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5 Great Post-Workout Snacks
Stress Eaters Beware
4 Weird Tricks to Feel Fuller
Secrets to Losing Weight and Keeping it Off
I'll Have What You're Having
Bone Building Nutrients
Weekend Splurging Done Right
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5 Great Post-Workout Snacks
If immediately after any workout, you reach for the next best snack, gulp down a big sugary drink or treat yourself to fatty foods, you may get instant gratification, but ...
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Personal Training
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Visualization Leads to Actualization
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Tips to Reach Your Fitness Goals for 2016
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Putting Positive Thinking Into Practice
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Benefits of a Sound Sleep
How Music Can Enhance Your Workout
Music fuels your workouts. It's no wonder many fitness buffs and endurance athletes put their headphones on and tune the world out during physical activity. No matter what their training ...
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Group Fitness 101
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The 34th Annual Fred & Adele Astaire Awards, which recognizes outstanding achievements in dance on Broadway and in film, takes place on Monday, May 16, 2016. (more...)
Paper Mill Playhouse is hosting its largest fundraising event of the year, the Paper Mill Playhouse Gala on Friday, May 6, at the renowned Hilton Short Hills. The reception and silent auction begin at 6:00pm followed by dinner and dancing featuring The Eddie Bruce Band. (more...)
MASTERWORKS BROADWAY proudly announces the release of the Original Broadway Cast Recording of ON YOUR FEET!. (more...)
Academy Award nominated actor, screenwriter and producer, Chazz Palminteri, will be joining Broadway Theatre Project's 2016 season for the first time as a guest artist. Gianna Palminteri, Chazz' wife, writer, actor, producer and vocal technique teacher, will also be joining Broadway Theatre Project. (more...)
Artistic Director Eric Schaeffer and Managing Director Maggie Boland announce the full lineup for Signature Theatre's 2016/17 season, including the previously announced production of Tom Kitt and Brian Yorkey's FREAKY FRIDAY. (more...)
Santa Susana High School, a award winning performing arts high school in Simi Valley, California presents: The Addams Family. Presented in our state of the art Performing Arts Center, the dates for the 6 show run are April 22, 23, 29 and 30. Santa Susana High School continues their tradition of producing top quality productions utilizing student producers, co directors, production orchestra and a student crew for sound, set, stage and tech support all led by our amazing teaching staff. In addition, this cast contain two actors nominated for 2015 Jerry Herman Awards (Grant Measures and Josh Ponce.) This is a can't miss event! (more...)
Just announced, the upcoming National Tour of SOMETHING ROTTEN will officially launch as part of Broadway In Boston's 2016-17 season! (more...)
The East Jefferson General Hospital Broadway in New Orleans 2016-17 season was announced today. Presented by the New Orleans Theatre Association, the new season will commence at the historic Saenger Theatre in fall 2016. (more...)
Past Washington appearances of note include performing on 'A Capitol Fourth' on PBS-TV in 2009 and at the National Christmas Tree Lighting Ceremony in 2011. (more...)
?Following a smash-hit run in Toronto, TRUWORTHY PRODUCTIONS (Sergio Trujillo & Jack Noseworthy) is thrilled to announce the South American Premiere of the musical ARRABAL at the Ibero-American Theatre Festival, for five performances this week from March 11-14, 2016. Directed and co-choreographed by Olivier Award winner Sergio Trujillo (On Your Feet!, Jersey Boys, Memphis), with music by Academy Award winner Gustavo Santaolalla (Brokeback Mountain, Babel) and a book by Tony Award nominee John Weidman (Contact, Assassins), ARRABAL is a rock 'n' roll-meets-tango dance musical-a unique piece of sexy, high energy theater which tells a powerful story, both personal and political, almost entirely through propulsive music and movement. (more...)
Gloria Estefan has sold over 100 million records and sold out stadiums around the world, they're about to sell some more. BroadwayWorld has just confirmed that the cast on ON YOUR FEET! will record a live album today. There's no word yet on when the album will be available, but check back here at BroadwayWorld for updates! (more...)
The New York Musical Festival (NYMF), previously known as The New York Musical Theatre Festival, announced today the initial lineup for NYMF 2016. Among the shows announced in the lineup are the Next Link Project musical selections, as well as invited production selections, developmental reading selections, and concerts. Now in its thirteenth year, the 2016 Festival will take place in July at locations around midtown Manhattan in New York City. (more...)
Jon Rua received the call that he would be going on as Alexander Hamilton the morning of the show. Without a proper put-in rehearsal, he rushed to the Richard Rodgers to take on the title role of the hottest ticket in town for the first time. (more...)
Joey Sorge and Vincenzo Faruolo are two of the New Jersey residents who are in the cast of 'A Bronx Tale: The Musical' at Paper Mill Playhouse. (more...)
The swing and assistant dance captain is the latest subject for Josh Lehrer's series of vintage photos. (more...)
Dates have been announced for the Cleveland return engagement of the Tony, Grammy and Olivier Award-winning hit musical JERSEY BOYS, the story of Frankie Valli and The Four Seasons. The musical will make its much-anticipated return to Cleveland to play the State Theatre at Playhouse Square, Sept. 20-25, 2016. Single tickets go on sale April 29. (more...)
Still not over Hamilton's performance at the Grammys? Gearing up for Waitress to hit Broadway in March? Didn't have time to check in on your favorite Broadway stars? Well don't worry BroadwayWorld has rounded up some of the biggest stories from this week from Broadway and beyond with BroadwayWorld's 'This Week in Pictures.' (more...)
Dates have been announced for the Seattle engagement of the Tony, Grammy and Olivier Award-winning hit musical JERSEY BOYS, the story of Frankie Valli and The Four Seasons, playing The Paramount Theatre from March 8 - 13, 2016. (more...)
'A Bronx Tale: The Musical' at Paper Mill Playhouse is a sensational new musical theater piece. It will be on the Millburn stage through March 6th. Co-directed by Jerry Zaks and Robert DeNiro, the book is by Chazz Palminteri based on his real-life experiences as a youth. With music by Alan Menken with lyrics by Glenn Slater and choreography by Sergio Trujillo, the all-star cast delivers exceptional performances. (more...)
Get your tickets now for what could become the hottest ticket of the 2016 theater season. (more...)
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VIDEO: Allison Janney Reprises Iconic WEST WING Role in the Actual West Wing
TV/MOVIES - VIDEO: Scarlett Johansson Performs 'Trust In Me' from Disney's THE JUNGLE BOOK
ART - Remembering Famed Photographer, Charles Gatewood
FITNESS - Race Review: 2016 BIG SUR INTERNATIONAL MARATHON - Breathtaking Views, Brutal Winds Make It a Course to be Reckoned With
MUSIC - VIDEO: Joss Stone Performs 'Let Me Breathe' on LATE LATE SHOW
CLASSICAL - The 2016 LOOK AND LISTEN FESTIVAL Kicks Off Tonight in Brooklyn - All Events are Free!
