Journal: Cells

Article Title: Epiregulin as an Alternative Ligand for Leptin Receptor Alleviates Glucose Intolerance without Change in Obesity

doi: 10.3390/cells11030425

Figure Lengend Snippet: EREG mediates glucose uptake via PI3K with transient activation of ERK. ( A ) FD-glucose uptake in 3T3-L3 preadipocytes treated with or without EREG (50 ng/mL) and in the presence of inhibitors for EGFR-I (AG1478, 10 µM), EGFR and ErbB2 (AST-1306 or CI-1033 10 µM), dual IR/IGF-1R inhibitor (BMS 536924, 1 µM), and SRC-I, AZM475271, 1 µM) for 30 min. Cells were starved for 90 min before stimulation. Dashed line shows glucose uptake in the presence of insulin (Ins, 10 µg/mL). ( B ) FD-glucose uptake was measured in mouse 3T3-L1 preadipocytes with or without EREG (50 ng/mL) and inhibitors of MEK1/2 and PI3K (MEK1/2-I, U0126 10 μM, and PI3K-I, wortmannin 200 nM). Data (mean ± SD, n = 6) are shown as a percentage of control (Veh 100%). Unpaired Student’s t -test. ( C ) 3T3-L1 preadipocytes were stimulated with EREG at different concentrations (0–100 ng/mL) for 5 or 15 min. The total and phosphorylated levels of AKT, STAT3, STAT5, and ERK were measured by Western blot in duplicates. Data are shown in a representative Western blot. ( D ) The kinetics of pERK expression was quantified based on the Western blots. pAKT, p-STAT3, and p-STAT5 analysis are described in . Pearson correlation analysis. ( E ) 3T3-L1 preadipocytes were stimulated with or without EREG or EGF (50 ng/mL, each) for 30 min in the presence and absence of EGFR inhibitor AST1306 (100 nM), and antibody against mouse LepR (Invitrogen, PA1-053, 10 μg/mL). For inhibition, cells were pre-treated 30 min before EREG and EGF stimulation. ( F ) FD-glucose uptake was measured in mouse 3T3-L3 preadipocytes pre-treated with either Veh (DMSO) or ERK inhibitors (U0126, SCH772984, or DEL 22379, each 10 µM in DMSO) for 40 min. Then, cells were treated with either Veh (PBS), mouse EREG (50 ng/mL), or mouse leptin (Lep, 200 ng/mL) for 80 min. Data are shown as a percentage of Veh-treated control (100%, n = 7 per group). Unpaired Student’s t -test. ns , not significant ( p > 0.05).

Article Snippet: Antibodies against mouse AKT (4691S), phosphorylated AKT (p-AKT, 9271S), STAT5 (94205S), p-STAT5 (4322S), STAT3 (9139S), ERK (4696S), and p-ERK (4370S) were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Control, Western Blot, Expressing, Inhibition

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutive activation of JAK2 in the mammary gland increases proliferation during pregnancy and overactivates Stat5 during gestation and lactation. (a) Ki67-positive cells (red) and (b) p-Stat5-positive cells (red) in the mammary glands of JAK2 V617F (control, left) or K14-Cre;JAK2 V617F (right) females at 10 days gestation. E-cadherin staining was used in (a) for visualisation of epithelial cells. Nuclei are in blue. Scale bar: 20 μm. Quantification of Ki67-positive cells (a, n=3 for each genotype) and p-Stat5-positive cells (b, n=5 for each genotype) in ducts and alveoli are shown in the corresponding graphs. (c) Western blot analysis of p-Stat5, p-Stat3 and p-Stat6 in the mammary glands of K14-Cre;JAK2 V617F and control mice (JAK2 V617F and K14 Cre;JAK2 wt) at 24 h lactation (first day post-partum). Total Stats were used for normalisation. Quantification of p-Stat5 signal compared to total Stat5 is shown in (d). Results are mean±S.D. *P<0.05, **P<0.01 versus control mice

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Activation Assay, Staining, Western Blot

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 in mammary epithelial cells induces Stat5 activation. (a) Representative immunofluorescence staining for JAK2 (red) in KIM-2 cells overexpressing JAK2 wt or JAK2 V617F. MIG (MSCV-IRES-GFP) represents cells carrying the empty vector. Nuclei are in blue. Scale bar: 50 μm. (b) Western blot analysis of JAK2 and the phosphorylation of different Stats in KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F. β-Actin and total Stats were used for normalisation. (c) Western blot of JAK2, Stat5 and p-Stat5 in cytosolic and nuclear protein extracts in MIG-, JAK2 wt- and JAK2 V617F-KIM-2 cells. α-tubulin and lamin A/C were used as markers of cytosolic and nuclear fractions, respectively. Experiments were carried out at least twice for two independent sets of retroviral infections

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Activation Assay, Immunofluorescence, Staining, Plasmid Preparation, Western Blot

