Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: ( a ) Eight-to-ten-week-old mice were fed chow or 60% HFD for 16 weeks, messenger RNA (mRNA) was extracted from the indicated tissues, and quantitative RT–PCR was performed for Map4k4 and normalized to 36b4. The data represent the mean±s.e.m. (* P <0.05, ** P <0.005, N =3–7). ( b – d ) Aortas were extracted from age-matched chow-fed wild-type or Apoe − / − mice or WD-fed Apoe − / − mice. ( b ) Immune-complex kinase assays were performed in Map4k4 immunoprecipitates using MBP as an exogenous substrate. Lysates were immunoblotted for tubulin as a loading control. ( c ) Densitometric quantification of 32 P MBP as normalized to tubulin. ( d ) Densitometric quantification of immunoprecipitated Map4k4 as normalized to tubulin (analysis of variance + P =0.05, * P <0.05, N =4–5). ( e ) mRNA was isolated from normal human arteries or atherosclerotic plaques, and quantitative RT–PCR was performed for MAP4K4 or GAPDH (* P <0.05, N =3–5).

Article Snippet: Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific) and immunoprecipitated with Bethyl MAP4K4 antibodies (503 A; 1 μg) or normal rabbit IgG (Cell Signaling 2729; 1 μg).

Techniques: Quantitative RT-PCR, Immune Complex Kinase Assay, Immunoprecipitation, Isolation

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: Map4k4 flox/flox animals were crossed with Cdh5(PAC)-ERT2-Cre animals and injected with tamoxifen for 5 consecutive days at 6–8 weeks of age. ( a ) Cre-mediated Map4k4 exon-7 deletion. ( b ) Schematic of injection and feeding scheme. ( c ) Messenger RNA was extracted and qRT–PCR was performed for Map4k4 in primary MLECs, the unselected, non-EC fraction of mouse lung cells and peripheral blood leukocytes. The data represent the mean±s.e.m. as normalized to 36b4 expression (* P <0.05, N =6–8). ( d ) Immunoblots were performed for Map4k4 or tubulin in immune-selected primary MLECs and the unselected, non-EC fraction of mouse lung cells. Western blots are representative of 6–8 animals per group. ( e – j ) Flox/flox and MAP4K4 ECKO mice were crossed with Apoe − / − mice and fed a WD for 16 weeks as in b . ( e ) Left, Oil Red-O-stained en face aortic preparations from flox/flox and MAP4K4 ECKO animals. Right, quantification of Oil Red-O-stained area. Data represent the mean±s.e.m. (*** P <0.0005, N =8–10). ( f – j ) Aortic root sections of flox/flox and MAP4K4 ECKO Apoe − / − animals stained with ( f ) haematoxylin and eosin (H&E) (scale bar, 250 μm), ( g ) Oil Red-O (scale bar, 250 μm), ( h ) trichrome (scale bar, 250 μm), ( i ) smooth muscle actin (scale bar, 250 μm (top image); scale bar, 100 μm (bottom image)) and ( j ) Cd68 (scale bar, 250 μm (top image); scale bar, 100 μm (bottom image)). Left panel, representative images. Right panels, quantification of stained area or as a percentage of lesion area. Data represent the mean±s.e.m. (* P <0.05, ** P <0.005, *** P <0.0005, N =8–11). DAPI, 4,6-diamidino-2-phenylindole.

Article Snippet: Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific) and immunoprecipitated with Bethyl MAP4K4 antibodies (503 A; 1 μg) or normal rabbit IgG (Cell Signaling 2729; 1 μg).

Techniques: Injection, Quantitative RT-PCR, Expressing, Western Blot, Staining

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: ( a ) Schematic of the transgenic construct used to generate MAP4K4 KD animals. The U6 promoter becomes reconstituted after cre-mediated recombination to drive tissue-specific shRNA expression. ( b , c ) Primary lung endothelial cells (MLECs) were derived from control and MAP4K4 KD animals. ( b ) Messenger RNA (mRNA) was extracted and quantitative RT–PCR was performed for Map4k4 in immune-selected or unselected cells. The data represent the mean±s.e.m. as normalized to 36b4 expression (* P <0.05, N =7). ( c ) Primary endothelial or unselected cell lysates were immunoblotted for Map4k4 and Vegfr2. Data represent the mean±s.e.m. as normalized to tubulin expression (* P <0.05, N =3–6). ( d – g ) MAP4K4 KD mice were crossed with Apoe − / − mice and fed a WD for 16 weeks. ( d ) Aortic root sections of control and MAP4K4 KD animals stained with haematoxylin and eosin (H&E) (top) and Oil Red-O (bottom). Scale bar, 250 μm. ( e ) Quantification of aortic root lesion area. Data represent the mean±s.e.m. (** P <0.005, N =9,11). ( f ) Oil Red-O-stained en face aortic preparations from control and MAP4K4 KD animals. ( g ) Quantification of Oil red-O-stained area. Data represent the mean±s.e.m. (* P <0.05, N =5–7). ( h – i ) mRNA was prepared from whole aortas, and qPCR was performed. ( h ) Macrophage markers F4/80 and Cd68 . ( i ) Chemokines Ccl2 , Cxcl1 , Ccl3 , Ccl4 , Ccl5 , Ccl7 , Cxcl9 and Cxcl10 . Data represent the mean±s.e.m. as normalized to 36b4 (* P <0.05, ** P <0.005, N =9–10).

Article Snippet: Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific) and immunoprecipitated with Bethyl MAP4K4 antibodies (503 A; 1 μg) or normal rabbit IgG (Cell Signaling 2729; 1 μg).