OPERA - Photo Flash: First Look at Caroline Bowman, Ramin Karimloo and More in Vancouver Opera's EVITA
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subset: commoncrawl


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Mitsubishi Lancer Questions General
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I just had my alternator and o2 sensors replaced and my cars stalls when out in park and idlea funny at stop lights
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your goal is to capture all throwing kisses the pretty girl from the house and dodge the ugly old pumps, plays with the arrows
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Fake Cop Tries to Pull Over Mayor
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Whitecraft by Woodard Bali SofaItem # HN-WD1933
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Photography News
ePHOTOzine brings you a daily round up of all the latest photography news including camera news, exhibitions, events, special offers, industry news, digital photography news, announcements and launches.
Seminars announced for Photo Fest Ireland
Canon & Nikon will be at Photo Fest Ireland, to be held this Sunday.
- 16 Apr 2010
Canon EOS 1D X DSLR Announced
Canon has announced the new Canon EOS 1D X - the camera features an 18 megapixel full frame sensor, and 12fps or 14fps shooting.
- 18 Oct 2011
- Canon (UK) Ltd
Fujifilm X-A2 Mirrorless Camera Announced
Fujifilm has announced the new X-A2, a new entry level mirrorless camera from Fujifilm, along with two updated lenses.
- 15 Jan 2015
- Fujifilm
- From £323.99
New flagship CanoScan 9950F introduced
World's first flat-bed scanner to offer 30-frame capacity for 35mm filmstrip
- 21 Sep 2004
Fujifilm Launches The X-M1
The X-M1 is a compact and lightweight CSC with the same APS-C X-Trans CMOS sensor as the X-Pro1, and is introduced with a new 16-50mm lens.
- 25 Jun 2013
- From £329.98
DSLR photo quality and full HD movies
The creative HD hybrid from the Lumix G Micro System.
- 4 Mar 2009
Nikon D800 And D800E DSLRs Announced
Nikon introduce the new 36.3 megapixel full frame Nikon D800 and D800E Digital SLRs.
- 7 Feb 2012
- Nikon
Nikon D40 digital SLR arrives
New lightweight SLR with an ease of use that's aimed at the family photographer and a collection of features from the D80 making it also fine for the enthusiast.
- 16 Nov 2006
- Nikon
- From £159.99
Canon Ixus 60 and Ixus 65
These two new ultra compact offerings from Canon each include a 3x zoom lens and 6 megapixel resolution and a large LCD screen.
- 21 Feb 2006
- Canon (UK) Ltd
Jobo Giga Vu Pro Evolution
Portable storage device available with a capacity of up to 120GB. RAW files are supported and an innovative dust detector feature find dust marks in your photos.
- 10 Mar 2006
- Intro 2020 Limited
Canons EOS-1D Mark III arrives
Canons EOS-1D Mark III -after much speculation the latest digital SLR from Canon is announced - the new benchmark for pro digital SLRS?
- 22 Feb 2007
- Canon (UK) Ltd
Canon Ixus 800
The new 8 megapixel Ixus from Canon sports 16 shooting modes including a manual mode, a 4x zoom lens with Image Stabiliser and widescreen stills recording.
- 21 Feb 2006
- Canon (UK) Ltd
HTC One Camera Phone With UltraPixel Announced
HTC has announced a new smartphone with a camera designed for optimum image quality with an UltraPixel sensor.
- 22 Feb 2013
Photographer Clicks His Way To Top Honour
A clever photo of a family inside small boxes has helped a Nottingham photographer pick up his first national award.
- 29 Jun 2011
- 3 Comments
Sony Lens Roadmap 2015 Plus New FE Lenses
Sony has shown a future lens roadmap at Photokina 2014, including details on four new FE Full-frame lenses.
- 15 Sep 2014
- Sony Corporation
ePHOTOzine Reviews All Panasonic Lumix G Lenses
ePHOTOzine has now published reviews of all Panasonic Lumix G lenses for the Micro Four Thirds camera system.
- 4 Nov 2014
- ePHOTOzine
Corel Announces Corel PaintShop Pro X4
Corel add 75 new features to the latest version of their PaintShop Pro software.
- 7 Sep 2011
- Corel UK Limited
- 6 Comments
Nikon Launch Interchangeable Lens Cameras
Nikon today introduces Nikon 1, a revolutionary digital camera system built from scratch to offer still image and movie recording without compromise.
- 21 Sep 2011
- Nikon
- 31 Comments
- From £149.00
Sony Introduce Four New Cyber-Shot Cameras
Sony introduce a series of small cameras which they say will give a 'big performance'.
- 12 Jan 2012
Jessops announce hit list
Following Jessops' plans to axe 81 of its shops as announced last Thursday, the photographic retail giant have unveiled exactly which stores it plans to close down.
- 25 Jun 2007
- 19 Comments
Canon Launch Three New PIXMA Printers
Canon have launched three design focused PIXMA printers, the iP7250, the MG5450 and the MG6350.
- 17 Sep 2012
- 3 Comments
HOYA Pro1 Digital Filter Fakes Found
Hoya filters have been faked, here's how to spot the difference and what you can do if you fall foul to these fresh filter fakes.
- 24 May 2012
- 3 Comments
- From £20.52
IGPOTY Announce Monochrome Winners
International Garden Photographer of the Year has announced the winners of the Monochrome Photo Project competition.
ePz Junior Member Scoops Scottish Nature Photography Award
ePHOTOzine member max_e has been awarded the Junior Scottish Nature Photographer of the Year accolade.
- 31 Mar 2016
- 5 Comments
Latest courses from Jessops Academy
Find out what is coming up over the next few weeks from the Jessops Training Academy.
- 13 Aug 2010
- Jessops Academy
Leica S (Typ 007) Medium Format Camera Captures 4K Video
Leica has announced the S (Typ 007) medium format camera with 37.5 megapixel CMOS microlens sensor.
- 25 Aug 2015
- Leica
£200 Offer for Nikon 1 Buyers At Jessops
Jessops are offering a £200 voucher to buyers of the new Nikon 1 series camera.
- 17 Oct 2011
- 7 Comments
- From £149.00
Park Cameras at The Gadget Show Live
Expert advice, show deals and brand representatives are all waiting for you at The Gadget Show Live.
- 7 Apr 2010
- Park Cameras Ltd.
Olympus Mju III 120 35mm compact announced
The Mju-III 120 is the first Olympus camera equipped with a Camera-Shake Indicator and has a weatherproof design.
- 31 Jan 2003
- Olympus UK
Canon Unveils PowerShot G3 X
Canon has announced the PowerShot G3 X, its first superzoom compact camera to feature a 1.0 type back-illuminated CMOS sensor.
- 18 Jun 2015
- Canon (UK) Ltd
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Photodex Goes Old School with ProShow StylePack
Slideshow tool provider Photodex has released StylePack Vintage, the latest installment in its popular ProShow StylePack product line.
- 4 May 2011


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Update (8:53 PM EST): The deal is now official, as Jackson has passed his physical, CSN Chicago's Dan Hayes reports. Mike Olt has been designated for assignment to make room for Jackson on the 40-man roster.
At long last, Austin Jackson has a home. MLB Network's Jon Heyman reports that the White Sox have agreed to a one-year, $5 million deal with the outfielder. Heyman adds that Jackson could have signed for more money elsewhere, but he valued the opportunity to continue playing center field rather than switching to left field.
