph node metastasis was found in the mice of MTA1 shRNA group. In contrast, the tumor cells were seen in the inguinal lymph nodes of the scramble shRNA infected mice. Reverse transcription PCR and Western blotting showed that the mRNA and protein levels of MTA1, β-catenin, matrix metalloproteinase-9 (MMP-9), cyclin D1 were obviously reduced in MTA1 shRNA infected mice compared with the scramble shRNA infected mice.

The inhibition of MTA1 gene could depress the growth and metastasis of laryngeal squamous cell carcinoma in nude mice.

The inhibition of MTA1 gene could depress the growth and metastasis of laryngeal squamous cell carcinoma in nude mice.

To construct a lentiviral vector (Lenti-GPER-shRNA) targeting G-protein coupled estrogen receptor (GPER) and explore the role of GPER in the effect of tamoxifen on cell proliferation and apoptosis in breast cancer associated fibroblasts (BCAFs).

The target sequence of GPER gene and negative control were cloned into lentiviral vectors. The recombinant lentivirus and control were extracted after HEK293T cells were transfected with the recombinant vector and helper vectors. After infection of BCAFs with the GPER lentiviral vector under the best interfering condition, GPER expression was detected by real-time quantitative PCR and Western blotting. BCAFs were divided into negative control group, GPER-RNAi group, negative control combined with tamoxifen (10(-8) mmol/L) group and GPER-RNAi combined with tamoxifen (10(-8) mmol/L) group. CCK-8 assay was used to detect the proliferation and annexin V-fluorescein isothiocyanate/propidium iodide (annexin V-FITC/PI) combined with flow cytometry was used to detect the apoptosis of BCAFs after the treatment of tamoxifen.

Lenti-GPER-shRNA significantly interfered the expression of GPER in BCAFs. Tamoxifen promoted the growth of BCAFs, which could be attenuated by knockdown of GPER. Moreover, the apoptosis of BCAFs was reduced by tamoxifen, which was also reversed by knockdown of GPER.

Lenti-GPER-shRNA could effectively silence the GPER expression in BCAFs. The ability of tamoxifen to accelerate cell proliferation and decrease cell apoptosis could be weakened by knockdown of GPER.

Lenti-GPER-shRNA could effectively silence the GPER expression in BCAFs. The ability of tamoxifen to accelerate cell proliferation and decrease cell apoptosis could be weakened by knockdown of GPER.

To investigate the expression of miR-199a-5p in airway smooth muscle cells (ASMCs) of rats in normoxia and hypoxia and its role in the regulation of proliferation and the expression of hypoxia inducible factor 1 alpha (HIF-1α) of ASMCs.

ASMCs were prepared by means of adherent culture in vitro. After ASMCs were cultured under normoxia and hypoxia conditions for 24 hours, the content of miR-199a-5p was detected by real-time quantitative PCR (qRT-PCR). The mimic or inhibitor of miR-199a-5p were artificially synthesized and transferred into ASMCs in hypoxia via liposomes. The expressions of miR-199a-5p and HIF-1α mRNA were detected by qRT-PCR. Western blotting and CCK-8 assay were applied to detect the expression levels of HIF-1α protein and the proliferation of ASMCs, respectively.

Compared with the normoxia group, hypoxia significantly promoted cell proliferation and increased the levels of HIF-1α mRNA and protein. The level of miR-199a-5p decreased in the hypoxia group compared with the normoxia group. The proliferation rate of ASMCs under hypoxia conditions was significantly attenuated by transfection of miR-199a-5p mimic, while it was significantly lifted by transfection of miR-199a-5p inhibitor. Compared with control group, the expression of HIF-1α protein was reduced in the mimic group and raised in the inhibitor group. There was no significant difference in the content of HIF-1α mRNA among groups under hypoxia conditions.

miR-199a-5p can inhibit the proliferation of ASMCs and the expression of HIF-1α protein in vitro under hypoxia conditions.

miR-199a-5p can inhibit the proliferation of ASMCs and the expression of HIF-1α protein in vitro under hypoxia conditions.

To explore the effects of interleukin 18 (IL-18) and tumor necrosis factor alpha (TNF-α) on the expression of IL-18 receptor beta (IL-18Rβ) in 3T3-L1 adipocytes and RAW264.7 macrophages.

The level of IL-18Rβ was determined by reverse transcription PCR and Western blotting in both 3T3-L1 adipocytes and RAW264.7 macrophages after processed by different concentrations of IL-18 and TNF-α.

The level of IL-18Rβ mRNA and protein significantly increased in 3T3-L1 adipocytes after treated with 10 ng/mL, 100 ng/mL TNF-α. The expression of IL-18Rβ was also raised in RAW264.7 macrophages after treated with 10 ng/mL, 100 ng/mL IL-18.

IL-18Rβ could be up-regulated by IL-18 or TNF-α in 3T3-L1 adipocytes and RAW264.7 macrophages. click here IL-18 may be an important factor involved in the inflammation of adipose tissues in diabetes and obesity.

IL-18Rβ could be up-regulated by IL-18 or TNF-α in 3T3-L1 adipocytes and RAW264.7 macrophages. IL-18 may be an important factor involved in the inflammation of adipose tissues in diabetes and obesity.

To establish murine macrophage RAW264.7 cell strain with stable expression of red fluorescent protein-green fluorescent protein-microtubule associated protein light chain 3 (RFP-GFP-LC3).

A lentiviral vector containing RFP-GFP-LC3 gene was constructed and then packaged in HEK293T cells with the packaging plasmids. The viral supernatant was collected to infect RAW264.7 cells. The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 was screened with puromycin and analyzed with flow cytometry and fluorescent microscopy for infection efficiency. The number of RFP-GFP-LC3 puncta was observed using florescence microscopy following starvation treatment.

The recombinant lentivirus pLV-CMV-RFP-GFP-LC3 was successfully constructed. The RAW264.7 cells with stable expression of RFP-GFP-LC3 were obtained by viral infection and puromycin screening. Fluorescent microscopy and flow cytometry demonstrated the expression rates of RFP and GFP reached to 100%. The number of autophagic puncta significantly increased after starvation treatment.

The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

The RAW264.7 cell strain with stable expression of RFP-GFP-LC3 has been successfully constructed, which provides a reliable cellular platform for autophagy research.

To investigate the expressions of Toll-like receptor 4 (TLR4), myeloid differentiation factor 88 (MyD88) and nuclear factor κBp65 (NF-κBp65) in the renal tissues of obese mice induced by high-fat diet (HFD).

Forty-five C57BL/6 male mice (8 weeks old) were randomized into HFD group (n=39) and control group (n=6). The mice of the HFD group were fed high-fat and high-glucose diet for 20 weeks, and the others were given the normal diet instead. The body mass was measured weekly. At the end of the 20th week, one-third mice of the HFD group with the highest mass gain were classified as diet-induced obese (DIO) mice (n=13). The DIO mice and control mice were then sacrificed and tissues were collected for further use. The white adipose tissues (epididymal and perirenal fat pads) were removed and weighed. The levels of the serum creatinine (Cre), blood urea nitrogen (BUN) and lipopolysaccharide (LPS) were measured. The morphology of the renal tissues in both groups was studied by HE staining and microscopy. The levels of TLR2, TLR4, MyD88, NF-κBp65 and tumor necrosis factorα (TNF-α) mRNAs in the renal tissues were detected by the real-time quantitative PCR (qRT-PCR), and the levels of TLR4, MyD88 and NF-κBp65 proteins were determined by Western blotting.