Techniques: Transgenic Assay, Construct, shRNA, Expressing, Derivative Assay, Quantitative RT-PCR, Staining

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: ( a , b ) Messenger RNA was prepared from whole aortas, and qPCR was performed. ( a ) Macrophage markers F4/80 and Cd68 . ( b ) Chemokines Ccl2 , Cxcl1 , Ccl3 , Ccl4 , Ccl5 , Ccl7 , Cxcl9 and Cxcl10 . Data represent the mean±s.e.m. as normalized to 36b4 (* P <0.05, ** P <0.005, N =9–10). ( c , d ) Homing of GFP leukocytes into atherosclerotic lesions 48 h after intravenous injection into control or MAP4K4 KD mice that were fed WD for 16 weeks. ( c ) Fluorescence micrograph of atherosclerotic plaque demonstrating four GFP leukocytes within the aortic arch. The dashed line indicates the plaque border. Inset, magnification of three GFP leukocytes. Left, 4,6-diamidino-2-phenylindole; middle, GFP; right, merge. Scale bars, 100 μm. ( d ) Quantification of GFP leukocytes per square millimetre of plaque. Data represent the mean±s.e.m. (** P <0.005, N =4,7).

Article Snippet: Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific) and immunoprecipitated with Bethyl MAP4K4 antibodies (503 A; 1 μg) or normal rabbit IgG (Cell Signaling 2729; 1 μg).

Techniques: Injection, Fluorescence

Journal: Nature Communications

Article Title: Endothelial protein kinase MAP4K4 promotes vascular inflammation and atherosclerosis

doi: 10.1038/ncomms9995

Figure Lengend Snippet: HUVECs were treated with scrambled or MAP4K4 siRNA, and cells were stimulated with 1 or 10 ng ml −1 TNF-α for the indicated times. ( a ) Cells were seeded onto transwell chambers. Confluent cells were treated overnight with 10 ng ml −1 TNF-α or left untreated, and FITC-labelled dextran that migrated through the HUVEC monolayer was measured. The data represent the mean fluorescence intensity±s.e.m. (analysis of variance (ANOVA) * P <0.05, N =4). ( b ) THP-1 monocytes were stained with calcein green and adhered to activated endothelium for 30 min. Fluorescence microscopy was performed to determine the number of adherent THP-1 monocytes per microscopic field (× 100). The data represent the mean±s.e.m. as normalized to the unstimulated control time point (ANOVA ** P <0.01, **** P <0.0001, N =5). ( c – f ) Messenger RNA was extracted and quantitative RT–PCR was performed for ( c ) MAP4K4 , ( d ) ICAM-1 , ( e ) VCAM-1 and ( f ) SELE . The data represent the mean±s.e.m. as normalized to RPLP0 (* P <0.05, ** P <0.005, # P <0.0001, N =5–7). ( g ) Immunoblots were performed for ICAM-1, VCAM-1 and E-selectin. ( h ) Densitometric analyses from g . The data represent the means±s.e.m. as normalized to VE-cadherin (* P <0.05, N =4–6). ( i ) Biochemical fractionations were performed, and nuclear fractions were immunoblotted for MAP4K4, p-p65, total p65, p50 and lamin-β1. ( j ) Densitometric analyses represent the mean±s.e.m. for the 60-min time point as normalized to lamin-β1 (* P <0.05, ** P <0.005, N =3). ( k ) NFκB-luciferase and SV40-Renilla were transfected into HUVECs after treatment with scrambled or MAP4K4 siRNA. Cells were left unstimulated or stimulated with 10 ng ml −1 TNF-α overnight before luciferase and Renilla measurement. The data represent the mean±s.e.m. from four independent experiments. ( l – n ) HUVECs were transfected with MAP4K4 siRNA and stimulated or not with 1 ng ml −1 TNF-α for 1 h. IgG, p65 and histone antibodies were used to immunoprecipitate chromatin and RT–PCR was used to amplify ( l ) E-selectin, ( m ) VCAM-1 and ( n ) IκBα promoters. The data represent the mean±s.e.m. as normalized to input (* P <0.05, N =3–4).

Article Snippet: Aortas were lysed in 1% NP-40, 50 mM Tris pH 7.4, 150 mM NaCl, 50 mM EDTA with 1 × HALT protease and phosphatase inhibitors (Thermo Scientific) and immunoprecipitated with Bethyl MAP4K4 antibodies (503 A; 1 μg) or normal rabbit IgG (Cell Signaling 2729; 1 μg).

Techniques: Fluorescence, Staining, Microscopy, Quantitative RT-PCR, Western Blot, Luciferase, Transfection, Reverse Transcription Polymerase Chain Reaction

Journal: Clinical Cancer Research

Article Title: Targeted Inhibition of Mammalian Target of Rapamycin Signaling Inhibits Tumorigenesis of Colorectal Cancer

doi: 10.1158/1078-0432.ccr-09-1249

Figure Lengend Snippet: Fig. 1. Expression of mTORC1 and mTORC2 in CRC tissues and cell lines. A and B, immunohistochemical analysis of mTOR, Raptor, Rictor, and pAkt in representative colorectal adenocarcinomas and adjacent normal mucosa (tissue microarray, 10× magnification; n = 45 cases; 90 tumor cores, 8 nonneoplastic cores). C, expression and activation of mTOR signaling pathway components in HCT116, KM20, SW480, and Caco-2 CRC cells treated with 20 nmol/L rapamycin for 24 h.

Article Snippet: Antibodies for mTOR, Raptor, and Rictor were obtained from Bethyl Labs.

Techniques: Expressing, Immunohistochemical staining, Microarray, Activation Assay