Jackson, 29, spent last season with the Mariners and the Cubs, hitting an aggregate .267/.311/.385 with nine home runs, 48 RBI, 56 runs scored, and 17 stolen bases in 527 plate appearances. The Mariners traded him to the Cubs at the end of August ahead of the waiver trade deadline.
Jackson joins an outfield that includes Adam Eaton, Avisail Garcia, and Melky Cabrera.
The Angels, Indians, and Brewers were previously rumored to have had interest in Jackson.


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Cancer Cell Biology
Phosphorylation of vasodilator-stimulated phosphoprotein Ser239 suppresses filopodia and invadopodia in colon cancer
Article first published online: 12 AUG 2011
DOI: 10.1002/ijc.26257
Copyright © 2011 UICC
International Journal of Cancer
Volume 130, Issue 11, pages 2539–2548, 1 June 2012
Additional Information
How to Cite
Zuzga, D. S., Pelta-Heller, J., Li, P., Bombonati, A., Waldman, S. A. and Pitari, G. M. (2012), Phosphorylation of vasodilator-stimulated phosphoprotein Ser239 suppresses filopodia and invadopodia in colon cancer. Int. J. Cancer, 130: 2539–2548. doi: 10.1002/ijc.26257
Publication History
- Issue published online: 27 MAR 2012
- Article first published online: 12 AUG 2011
- Accepted manuscript online: 23 JUN 2011 08:51AM EST
- Manuscript Accepted: 30 MAY 2011
- Manuscript Received: 20 JAN 2011
Funded by
- NIH. Grant Numbers: R03CA133950, CA75123, CA95026, RC1CA112147
- Elsa U. Pardee Foundation, Merck
- colorectal cancer;
- guanylyl cyclase C;
- cyclic GMP;
- vasodilator-stimulated phosphoprotein;
- invadopodia;
- filopodia
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
In colorectal cancer, the antitumorigenic guanylyl cyclase C (GCC) signalome is defective reflecting ligand deprivation from downregulation of endogenous hormone expression. Although the proximal intracellular mediators of that signal transduction system, including cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG), are well characterized, the functional significance of its distal effectors remain vague. Dysregulation of ligand-dependent GCC signaling through vasodilator-stimulated phosphoprotein (VASP), an actin-binding protein implicated in membrane protrusion dynamics, drastically reduced cGMP-dependent VASP phosphorylation levels in colorectal tumors from patients. Restoration of cGMP-dependent VASP phosphorylation by GCC agonists suppressed the number and length of locomotory (filopodia) and invasive (invadopodia) actin-based organelles in human colon cancer cells. Membrane organelle disassembly reflected specific phosphorylation of VASP Ser239, the cGMP/PKG preferred site, and rapid VASP removal from tumor cell protrusions. Importantly, VASP Ser239 phosphorylation inhibited the proteolytic function of invadopodia, reflected by suppression of the cancer cell ability to digest DQ-collagen IV embedded in Matrigel. These results demonstrate a previously unrecognized role for VASP Ser239 phosphorylation, a single intracellular biochemical reaction, as an effective mechanism which opposes tumor cell shape promoting colon cancer invasion and metastasis. Reconstitution of physiological cGMP circuitry through VASP, in turn, represents an attractive targeted approach for patients with colorectal cancer.
Despite considerable advancements in elucidating pathognomonic genetic and molecular alterations, the causal mechanisms underlying initiation and progression of colorectal cancer, the third most common and deadly neoplasm in the western world, remain unknown. In the paracrine hormone hypothesis of colon cancer,1 tumorigenesis is characterized by a state of guanylyl cyclase C (GCC) ligand insufficiency, in which expression of endogenous hormones guanylin and uroguanylin, secreted by and activating GCC receptors on intestinal epithelial cells in an autocrine and paracrine fashion, is markedly reduced after a yet unidentified event early during transformation.2, 3 Ligand-induced GCC activation physiologically regulates intestinal fluid and electrolyte homeostasis through intracellular accumulation of the second messenger cyclic guanosine monophosphate (cGMP) and cGMP-dependent protein kinase (PKG) phosphorylation of ion channels.4 Activation of GCC also promotes the transition from the proliferative crypt phenotype to the differentiated colonocyte along intestinal mucosal surfaces by imposing cytostasis5 and oxidative metabolic reprogramming,6 effects presumably underlying GCC-mediated colorectal antitumorigenesis.7 Thus, the GCC ligandopenia associated with neoplastic transformation may promote colorectal carcinogenesis by producing a repressed GCC and cGMP pathway devoid of tumor suppressor activities. However, the exact consequences at the molecular level of the absence of GCC activation in colon cancer cells are unclear.
A poorly investigated target for the antitumorigenic GCC pathway is the vasodilator-stimulated phosphoprotein (VASP).8 A mammalian member of the highly conserved Ena/VASP family proteins, VASP acts as a molecular scaffold to bring together growing actin filaments with regulatory proteins at the cell leading edge.9 VASP controls cell spreading and migration by regulating the formation and stability of protrusive membrane structures driven by actin polymerization, including lamellipodia and filopodia, and the integrity of cell–matrix interactions at adhesion complexes.9 In that context, VASP exerts a pivotal role in filopodial dynamics, reflected by its localization at the filopodial tip complex where it promotes filopodia formation and elongation by recruiting the initial nanomachinery and imposing anticapping pressure on actin filament barbed ends.10 Three critical domains mediate VASP/actin interactions9: (i) the Ena/VASP homology 1 (EVH1) domain at the N-terminus, which binds to proteins with poly-proline II helix, including vinculin and zyxin; (ii) the central prolin-rich region, which binds to proteins containing SH3 and WW domains and the globular (G)-actin-binding profiling; (iii) the EVH2 domain at the C-terminus, which binds to both G- and filamentous (F)-actin and mediates VASP oligomerization, thereby promoting F-actin bundling and stabilization. Importantly, VASP harbors 3 phosphorylation sites (Ser157, Ser239 and Thr278), which are targeted with different affinities by PKG, cAMP-dependent protein kinase and AMP-activated protein kinase, and modulate its localization and function.11–13 Phosphorylation of VASP Ser239, adjacent to the G-actin-binding site in the EVH2 domain, is selectively regulated by cGMP-dependent signaling through PKG,11 an event that disrupts VASP anticapping and filament-bundling activities and inhibits membrane protrusion formation.9, 14
Although its engagement in migration and membrane protrusion dynamics in normal cells is well-established, a similar role for VASP Ser phosphorylation in cancer cells has not emerged. However, actin cytoskeletal remodeling at dynamic membrane regions determines oncogenic behaviors, from epithelial-to-mesenchymal transition to tissue invasion and metastasis.15, 16 In principle, PKG-mediated VASP phosphorylation could represent one of the biochemical reactions of the antitumorigenic GCC/cGMP signalome.1 Here, human colon tumors exhibited reduced GCC ligand expression associated with depletion of cGMP-dependent VASP phosphorylation, indicating interruption of downstream signaling by the dysregulated GCC pathway. Restoration of cGMP-mediated phosphorylation of VASP Ser239 by ligand-induced GCC signaling suppressed the invasive cancer cell phenotype, with disassembly of protrusive membrane organelles (filopodia and invadopodia) and inability to promote DQ-collagen IV degradation. Together, these observations reveal a previously unrecognized suppressor of invasive membrane protrusions and therapeutic target, VASP Ser239 phosphorylation, for colorectal cancer.
