Journal: Scientific Reports

Article Title: Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-β1

doi: 10.1038/srep42487

Figure Lengend Snippet: Endothelial cells transduced with a constitutively active mutant of MEK5 (MEK5D) produce lower level of ( A ) ROS and ( B ) NO metabolites than the vector control (N = 3). TGF-β1 does not alter the effects of ERK5 on downregulating the generation of ROS and NO metabolites (N = 3). ( C ) Activation of ERK5 pathway downregulates the induced expression of SELE, CXCL8 and CCL2 , but does not repress the elevation of ICAM1 and VCAM1 (N = 3). ( D ) ERK5 activation does not suppress the protein expression of ICAM-1 (N = 3). Scale bar represents 50 μm. ( E ) TGF-β1 intensifies the induced expression of VCAM-1 by ERK5 (N = 4). ( F ) Activation of ERK5 pathway suppresses the secretion of IL-8 (N = 4). *p < 0.05, **p < 0.01, ***p < 0.001 & ****p < 0.0001.

Article Snippet: Antibodies against human VCAM-1 (1:100; Santa Cruz Biotechnology, USA; sc-8304), ERK5 (1:500; Upstate Cell Signaling Solutions, USA; #07–039), KLF4 (1:500; Santa Cruz Biotechnology; sc-20691), KLF2 (1:500; Santa Cruz Biotechnology; sc-28675), p-SMAD2 (Ser465/467; 1:200; Cell Signaling Technology, USA; #3108), SMAD2/3 (1:200; Cell Signaling Technology; #3102) and GAPDH (1:2000; Abcam, UK; ab9484) were used for detection of target proteins.

Techniques: Transduction, Mutagenesis, Plasmid Preparation, Control, Activation Assay, Expressing

Journal: Scientific Reports

Article Title: Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-β1

doi: 10.1038/srep42487

Figure Lengend Snippet: ( A ) Immunoblot analyses of ERK5 activation and the expression of KLF2 and KLF4 induced by shear stress and MEK5D mutant (representative blots are shown). ( B ) Shear stress attenuates the phosphorylation of SMAD2 induced by TGF-β1. This suppression is not caused by the reduced level of total SMAD2 (N = 6). ( C ) ERK5 activation under static condition does not suppress the SMAD2 activation. ERK5 activation has no effect on the expression of total SMAD2 (N = 4). ( D ) Shear stress upregulates the expression of SMAD6 and SMAD7 . TGF-β1 has no effects on these upregulations (N = 4). ( E ) ERK5 activation under static condition upregulates the expression of SMAD6 and SMAD7 . TGF-β1 has no effects on these upregulations (N = 2). *p < 0.05, **p < 0.01, ***p < 0.001 & ****p < 0.0001.

Article Snippet: Antibodies against human VCAM-1 (1:100; Santa Cruz Biotechnology, USA; sc-8304), ERK5 (1:500; Upstate Cell Signaling Solutions, USA; #07–039), KLF4 (1:500; Santa Cruz Biotechnology; sc-20691), KLF2 (1:500; Santa Cruz Biotechnology; sc-28675), p-SMAD2 (Ser465/467; 1:200; Cell Signaling Technology, USA; #3108), SMAD2/3 (1:200; Cell Signaling Technology; #3102) and GAPDH (1:2000; Abcam, UK; ab9484) were used for detection of target proteins.

Techniques: Western Blot, Activation Assay, Expressing, Shear, Mutagenesis, Phospho-proteomics

Journal: Scientific Reports

Article Title: Suppression of TAK1 pathway by shear stress counteracts the inflammatory endothelial cell phenotype induced by oxidative stress and TGF-β1

doi: 10.1038/srep42487

Figure Lengend Snippet: ROS derived from mitochondria reduce NO bioavailability, impair NO-mediated signalling and induce the activation of inflammatory signalling, such as NFκB and p38 MAPK pathways. Shear stress attenuates ROS generation and increases NO bioavailability via the ERK5 pathway. Shear stress represses TGF-β1-induced inflammatory activation by suppressing p38 MAPK and NFκB signalling which act downstream of TAK1, in a manner independent of ERK5, redox poise and ALK5 kinase activity. Shear stress may attenuate the canonical ALK5/SMAD pathway via the upregulation of inhibitory SMADs, i.e. SMAD6 and SMAD7.

Article Snippet: Antibodies against human VCAM-1 (1:100; Santa Cruz Biotechnology, USA; sc-8304), ERK5 (1:500; Upstate Cell Signaling Solutions, USA; #07–039), KLF4 (1:500; Santa Cruz Biotechnology; sc-20691), KLF2 (1:500; Santa Cruz Biotechnology; sc-28675), p-SMAD2 (Ser465/467; 1:200; Cell Signaling Technology, USA; #3108), SMAD2/3 (1:200; Cell Signaling Technology; #3102) and GAPDH (1:2000; Abcam, UK; ab9484) were used for detection of target proteins.

