Journal: Brain : a journal of neurology

Article Title: Connexin43 mimetic peptide reduces vascular leak and retinal ganglion cell death following retinal ischaemia.

doi: 10.1093/brain/awr338

Figure Lengend Snippet: Figure 3 Three hours of hypoxia and 6 h of reperfusion lead to significant endothelial cell death in vitro in rat brain microvascular endothelial cells (RBMVEC; A). Non-specific gap junction blocker carbenoxolone, non-specific hemichannel blocker LaCl3, and connexin43 (Cx43) mimetic peptide protected endothelial cells against hypoxic injury, with the number of viable cells significantly higher than no treatment, while scrambled peptide did not have any protective effects. The number of viable cells was expressed as percentage of the control without hypoxia. Hypoxia and reperfusion also lead to significant propidium iodide dye uptake into primary rat brain microvascular endothelial cells indicating open hemichannels (B). Carbenoxolone, LaCl3 and connexin43 mimetic peptide significantly prevented dye uptake compared to no treatment, indicating hemichannel closure, while scrambled peptide did not have any effect. Stars denote statistical significance when compared to the control group or compared between groups in brackets; P 5 0.05.

Article Snippet: Rat brain microvascular endothelial cells (R840K-05a, Cell Applications) were plated into 24-well plates (1 105 cells/well) in rat brain microvascular endothelial cell growth media (R819K-500, Cell Applications) and allowed to settle for 16 h. Medium was then removed and replaced with Dulbecco’s Modified Eagle’s Medium/F12 containing 0.5% foetal bovine serum and 1% glutamine.

Techniques: In Vitro, Control

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Construction, purification, and characterization of FGF21-164. ( A ) FGF21-164 sequence and FGF21 sequence. In the FGF21-164 sequence, red marked the mutated amino acid and blue represents the TR acid sequence from hCD164. In the FGF21 sequence, the red-labeled amino acid represents the amino acid sequence whose n-terminal is deleted in FGF21-164. ( B ) Subcloned cell culture supernatants expressing FGF21-164 and FGF21 were analyzed using Western blotting (Lane M: molecular weight marker. Lane 1~7: FGF21-164 clones 1, 2, 3, A4, B2, F3, and H4. Line 8: FGF21 clone2). ( C ) The purified FGF21-164 and FGF21 were assayed using SDS-PAGE and Western blotting (Lane M: molecular weight marker. Lane 1: purified FGF21-164. Line 2: purified FGF21). ( D ) Purity and hydrodynamic radius of FGF21-164 were analyzed using SEC-HPLC. ( E ) MALDI-TOF mass-spectrometry analysis of FGF21-164. ([M + H] + denotes the singly charged ionic forms, and [M + 2H] 2+ denotes doubly charged ionic forms). ( F ) Circular Dichroism (CD) spectra of FGF21-164 and FGF21.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Purification, Sequencing, Labeling, Cell Culture, Expressing, Western Blot, Molecular Weight, Marker, Clone Assay, SDS Page, Mass Spectrometry, Circular Dichroism

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Glycan forms analysis of the protein FGF21-164. ( A ) O-linked glycan forms of FGF21-164. ( B ) N-linked glycan forms of FGF21-164.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of FGF21-164 in adipocytes induced by 3T3-L1. ( A ) The process of inducing 3T3-L1’s differentiation into adipocytes. ( B ) Reduction in glucose in medium stimulated by FGF21-164 in adipocytes induced by 3T3-L1. Data are means ± SEM (n = 6). * p < 0.05, *** p < 0.001, and **** p < 0.0001 compared to 0 μg/mL FGF21-164. ( C ) Phospho-Erk1/2-specific bands (Thr202/Tyr204; 44–42 kDa) were detected in 3T3-L1-derived adipocytes after FGF21-164 stimulation. ( D ) Evaluation of lipid droplet accumulation in adipocytes induced by 3T3-L1 with or without FGF21-164. The scale bar represents 50 μm.Data are the means ± SEM (n = 6). * p < 0.05, **** p < 0.0001.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Pharmacokinetics of FGF21-164. FGF21-164 plasma levels in C57BL/6 mice as determined via targeted LC-MS after i.v. (tail vein) bolus injection of FGF21-164 protein.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Liquid Chromatography with Mass Spectroscopy, Injection