Material and Methods
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
E. coli heat-stable enterotoxin (ST) was prepared as described,5 whereas 8-br-cGMP was obtained from Sigma-Aldrich (St. Louis, MO). Antibodies to human VASP, phosphorylated VASP at Ser157, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), villin and cortactin were from Santa Cruz Biotechnology (Santa Cruz, CA). Two antiphoshorylated VASP at Ser239 antibodies were purchased, including clone 16C2 from Abcam (Cambridge, MA) and a rabbit antibody from Sigma-Aldrich. The anti-human guanylin antibody was from BioDesign International (Saco, ME), whereas the anti-human PKG was obtained from Assay Designs (Ann Arbor, MI). The antibody to the human GCC was obtained from Rockland Immunochemicals (Gilbertsville, PA). DQ-collagen IV, Alexa fluor 555 anti-rabbit IgG, Alexa fluor 633 anti-mouse IgG, Oregon Green 488 phalloidin and Slowfade Gold with 4′,6-diamidino-2-phenylindole (DAPI) were obtained from Invitrogen (Carlsbad, CA). Matrigel was from BD Bioscience (Bedford, MA), the general matrix metalloproteinase (MMP) inhibitor GM1006 from Calbiochem (San Diego, CA) and all the reagents for cell culture from Mediatech (Herndon, VA).
Clinical specimens and immunohistochemistry
Paraffin-embedded specimens from seven patients (Supporting Information Table 1) with histologically confirmed adenocarcinomas of the colo-rectum were obtained from the Department of Pathology, Anatomy and Cell Biology of Thomas Jefferson University (Philadelphia, PA) under a protocol approved by the Institutional Review Board and subjected to immunohistochemistry (IHC). Briefly, after deparaffinization and rehydratation (with xylene/ethanol/water washes), antigens in tissue sections (5 μm) were unmasked by two consecutive heating cycles (100°C for 5 min in 10 mM citric buffer, pH 6.0). Then, specimens were incubated overnight (4°C) with antibodies (1:100 dilution, unless otherwise indicated) against human guanylin, GCC, PKG, VASP, and phosphorylated VASP at Ser157 or Ser239 followed by incubation with the respective secondary antibody and DAB substrate (Avidin–Biotin Kit; Vector Laboratory, Burlingame, CA). Staining intensity was quantified by a blinded pathologist on a 1–3 scale (1: low or absent; 2: medium; 3: high).
Cell culture
All the human colon carcinoma cell lines investigated were purchased from the American Type Culture Collection (ATCC; Manassas, VA) and used within 6 months from resuscitation. Methods of cell authentication by ATCC include analyses of morphological and growth characteristics, isoenzymology and DNA profiles. T84 and HCT-116 human colon carcinoma cells were cultured (37°C, 5% CO2) with 10% fetal bovine serum in Dulbecco's modified Eagle's medium (DMEM)/F12 or DMEM, respectively. Cells (2–20 passages) were fed with fresh medium every third day and split when subconfluent.
Plasmids and cell transductions
Full-length VASP cDNA (Invitrogen) was subcloned into the Xho1-EcoR1 multiple cloning site of mouse stem cell virus (MSCV)-puro retroviral vector. Point mutations of VASP serines 157 (tcc gcc) and 239 (agc gcc) were performed using the QuikChange XL Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA) to generate VASP mutants with alanine substitutions at Ser157 (S157A), Ser239 (S239A) or both (AA). Green fluorescent protein (GFP)–VASP constructs were generated by subcloning wild-type VASP or the VASP mutant S239A in-frame to the C-terminus of GFP using the pRetroQ-AcGFP-Cl vector (Stratagene). HEK 293T17 cells (from ATCC) were transfected with 1 μg of each purified MSCV construct plus 1 μg of the packaging vector pCl-Ampho using Fugene transfection reagent (Roche, Basel, Switzerland). T84 cells were transduced (for 72 hr) with viral supernatants supplemented with 4 μg/ml polybrene, then selected and maintained in 5 μg/ml puromycin. In this way, the following T84 clones stably expressing the MSCV-driven gene were generated: VASP, VASP–S157A, VASP–S239A, VASP–AA, GFP, GFP–VASP and GFP–VASP–S239A.
Immunoblot analysis
Protein samples (40 μg in sodium dodecyl sulfate (SDS) loading buffer) were separated by electrophoresis on 10% acrylamide Tris–glycine gels, transferred to nitrocellulose membranes, and probed (1:1,000 each) with rabbit polyclonal antibodies against VASP, phosphorylated VASP at Ser157, GAPDH, villin or mouse monoclonal antibody against phosphorylated VASP at Ser239 in Tris buffered saline (TBS)–Tween (5% milk) overnight at 4°C. After washing the primary antibody, membranes were probed with the appropriate secondary antibody (1:2,000; Santa Cruz Biotechnology) for 1 hr at room temperature. Immunostained bands were imaged with a Kodak Image Station 4000R and quantified by densitometry.
Immunofluorescence and imaging
Filopodia dynamics were studied in cancer cells cultured on Lab-Tek glass microscope-chambered slides and treated for 2 hr with phosphate buffered saline (PBS) (control), the GCC ligand ST (1 μM) or the cGMP analog 8-br-cGMP (5 mM). Invadopodia were examined using glass coverslips coated with 25 μl Matrigel and incubated at 37°C (10 min) to solidify. Here, cancer cells were plated dropwise directly onto Matrigel, allowed to firmly adhere (1 hr) and equilibrate (24 hr) in their culturing medium, and finally treated (2 hr) with PBS or ST (1 μM). After treatments, cells were fixed (15 min) with ice-cold paraformaldehyde (4%, in PBS) and permeabilized (10 min) with 0.1% Triton X-100. After overnight incubations (4°C; in PBS containing 2% bovine serum albumin) with mouse anti-cortactin and/or rabbit anti-VASP antibodies (1:100 each) and 5 units/ml Oregon Green 488 phalloidin, cells were incubated (60 min at room temperature) with Alexa fluor 633 anti-mouse IgG and/or Alexa fluor 555 anti-rabbit IgG. Specificity of antigen/antibody reactions was assessed using the secondary antibody alone. Some preparations were counterstained with Slowfade Gold antifade reagent with DAPI to identify cell nuclei. Images (63×) were acquired with a confocal laser scanning microscope (LSM 510, Carl Zeiss, Jena, Germany) equipped with a monochrome CCD camera and pseudo-colored with image-analysis software.