Techniques: Derivative Assay, Activation Assay, Shear, Activity Assay

Journal: Angiogenesis

Article Title: The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence

doi: 10.1007/s10456-015-9485-2

Figure Lengend Snippet: Primer sequences used in the study

Article Snippet: The following antibodies were used: NOS3/eNOS (1:1000, BD Biosciences, San Jose, CA, USA, 610299), MAPK7/Erk5 (1:500, Upstate/Merck Millipore, Billerica, MA, USA, 07-039), EZH2 (1:1000, Cell Signaling, Danvers, MA, USA, 5246), GAPDH (1:1000, Abcam, Cambridge, UK, ab9485 or ab9484), KLF2 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-28675), KLF4 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-20691), cyclin A (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-751), cyclin B1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-s45), cyclin E (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-247), anti-rabbit IgG IRDye-680LT (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-68021), and anti-mouse IgG IRDye-800CW (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-32210).

Techniques: Sequencing

Journal: Angiogenesis

Article Title: The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence

doi: 10.1007/s10456-015-9485-2

Figure Lengend Snippet: Protein expression of EZH2 is decreased along MAPK7 activation. The constitutively active MEK5 mutein (MEK5D) was expressed in HUVEC. a Representative western blotting images. b Protein expression of EZH2 in cells expressing MEK5D, obtained through the densitometry of the western blotting data, n = 6, * p < 0.05, t test and c gene expression of EZH2 in cells expressing MEK5D, n = 4

Article Snippet: The following antibodies were used: NOS3/eNOS (1:1000, BD Biosciences, San Jose, CA, USA, 610299), MAPK7/Erk5 (1:500, Upstate/Merck Millipore, Billerica, MA, USA, 07-039), EZH2 (1:1000, Cell Signaling, Danvers, MA, USA, 5246), GAPDH (1:1000, Abcam, Cambridge, UK, ab9485 or ab9484), KLF2 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-28675), KLF4 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-20691), cyclin A (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-751), cyclin B1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-s45), cyclin E (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-247), anti-rabbit IgG IRDye-680LT (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-68021), and anti-mouse IgG IRDye-800CW (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-32210).

Techniques: Expressing, Activation Assay, Western Blot

Journal: Angiogenesis

Article Title: The decrease in histone methyltransferase EZH2 in response to fluid shear stress alters endothelial gene expression and promotes quiescence

doi: 10.1007/s10456-015-9485-2

Figure Lengend Snippet: EZH2 levels determine the activation capacity of MAPK7. a Gene expression of MAPK7 in cells depleted of EZH2. Anti-EZH2 shRNA was expressed in HUVEC for 7 days by means of lentiviral delivery. Scrambled shRNA was used as control, n = 3. b Representative images of western blotting showing the enhanced activation of MAPK7 in static conditions upon 7-day knockdown of EZH2 in HUVEC. c Densitometry results showing the enhanced activation of MAPK7 in static conditions upon the knockdown of EZH2, derived from the western blotting data, normalized to β-actin (ACTB), n = 3, ** p < 0.01, t test. d Representative western blotting images, showing enhanced activation of MAPK7 in EZH2-depleted cells compared to control, both in static and in FSS-exposed cultures. Dashed line indicates where the images were artificially connected: they are parts of the same membrane (one image) and were only moved to depict the lanes in the order which is easier for interpretation. Control and EZH2-depleted HUVEC were cultured under FSS for 3 days. e Densitometry results of the western blotting data showing the protein expression levels of EZH2, n = 3, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s post hoc comparisons between all pairs of means. f , g Densitometry results of the western blotting data showing the total phosphorylation levels of MAPK7 (normalized to GAPDH) and the ratio of phosphorylated MAPK7 to total expressed MAPK7 (both normalized to GAPDH), respectively, n = 3, ** p < 0.01, *** p < 0.001, one-way ANOVA with Tukey’s post hoc comparisons between all pairs of means. h Representative western blotting images showing rapid dephosphorylation of MAPK7 upon the cessation of the flow. Cells were cultured for 72 h in static conditions or under FSS; afterwards one group was kept for an additional 1 h in static conditions before cells lysis (“1-h stop FSS”) and i , j Densitometry results showing the levels of MAPK7 phosphorylation and EZH2 protein expression, respectively, normalized to GAPDH. The “stop” caption refers to the 1-h stop FSS condition (see h ), n = 3, *** p < 0.001, one-way ANOVA with Tukey’s post hoc comparisons between all pairs of means

Article Snippet: The following antibodies were used: NOS3/eNOS (1:1000, BD Biosciences, San Jose, CA, USA, 610299), MAPK7/Erk5 (1:500, Upstate/Merck Millipore, Billerica, MA, USA, 07-039), EZH2 (1:1000, Cell Signaling, Danvers, MA, USA, 5246), GAPDH (1:1000, Abcam, Cambridge, UK, ab9485 or ab9484), KLF2 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-28675), KLF4 (1:250, Santa Cruz Biotechnology, Dallas, TX, USA, sc-20691), cyclin A (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-751), cyclin B1 (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-s45), cyclin E (1:500, Santa Cruz Biotechnology, Dallas, TX, USA, sc-247), anti-rabbit IgG IRDye-680LT (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-68021), and anti-mouse IgG IRDye-800CW (1:10 000, Li-COR Biosciences, Lincoln, NE, USA, 926-32210).

Techniques: Activation Assay, Expressing, shRNA, Western Blot, Derivative Assay, Cell Culture, De-Phosphorylation Assay, Lysis