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: The results of an oral glucose tolerance test applied to ob / ob mice after a single FGF21-164 treatment. ( A ) Blood glucose levels were measured at 0, 2, 3, 4, 5, 6, 7, and 8 h after the administration of FGF21-164. Data are the means ± SEM (n = 4–5). * p < 0.05 and ** p < 0.01 compared with the model. ( B ) The decrease in blood glucose levels post-administration relative to the baseline measurement at 0 h. Data are the means ± SEM (n = 4–5). * p < 0.05 compared with the model.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of a single administration of FGF21-164 on ob / ob mice. ( A , D , G ) Fasting glucose levels. Blood glucose levels were measured at 7, 21, and 28 days after a single dose of FGF21-164 was administered. Data are the means ± SEM (n = 4–5). ns: no significance. * p < 0.05, ** p < 0.01, and **** p < 0.0001. ( B , E , H ) The OGTT at 7, 21, and 28 days. After oral glucose administration, blood glucose levels were measured. Data are the means ± SEM (n = 4–5). * p < 0.05, *** p < 0.001, and **** p < 0.0001 at 6 mg·kg −1 vs. model. # p < 0.05, ## p < 0.01 and #### p < 0.0001 at 12 mg·kg −1 vs. model. ( C , F , I ) The glucose AUC during the OGTT process. Data are the means ± SEM (n = 4–5). ns: no significance. * p < 0.05, *** p < 0.001. ( J ) Body weight measured at 0, 7, 21, and 28 days. Data are the means ± SEM (n = 4–5). ns: no significance.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: A Novel Recombinant Human FGF21 Analog with High Glycosylation Has a Prolonged Half-Life and Affects Glycemic and Body Weight Control

doi: 10.3390/ijms26062672

Figure Lengend Snippet: Effect of repeated administration of FGF21-164. ( A ) Body weight changes. The DIO mice were treated with PBS or FGF21-164 once every 2 days. Data are the means ± SEM (n = 5–7). **** p < 0.0001 vs. Ctrl. #### p < 0.0001 for FGF21-164 vs. the model. ( B ) The liver sections were stained with Oil-red-O. The scale bar represents 100 μm. DIO mice showed many red lipid droplets in hepatic tissue (indicated by the black arrow), while lipid droplet levels were lower after the FGF21-164 treatment.

Article Snippet: Here, we describe a novel human FGF21 analog, designated as FGF21-164, which was generated through fusion protein technology and co-encoded by a mutated FGF21 gene and the TR gene of hCD164.

Techniques: Staining

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 2 Klf9 regulation of BA synthesis has a nonhepatic basis. a, b Images of gallbladder and BA levels in the gallbladder, faeces and serum of the control (Klf9flox/flox) and liver-specific Klf9-KO (Klf9alb−/−) mice. c mRNA expression levels of Klf9, Cyp7a1, Cyp8b1, Cyp27a1, Shp, Fxr, Oatp, Mdr2, Bsep, Ntcp and Ost-α in the livers of the Klf9flox/flox and Klf9alb−/−mice. Gene expression was normalised to 36B4 expression. d mRNA expression levels of Klf9, Fgf15, Asbt, Fxr, and Ost-α in the intestines of the Klf9flox/flox and Klf9alb−/−mice. Gene expression was normalised to 36B4 expression. e Protein expression levels of Klf9 and Cyp7a1 in the livers of the Klf9flox/flox and Klf9alb−/−mice. Actin served as the loading control. f Protein expression levels of Klf9, Asbt and Fgf15 in the intestines of the Klf9flox/flox and Klf9alb−/−mice. β-actin served as the loading control. Data are represented as the mean ± SEM. **P < 0.01 (b–d).

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Control, Expressing, Gene Expression

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 3 Klf9 affects BA metabolism in the intestine. a Images of gallbladders in the control (Klf9flox/flox) and intestine-specific Klf9-KO (Klf9vil−/−) mice. b BA levels in the gallbladder, serum and faeces of the Klf9flox/flox and Klf9vil−/−mice. c mRNA expression levels of Klf9, Fgf15, Asbt, Fxr and Ost-α in the intestines of the Klf9flox/flox and Klf9vil−/−mice. Gene expression was normalised to 36B4 expression. d mRNA expression levels of Klf9, Cyp7a1, Cyp8b1, Cyp27a1, Shp, Fxr, Oatp, Mdr2, Bsep, Ntcp and Ostα in the liver tissues of the Klf9flox/flox and Klf9vil−/−mice. Gene expression was normalised to 36B4 expression. e Protein expression levels of Klf9 and Cyp7a1 in the intestines of the Klf9flox/flox and Klf9vil−/−mice. β-actin served as the loading control. f Protein expression levels of Klf9, Fgf15, and Asbt in the livers of the Klf9flox/flox and Klf9vil−/−mice. β-actin served as the loading control. Data are represented as the mean ± SEM. *P < 0.05, **P < 0.01 (b–d).

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Control, Expressing, Gene Expression

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 4 Klf9 induces Asbt expression in the terminal ileum. Primary small intestinal epithelial cells (a, b) and Caco-2 cells (c, d) were infected with adenovirus with Klf9 overexpression (Klf9) or Klf9 KO (siKlf9). The mRNA expression levels of Klf9, Asbt, and Fgf15 were analysed by RT- qPCR. Gene expression was normalised to 36B4 expression. Dual-luciferase reporter gene assays of the (e) Fgf15 promoter (–2500 bp) and (f) ABST promoter (−2500 bp) were performed in HEK-293A cells. g The wild-type putative Klf9-binding element and its mutant sequence in the Asbt promoter region. A series of Asbt promoters fused to the luciferase reporter gene (–2500, –1850, –700, and –150 bp) were cotransfected into HEK-293A cells together with Klf9 expression plasmids or pc-DNA3.1 (control, Ctl). h ChIP assays were performed to confirm Klf9 binding to the Asbt gene promoter. Data are represented as the mean ± SEM. *P < 0.05 (a–f).