For filopodia measurements, cells were analyzed by differential interference contrast (DIC) microscopy. Filopodial protrusions (≥5 μM) were examined on eight to ten random cell colonies per treatment (∼50–100 filopodia/treatment) by quantifying the number (per cell colony perimeter) and length (the average distance from the cell base to the tip) of filopodia. The cancer cell colony perimeter (∼264 ± 41 μM) was determined to ensure that results from colonies with different sizes could be directly compared. Measurements were performed using the Zeiss LSM Image Browser software (Carl Zeiss Microimaging, Thornwood, NY). For invadopodia analyses, the number and length of individual invadopodia (the baso-lateral actin-based membrane projections) per cancer cell were quantified (with the Zeiss LSM Image Browser software) on eight to ten random cells per treatment from three-dimensional (3D) reconstructions created from orthogonal X, Y and Z sections of complete Z-stack cell images (at 1 μm increments) of the confocal microscope. Finally, live cell microscopy was performed on culture dishes (fitted on a heated stage) of GFP–VASP (n = 6) or GFP–VASP–S239A (n = 7) cells by acquiring time-lapse images at 0, 1, 5, 10 and 15 min after treatment with the confocal microscope. Analysis of filopodia was performed with Image J (NIH, Bethesda, MD). The GFP-based fluorescent signal was quantified at filopodia tips (the distal 1/4 of each filopodia), along the entire length of each filopodium and throughout the cell body, and normalized to the background fluorescence.
DQ-collagen IV degradation
Glass coverslips were coated with 20-μl Matrigel containing 25 μg/ml DQ-collagen IV and incubated at 37°C (10 min) to solidify. Cancer cells were plated dropwise directly onto these Matrigel scaffolds, allowed to firmly adhere (1 hr), and treated (24 hr) in their culturing medium with PBS or ST (1 μM). Then, cells were fixed in methanol (-20°C for 15 min) and processed by confocal microscopy to generate Z-stack images, as described for invadopodia analyses above. DQ-collagen IV degradation was quantified in eight to ten cells per treatment with the NIH Image J as the mean fluorescent intensity of the fluorescent cleavage product (from DQ-collagen IV) over the cell and pericellular area per cell area. Results were normalized to the background fluorescence.
Statistical analysis
Unless otherwise indicated, data are mean ± SEM of ≥3 independent experiments. Statistical analyses were performed with the Student's t test.
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
VASP is an intracellular effector of the GCC pathway in colon cancer cells
VASP is expressed in colon cancer cells (46-kDa band; Supporting Information Fig. 1a) and distributes to dynamic membrane domains, including cell leading edges (Fig. 1a) and invadopodia (Fig. 1b), where it colocalizes with cortactin and actin (Fig. 1b, right panels). Invadopodia identity was confirmed by assessing their ability to focally degrade DQ-collagen IV (Fig. 1c), a fluorogenic analog of the basement membrane component collagen IV. In addition, VASP is an intracellular target of ligand-dependent GCC signaling in human colon cancer cells,8 and the potent GCC agonist ST4 induced rapid phosphorylation of VASP at both Ser157 and Ser239 (Supporting Information Fig. 1b). ST effects on VASP were mimicked by the membrane-permeant cGMP analog 8-br-cGMP (Figs. 1d and 1e and Supporting Information Fig. 2a) and blocked by the selective PKG peptide inhibitor DT217 (Fig. 1e and Supporting Information Fig. 2b), confirming that ST induces VASP Ser phosphorylation by activating the GCC/cGMP/PKG pathway in human colon cancer cells.
GCC signaling through VASP is dysregulated in colon cancer
In the human colon, the GCC pathway is constitutively activated by the paracrine hormone guanylin, which induces cGMP- and PKG-dependent phosphorylation of specific substrates.1, 4 Accordingly, normal colonic mucosa from patients exhibited epithelial cells with VASP Ser157 and Ser239 phosphorylation in the context of an intact guanylin–VASP signaling axis (Fig. 2a, upper panel). However, after transformation and loss of endogenous ligand expression2, 3 this GCC pathway becomes dysregulated (Fig. 2a, upper panel). Although GCC and PKG exhibited mixed trends with no significant changes, expression of guanylin and all the VASP species were significantly reduced in colorectal adenocarcinomas compared to their respective normal counterparts (Fig. 2a). Of significance, VASP phosphorylation at Ser157 and Ser239 were the most compromised signals of the GCC pathway, reflected by their downregulation in all tumors examined (Fig. 2a, middle panel) and maximal loss of expression (Fig. 2a, lower panel). Thus, loss of VASP Ser phosphorylation in colon cancer reflects interruption of effective induction and transmission of GCC–VASP signaling, beyond the mere changes in VASP expression (Supporting Information Fig. 3).
VASP Ser239 phosphorylation by GCC activation inhibits cancer cell shape mediating migration and invasion
As VASP regulates membrane protrusion geometry18 and is present at cell leading edges and invadopodia (Figs. 1a and 1b), the functional consequences of ligand-dependent GCC signaling through VASP Ser phosphorylation on the membrane architecture of colon cancer cells were examined. GCC signaling through cGMP significantly reduced the number and length of migratory (filopodia; Fig. 2b) and invasive (invadopodia; Fig. 2c) actin-based protrusions. Filopodia identity was confirmed by specific immunostaining with the filopodia marker fascin (data not shown). Importantly, cGMP inhibitory effects were specifically mediated by induction of VASP Ser239 phosphorylation, as demonstrated by using tumor cells stably expressing distinct VASP phosphomutants (Supporting Information Fig. 4a). Thus, cancer cells with VASP–S239A or VASP–AA, which contain point mutations at Ser239 and do not accept phosphorylation at that site (Supporting Information Fig. 4b), were resistant to GCC-mediated inhibition of filopodia (Fig. 3) and invadopodia (Fig. 4). By contrast, control cells expressing GFP or overexpressing VASP and tumor cells with the phosphomutant VASP–S157A, which harbors a point mutation at Ser157 only and does not accept phosphorylation at that site (Supporting Information Fig. 4b), were sensitive to suppression of filopodia (Fig. 3) and invadopodia (Fig. 4) upon ligand-dependent GCC signaling.
VASP Ser239 phosphorylation suppresses invasive membrane protrusions by inducing intracellular VASP redistribution
The molecular mechanism mediating inhibition of invasive cell shape by VASP Ser239 phosphorylation was investigated with live imaging microscopy in cells stably expressing GFP–VASP constructs (Fig. 5). Colon cancer cells with GFP–VASP, but not mutant cells expressing GFP–VASP–S239A, exhibited disassembly of filopodia (Fig. 5a) and invadopodia (Fig. 5b) upon ligand-dependent GCC activation. Protrusion retraction by GCC (Fig. 5c) reflected rapid removal of VASP from membrane projections (Fig. 5d), with a calculated half-life of 8.11 min (Supporting Information Fig. 5), and VASP redistribution to inner cell compartments away from membrane tips (Fig. 5e). However, the GFP–VASP–S239A mutant (resistant to Ser239 phosphorylation), was insensitive to GCC signaling (Fig. 5c), and retained its localization (Fig. 5d) and distribution (Fig. 5e) at locomotory membrane extensions, suggesting that VASP Ser239 phosphorylation represents a disassembly signal for invasive migratory organelles by uncoupling VASP from actin-based microregions at colon cancer cell membranes.