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 increases the expression of differentiation markers in mammary epithelial cells in vitro. (a) β-Casein and whey acidic protein (WAP) mRNA expression in cells transiently or stably transduced with empty vector (MIG), JAK2 wt or JAK2 V617F was determined by RT-PCR (representative experiment, n=2). Cyclophilin A was used for normalisation. (b–d) Western blot analysis of β-casein (b), WAP (c), p-Stat5 and Elf5 (d) in KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F and incubated with lactogenic hormones for the indicated times (representative images, n=3). β-Actin or total Stat5 were used for normalisation

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Expressing, In Vitro, Stable Transfection, Transduction, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Incubation

Journal: Cell Death and Differentiation

Article Title: Constitutive activation of JAK2 in mammary epithelium elevates Stat5 signalling, promotes alveologenesis and resistance to cell death, and contributes to tumourigenesis

doi: 10.1038/cdd.2011.122

Figure Lengend Snippet: Constitutively active JAK2 enhances mammary tumourigenesis. (a) Morphology of KIM-2 cells stably expressing empty vector (MIG), JAK2 wt or JAK2 V617F. Scale bar: 100 μm. (b) Number of colonies in soft agar formed by KIM-2 cells harbouring MIG, JAK2 wt or JAK2 V617F after 3 weeks at the indicated temperatures. Bars represent mean±S.D. of three independent experiments. **P<0.01 versus JAK2 V617F-KIM-2 cells. (c) JAK2 mRNA expression in MCF-7 cells stably transduced with empty vector (MIG), JAK2 wt or JAK2 V617F was determined by RT-PCR (representative experiment, n=2). GAPDH mRNA expression was used for normalisation. (d) Western blot analysis of the phosphorylation of Stat5 in MCF-7 cells harbouring MIG, JAK2 wt or JAK2 V617F (representative images, n=2). Total Stat5 was used for normalisation. (e) Final tumour volume of MIG-, JAK2 wt- and JAK2 V617F-MCF-7-derived xenografts. Mean of each experimental group (n=5) is represented with a horizontal bar and P-values versus JAK2 V617F-MCF-7 xenografts are indicated. (f) Images of MIG-MCF-7- and JAK2 V617F-MCF-7-derived xenografts 6 weeks after the injection of the cells. (g) Representative H&E sections from MIG-MCF-7- and JAK2 V617F-MCF-7-derived xenografts showing well-defined tumour boundary in MIG-MCF-7-derived tumours and invasive clusters of neoplastic cells (marked by an arrow) in JAK2 V617F-MCF-7-derived tumours. Dashed lines indicate tumour margins. Scale bar: 300 μm

Article Snippet: Primary antibodies against E-cadherin (BD Biosciences), Ki67 (Leica), p-Stat5 and JAK2 (Cell Signaling Technology, Danvers, MA, USA), CK-14 (Abcam, Cambridge, UK), CK18 (Progen, Heidelberg, Germany) and β -casein (kindly donated by Bert Binas, Texas A & M University, College Station, TX, USA) were used.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transduction, Reverse Transcription Polymerase Chain Reaction, Western Blot, Derivative Assay, Injection

Journal: Journal of Virology

Article Title: GCRV-II invades monocytes/macrophages and induces macrophage polarization and apoptosis in tissues to facilitate viral replication and dissemination

doi: 10.1128/jvi.01469-23

Figure Lengend Snippet: Polarization of M1 and M2 macrophages in vitro. (A) SDS-PAGE analysis of purified recombinant intact CiIFN-γ2 (17.6 kDa), CiIL-4/13A (17.2 kDa), and CiIL-4/13B (17.1 kDa) proteins. (B and C) Measurement of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B protein activity by determining the phosphorylation levels of STAT5, ERK, and mTOR. (C) Measurement of phosphorylation levels of STAT5, ERK, and mTOR in three separate experiments by determining the gray value of the WB bands of CiIFN-γ2, CiIL-4/13A, and CiIL-4/13B proteins using the ImageJ software. (D and E) Verification of M1 macrophage polarization by mRNA expression levels of IL-1β, CXCR 3.1, and iNOS (D), as well as enzyme activity of iNOS (E). (F and G) Verification of M2 macrophage polarization by mRNA expression levels of IL-10, CXCR 3.2, and arginase-2 (F), as well as arginase-2 enzyme activity (G). Relative mRNA expression level was normalized to that of EF1a (n = 3) (*P < 0.05 and **P < 0.01).

Article Snippet: Other antibodies used in the study included β-actin mouse mAb (1:10,000, AC004, ABclonal), arginase-2 rabbit pAb (1:500, A6355, ABclonal), iNOS rabbit mAb (1:500, A3774, ABclonal), Bcl-2 rabbit pAb (1:5,000, A0208, ABclonal), HRP rabbit anti-goat IgG (1:5,000, AS029, ABclonal), p-STAT5 rabbit mAb (1:2,000, AP0758, ABclonal), p-ERK rabbit pAb (1:500, AP0472, ABclonal), and p-mTOR rabbit pAb (1:500, AP0094, ABclonal).

Techniques: In Vitro, SDS Page, Purification, Recombinant, Activity Assay, Software, Expressing