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Expressing, Infection, Over Expression, Quantitative RT-PCR, Gene Expression, Luciferase, Binding Assay, Mutagenesis, Sequencing, Control

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 5 Klf9 overexpression promotes BA reabsorption. a Images of gallbladders in the control (Rosa) and intestine-specific Klf9- overexpressing (Klf9Rosa+/+) mice. b BA levels in the gallbladder, serum and faeces of the Rosa and Klf9Rosa+/+ mice. c mRNA expression levels of Klf9, Fgf15, Asbt, Fxr and Ost-α in the intestines of the Rosa and Klf9Rosa+/+ mice. Gene expression was normalised to 36B4 expression. d mRNA expression levels of Klf9, Cyp7a1, Cyp8b1, Cyp27a1, Shp, Fxr, Oatp, Mdr2, Bsep, Ntcp and Ost-α in the livers of the Rosa and Klf9Rosa+/+

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Over Expression, Control, Expressing, Gene Expression

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 6 Changes in small intestine morphology. a HE staining of the jejunum, ileum and colon in flox/flox mice, Klf9vil−/−mice, Rosa mice and Klf9Rosa+/+ mice; images are shown at ×200 magnification; scale bars, 100 μm. b Fgf15 protein concentration in blood from the four groups. c Immunohistochemical staining of Asbt in the ileums of the four groups; images are shown at ×400 magnification; scale bars, 50 μm. d Representative immunofluorescence staining of Fgf15 in the intestinal tissues in the four groups; images are shown at ×400 magnification; scale bars, 50 μm. *P < 0.05.

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Staining, Protein Concentration, Immunohistochemical staining

Journal: Acta pharmacologica Sinica

Article Title: Transcription factor Klf9 controls bile acid reabsorption and enterohepatic circulation in mice via promoting intestinal Asbt expression.

doi: 10.1038/s41401-021-00850-x

Figure Lengend Snippet: Fig. 7 Proposed model of Klf9 induction of BAs absorption. Proposed model of Klf9 induction of BA absorption. Klf9 promotes Asbt expression in the ileum, and Asbt promotes BA reabsorption. The reabsorbed BAs activate the Fxr/Fgf15 signalling pathway. Fgf15 is secreted from the intestine to repress Cyp7a1 expression in the liver and reduce BA production.

Article Snippet: A mouse FGF15 ELISA kit (catalogue: CSB-EL522052MO) was purchased from CUSABIO (Wuhan, China).

Techniques: Expressing

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: In vitro analysis also shows a decreased response to hedgehog protein in mouse embryonic fibroblasts (MEFs). Wild-type, but not mutant, MEFs show increased cell growth upon treatment with SHH (A; *p = 0.001 for wt; n = 4). Quantitative RT-PCR analysis also shows a reduced response of Gli1 (B; *p = 0.002 for wt, p = 0.01 for rud ; n = 3) and Patched (C; *p = 0.001 for wt; n = 3) upon SHH treatment in mutant MEFs. β-galactosidase accumulates in SHH-treated wild-type; Ptc-lacZ MEFs, but not rud;Ptc-lacZ MEFs (D; *p = 0.005 for wt; n = 4). (E) PzP53Med ( Ptc-lacZ;p53 medulloblastoma) cells infected and clonally selected with control plKO.1 lentivirus respond to SHH treatment with accumulation of β-galactosidase (* p = 0.07 for wt; n = 4). Two independent clonal lines infected with Hsd17b7 RNAi constructs do not respond as robustly to SHH treatment.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: In Vitro, Mutagenesis, Quantitative RT-PCR, Infection, Control, Construct

Journal: PLoS Genetics

Article Title: Cholesterol Metabolism Is Required for Intracellular Hedgehog Signal Transduction In Vivo

doi: 10.1371/journal.pgen.1002224

Figure Lengend Snippet: Wild-type (A–I) and rudolph (J–R) mutant MEFs were transfected with a SMO-GFP plasmid and acetylated α-tubulin immunocytochemistry was used to identify the primary cilium. SMO-GFP in untreated cells is seen outside the cilium (A–C, J–L), accumulating at the base of the axoneme (not shown), or throughout the cilium (D–F, M–O). Treatment with SHH protein localizes SMO-GFP throughout the cilium in the majority of cells observed (G–I, P–R). All images were taken at the same magnification and the scale bar in C represent 10 µm.

Article Snippet: Mouse embryonic fibroblasts were generated using standard methods and plated at a density of 20,000 cells/cm 2 in the presence or absence of 200 ng/mL SHH amino terminal peptide (R&D Systems).

Techniques: Mutagenesis, Transfection, Plasmid Preparation, Immunocytochemistry