VASP Ser239 phosphorylation prevents DQ-collagen IV degradation by colon cancer cells
Metastatic cancer dissemination requires degradation and remodeling of the surrounding environment,19 a malignant attribute conferred to colon cancer cells by their invadopodia (Fig. 1c). Strikingly, activation of GCC signaling through VASP abolished the cancer ability to degrade DQ-collagen IV, with similar magnitude to that of a general inhibitor of MMPs (Fig. 6a), enzymes mediating tumor matrix degradation.20 However, colon cancer cells with VASP–S239A or VASP–AA, but not VASP–S157A, did continue to proteolytically digest DQ-collagen IV after ligand-dependent GCC signaling (Fig. 6a), indicating that VASP Ser239 phosphorylation mediates arrest of invadopodia-mediated proteolysis by the GCC pathway. Thus, VASP Ser239 phosphorylation is a unique biochemical reaction that suppresses membrane protrusions promoting tumor invasion in colorectal cancer (Fig. 6b).
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
The antitumorigenic GCC signalome is deregulated during colorectal transformation reflecting loss of endogenous ligand expression.1–3 Present findings reveal that this alteration affects a cGMP-dependent intracellular pathway comprising VASP as the distal effector, with subsequent silencing of an important biochemical reaction, VASP Ser239 phosphorylation, which opposes malignant cell shape. Interruption of GCC–VASP signaling, in turn, results in specific depletion of the VASP phosphospecies, which represent previously undescribed biomarkers for GCC deregulation and tumorigenesis in colon cancer. Conversely, restoration of GCC signaling by ligand replacement therapy may provide a rationale strategy for reconstituting cGMP–VASP circuitry and preventing metastasis. Indeed, ligand-dependent GCC activation compromises motility and metastasis of colon cancer cells,21 presumably by targeting VASP signaling suppressing invasive membrane protrusions (Fig. 6b) described herein.
Membrane protrusion extension principally reflects the balance of antagonistic forces at actin filament barbed ends imposing persistence (anticapping proteins) or arrest (capping proteins) of actin polymerization.10 VASP is a critical anticapping protein that initiates and maintains dynamic membrane regions by orchestrating a cytoskeletal assembly-line promoting simultaneous F-actin elongation and bundling.10, 22 In this way, Ena/VASP family members control migration and membrane protrusion formation underlying important physio(patho)logical processes, from wound-healing, platelet aggregation and neural development9 to inflammation, cancer spreading and metastasis.9, 23, 24 Accordingly, expression of Ena/VASP proteins positively correlates with disease progression in patients with lung25 or breast26 cancer, suggesting their involvement in carcinogenesis and metastasis. Our study underscores the critical role of VASP in organizing cancer functions associated with the malignant membrane shape. In that context, although with unclear activities VASP is a molecular component of invadopodia,27 cancer cell projections mediating focal matrix proteolysis. Here, colon cancer cells required VASP for invadopodia stability and function, indicating that VASP maintains the structural integrity of these invasive protrusions. Indeed, intracellular VASP redistribution and removal from protrusive tips, upon GCC-dependent phosphorylation, destabilizes actin-driven membrane extensions (Fig. 5) by presumably depleting actin filament plus-ends of VASP-mediated anticapping forces,22 resulting in protrusion retraction from the persistent actin retrograde flow. Importantly, invadopodia represent attractive antimetastatic targets since they function as critical hubs of invasive cell behavior, spatially and temporally organizing the activities of actin nucleators and regulators (Arp2/3, N-WASP and cortactin), signaling enzymes (Src, Erk1/Erk2 and FAK), adhesive receptors (integrins β1/β3 and CD44) and proteases (MT1-MMP, MMP2 and MMP9).27, 28 Thus, the ability of cGMP-dependent VASP phosphorylation to dismantle invadopodia in colon cancer cells (Fig. 4) offers an innovative anti-invadopodial strategy to be exploited against tumor metastasis in patients.
Tumor suppressors inhibiting metastatic cell dissemination are scarce. Their critical value resides in the potential to serve as biomarkers to improve clinical staging and targets to reduce cancer mortality. VASP Ser239 phosphorylation, a simple intracellular biochemical reaction, could represent a unique invasion suppressor with sophisticated regulatory dynamics, an inducible mechanism embedded into signal transduction networks shaping tumor cell metastasis. Further, inhibition of metastasis by VASP Ser239 phosphorylation could be a general strategy, as VASP is ubiquitously expressed across human tissues.9 Of significance, cGMP-dependent VASP Ser157 phosphorylation does not appear to affect the invasive phenotype of colon cancer cells (Figs. 3, 4 and 6). As VASP Ser239 is selectively regulated by PKG-dependent phosphorylation,9, 14 while VASP Ser157 is a more promiscuous site phosphorylated by PKG, cAMP-dependent protein kinase, and protein kinase C-dependent and -independent mechanisms,11, 29 present observations suggest a unique role of the GCC/cGMP/PKG pathway in suppressing colorectal cancer invasion through VASP Ser239. Reduced levels of VASP Ser157 phosphorylation in colon cancer (Fig. 2a), in turn, may reflect more complex deregulation of tumor intracellular networks, including cGMP-dependent and -independent pathways, probably affecting carcinogenetic processes beyond GCC-regulated invasive cell shape, a model currently being explored in this laboratory.
Present findings also support the existence of a biological divergence between cGMP and cAMP signaling on Ena/VASP activities, an established paradigm in neurons where the cAMP-VASP axis mediates attractive guidance cues by increasing the number and length of filopodia, while the cGMP/PKG/VASP pathway promotes repulsive guidance cues.30, 31 However, the precise consequences of cyclic nucleotide signaling through VASP on the actin cytoskeleton appear to be cell and tissue specific, as demonstrated by identical cGMP and cAMP inhibitory actions on platelet aggregation, reflecting respective VASP phosphorylation mechanisms.32 Regardless, VASP Ser239 phosphorylation induced by cGMP is emerging as a pivotal mechanism mediating VASP redistribution and disassembly of VASP-regulated membrane protrusions, including filopodia and lamellipodia.14, 33, 34 In that context, Ser239 is strategically located within the EVH2 domain of VASP, immediately adjacent to the G-actin-binding site, which localizes VASP at filopodial tips and coordinates the transfer of profilin-bound G-actin to the barbed end of the elongating F-actin filament.33 Phosphorylation of VASP Ser239 interferes with EVH2-dependent processes at membrane leading edges, destabilizing VASP-actin interactions and organelle protrusive dynamics.14, 33 Moreover, VASP Ser239, but not VASP Ser157, phosphorylation inhibits VASP-driven F-actin polymerization.13 Our study demonstrates VASP Ser239 phosphorylation suppresses the invasive actin cytoskeleton of tumor cells and represents an unappreciated prototype to be exploited in innovative diagnostic and therapeutic approaches for colorectal cancer. Specifically, ligand-dependent GCC signaling may prevent colon cancer metastasis by neutralizing oncogenic VASP functions at the invasive cancer front.
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
This work was supported by grants to G.M.P. from NIH and Elsa U. Pardee Foundation, and to S.A.W. from NIH. The National Institute of Health specifically disclaims responsibility for any analyses, interpretations or conclusions. S.A.W. is the Samuel M.V. Hamilton Professor of Medicine, and a paid consultant to Merck and the Chair (uncompensated) of the Scientific Advisory Board of Targeted Diagnostics and Therapeutics. G.M.P. receives research salary support from Merck. All the other authors have no conflict of interest to disclose.
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
- 1The paracrine hormone hypothesis of colorectal cancer. Clin Pharmacol Ther 2007; 82: 441–7., , , , , , , .
- 2Expression of guanylin is downregulated in mouse and human intestinal adenomas. Biochem Biophys Res Commun 2000; 273: 225–30., , , , , , .
- 3Transcriptional gene expression profiles of colorectal adenoma, adenocarcinoma, and normal tissue examined by oligonucleotide arrays. Cancer Res 2001; 61: 3124–30., , , .
- 4Guanylyl cyclases and signaling by cyclic GMP. Pharmacol Rev 2000; 52: 375–414., , , , , , , .
- 5Guanylyl cyclase C agonists regulate progression through the cell cycle of human colon carcinoma cells. Proc Natl Acad Sci USA 2001; 98: 7846–51., , , , .
- 6The hormone receptor GUCY2C suppresses intestinal tumor formation by inhibiting AKT signaling. Gastroenterology 2010; 138: 241–54., , , , , , , , , .
- 7Guanylyl cyclase C suppresses intestinal tumorigenesis by restricting proliferation and maintaining genomic integrity. Gastroenterology 2007; 133: 599–607., , , , , , , , , .
- 8Vasodilator-stimulated phosphoprotein (VASP) phosphorylation provides a biomarker for the action of exisulind and related agents that activate protein kinase G. Mol Cancer Ther 2002; 1: 803–9., , , , , .
- 9Ena/VASP proteins: regulators of the actin cytoskeleton and cell migration. Annu Rev Cell Dev Biol 2003; 19: 541–64., , , , .
- 10Lamellipodial versus filopodial mode of the actin nanomachinery: pivotal role of the filament barbed end. Cell 2004; 118: 363–73., , , , , .
- 11cAMP- and cGMP-dependent protein kinase phosphorylation sites of the focal adhesion vasodilator-stimulated phosphoprotein (VASP) in vitro and in intact human platelets. J Biol Chem 1994; 269: 14509–17., , , , , , .
- 12AMP-activated protein kinase impairs endothelial actin cytoskeleton assembly by phosphorylating vasodilator-stimulated phosphoprotein. J Biol Chem 2007; 282: 4601–12., , , , , .
- 13Differential VASP phosphorylation controls remodeling of the actin cytoskeleton. J Cell Sci 2009; 122: 3954–65., , , , , , , , .
- 14Modulation of lamellipodial structure and dynamics by NO-dependent phosphorylation of VASP Ser239. J Cell Sci 2007; 120: 3011–21., , , , .
- 15EMT, the cytoskeleton, and cancer cell invasion. Cancer Metastasis Rev 2009; 28: 15–33., .
- 16Regulation of the actin cytoskeleton in cancer cell migration and invasion. Biochim Biophys Acta 2007; 1773: 642–52., .
- 17Highly specific, membrane-permeant peptide blockers of cGMP-dependent protein kinase Ialpha inhibit NO-induced cerebral dilation. Proc Natl Acad Sci USA 2000; 97: 14772–7., , , , , .
- 18Ena/VASP: towards resolving a pointed controversy at the barbed end. J Cell Sci 2009; 122: 1947–53., .
- 19Tumor metastasis: mechanistic insights and clinical challenges. Nat Med 2006; 12: 895–904..
- 20The matrix corroded: podosomes and invadopodia in extracellular matrix degradation. Trends Cell Biol 2007; 17: 107–17..
- 21Guanylyl cyclase C prevents colon cancer metastasis by regulating tumor epithelial cell matrix metalloproteinase-9. Cancer Res 2009; 69: 3529–36., , , , , , , .
- 22Ena/VASP proteins have an anti-capping independent function in filopodia formation. Mol Biol Cell 2007; 18: 2579–91., , , , , .
- 23A Mena invasion isoform potentiates EGF-induced carcinoma cell invasion and metastasis. Dev Cell 2008; 15: 813–28., , , , , , , , , , , , et al.
- 24Positive regulation of migration and invasion by vasodilator-stimulated phosphoprotein via Rac1 pathway in human breast cancer cells. Oncol Rep 2008; 20: 929–39., , , , , , , , , , .
- 25Differential expression of VASP in normal lung tissue and lung adenocarcinomas. Thorax 2005; 60: 576–81., , , , , .
- 26EVL (Ena/VASP-like) expression is up-regulated in human breast cancer and its relative expression level is correlated with clinical stages. Oncol Rep 2008; 19: 1015–20., , , .
- 27Invadopodia: specialized cell structures for cancer invasion. Clin Exp Metastasis 2006; 23: 97–105..
- 28Invadopodia: at the cutting edge of tumour invasion. J Clin Neurosci 2008; 15: 725–37., , .
- 29Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets. Biochem J 2006; 393: 555–64., , .
- 30Critical role of Ena/VASP proteins for filopodia formation in neurons and in function downstream of netrin-1. Neuron 2004; 42: 37–49., , , , , , , .
- 31The great escape; phosphorylation of Ena/VASP by PKA promotes filopodial formation. Neuron 2004; 42: 1–3., .
- 32The vasodilator-stimulated phosphoprotein (VASP) is involved in cGMP- and cAMP-mediated inhibition of agonist-induced platelet aggregation, but is dispensable for smooth muscle function. EMBO J 1999; 18: 37–48., , , , , , , , , .
- 33Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping proteins. J Biol Chem 2005; 280: 28653–62., , , , , , .
- 34NO-dependent osteoclast motility: reliance on cGMP-dependent protein kinase I and VASP. J Cell Sci 2005; 118: 5479–87., , , , , , , , .
Supporting Information
- Top of page
- Abstract
- Material and Methods
- Results
- Discussion
- Acknowledgements
- References
- Supporting Information
Additional Supporting Information may be found in the online version of this article.
|Supporting Figure 1 Ligand-dependent GCC signaling induces rapid VASP Ser phosphorylation in colon cancer cells. (a) VASP-specific immunoblots of representative experiments (repeated 3 times) analyzing total cell lysates from various human colon carcinomas. GAPDH, the loading control. (b) Immunoblots of representative time-course experiments with T84 human colon carcinoma cells treated with the GCC agonist (1 μM). pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively. Villin, the loading control.
|Supporting Figure 2 GCC signaling trough PKG induces VASP Ser phosphorylation in colon cancer cells. Immunoblots of representative experiments with HCT116 (a) and T84 (b) human colon carcinoma cells. Treatments were: the cGMP analog 8-br-cGMP (5 mM, 30 min); the GCC ligand ST (1 μM, 5 min), and the selective PKG inhibitor DT2 (5 μM, 30 min; ref. 17 in manuscript). pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively; GAPDH, the loading control.
|Supporting Figure 3 GCC signaling through VASP Ser phosphorylation is dysregulated in colon cancer. IHC images (magnification, 20x) of serial sections from primary tumor (Tumor) and matched normal adjacent tissue (NAT) of a patient with colorectal cancer. Tissues were stained with the specific primary antibody (brown) and hematoxylin (blue, nuclei). A rabbit primary antibody (1:600 dilution) from Sigma-Aldrich was employed for VASP phosphorylated at Ser239. pS157-VASP and pS239-VASP, VASP phosphorylated at Ser157 and Ser239, respectively.
|Supporting Figure 4. Colon cancer cells with VASP phosphomutants are resistant to GCC-mediated phosphorylation. Plasmid constructions and cell transductions were performed as detailed in Materials and Methods. (a) Representative immunoblots of total VASP (VASP) and the loading control (GAPDH) from T84 cells stably expressing the indicated MSCV-driven genes, including VASP-S157A (S157A), VASP-S239A (S239A) and VASP-AA (AA). Wild-type (WT) T84 cells, and cells expressing GFP or VASP represent the control conditions. (b) Representative immunoblots and associated graphs (reflecting mean ± SEM of 3 independent experiments) of VASP phosphorylated at Ser157 (pS157-VASP; left) and VASP phosphorylated at Ser239 (pS239-VASP; right) in T84 cells (genetically manipulated as in a) treated for 5 min with ST (1 μM). Values in graphs are relative levels normalized to respective loading (villin) and vehicle (PBS) controls. *, P < 0.05, versus respective PBS control.
|Supporting Figure 5 GCC signaling induces rapid VASP removal from colon cancer cell filopodia. Exponential decay curve fitted to the data presented in Fig. 5d, examining the effects of the GCC agonist ST (1 μM) on VASP localization at filopodia. Dotted lines are 95% confidence limits. The curve was generated employing the software Prism 5.0 (GraphPad).
|Supporting Table 1.
Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.


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The National Retail Federation had some interesting numbers from Thanksgiving/Black Friday weekend.
Low prices helped keep Americans' budgets in check this weekend: on average, shoppers spent $407.02 from Thursday through Sunday, down from $423.55 last year.
That's one way to look at it. Here's another interpretation: consumers on average spent less than they did than the same period a year ago.
More than 141 million unique shoppers have already or will have shopped by the end of the big Thanksgiving weekend, up from 139 million.
More shoppers are always a good thing. But when you factor in how much money they spent, you get this: the number of shoppers over the weekend grew just 1.4 percent while average spending fell 4 percent.
Yikes. A 1.4 percent gain in shoppers hardly equals out a 4 percent decline in spending. Retailers are essentially brawling over a barely growing pool of Black Friday shoppers who are actually spending less on average than did a year ago.
In addition, there is one less shopping week this year because Thanksgiving fell on the last week of November. Suddenly, a season with an already low margin of (profit) error just got a little more perilous.
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- Trump virtually clinches GOP nomination; Cruz drops out
- Minnesota Poll: Most favor Supreme Court nominee, Senate vote
- Prince died amid frantic plans for drug addiction treatment
- Meyer's outing goes south, then he's sent down after Twins lose
- Preservation Commission says no to proposed 40-story Mpls. condo tower
- Bike lanes to be added along Cleveland Avenue in St. Paul


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Check BNN and Bulbanews for up-to-date Pokémon news and discuss it on the forums or in our IRC channel #bulbagarden on irc.systemnet.info.
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Delighting in the Law of the Lord: God's Alternative to Legalism and Moralism - eBook
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Our culture has a few catch phrases: Be who you want to be; Do what makes you happy. We are told to do our own thing and follow our own rules, which often makes the Bible appear to be oppressively restrictive and hopelessly outdated, even to Christians! Responding to the misdirection of our society and misperceptions the church has of Gods law, Professor Jerram Barrs helps readers recognize the beauty in and purpose of Gods rules for living healthy, productive, and fulfilling lives. From the Old Testament to the New, Barrs demonstrates how Gods commands to his people were intended to protect them from sin and direct them in godliness. Rejecting the idea that we can earn Gods favor through good works, Barrs nevertheless highlights how Gods commands should spur us to obedience and ultimately remind us of grace.
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Amid rumors of ongoing negotiations between Netanyahu and Herzog on national unity government, Habayit Hayehudi chairman says his party views justice portfolio as key to its agenda.09:39 03.05.16 | 1 comments
isn't it humiliating for an israeli elite commando to be attacked by civilians with such ridiculous weapons? why is it that they insist saying that they were attacked. weren't the commandos who stormed a passenger ship in international waters? if it is they that stormed and attacked the ship, how come that they now say it was the passengers that attacked them? it's a cowardly excuse for a humiliating military action badly planned and miserably executed. once again.


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Here's the question you clicked on:
In their advertisements, the manufacturers of a certain brand of cereal would like to claim that eating their oatmeal for breakfast daily will produce a mean decrease in cholesterol of more than 10 mg/dl in one month for people with cholesterol levels over 200 mg/dl. In order to determine if this is a valid claim, they hire an independent testing agency, which then randomly selects 25 people with a cholesterol level over 200 for inclusion in a study. The 25 people in the study eat this cereal for breakfast daily for a month. Which of the following is the alternative hypothesis of interest in this study? A. Ha: μ < 10 B. Ha: μ > 10 C. Ha: μ 10 D. Ha: > 10
@jim_thompson5910 @kropot72
I'm assuming B?
B looks good, the claim is a decrease in cholesterol of MORE than 10 mg/dl


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Sleep Inn North, Phoenix
- Free buffet breakfast, free WiFi, and free parking
Main amenities
- 61 guestrooms
- Outdoor pool
- Fitness center
- 24-hour business center
- 24-hour front desk
- Coffee/tea in the lobby
- Air conditioning
- Daily housekeeping
- Laundry service
- Multilingual staff
What's around
- In Deer Valley
- University of Phoenix-Northwest Learning Center (0.5 mi / 0.8 km)
- Peoria Sports Complex (7.9 mi / 12.7 km)
- Cave Creek Golf Course (3.5 mi / 5.6 km)
- Glendale Aquatic Center (3.7 mi / 5.9 km)
- Deer Valley Rock Art Center (4.3 mi / 6.9 km)
Sleep Inn North, Phoenix
Hotel Info: 866-539-8117from $62
- Standard Double Room, 2 Double Beds, Non Smoking
- Business Room, 1 King Bed, Non Smoking
- Business Room, 1 King Bed, Smoking
- Standard Double Room, 2 Double Beds, Non Smoking
Also popular with travelers from the USA
At a glance
Key facts
Hotel size
- This hotel has 61 rooms
- This hotel is arranged over 2 floors
- Check-in time starts at 3 PM
- Check-out time is noon
Required at check in
- Credit card or cash deposit required
- Government-issued photo ID required
Travelling with others
- Children (18 years old and younger) stay free when occupying the parent or guardian's room, using existing bedding
- Pets allowed (dogs and cats only) *
- 2 per room (up to 35 lb)
Extra people
- Roll-away beds available, charges may apply*
- Free WiFi in public areas
- Free WiFi in rooms
- Free self parking
Nearby Two Star Phoenix hotels
In the hotel
- Free buffet breakfast
- Coffee/tea in the lobby
- Outdoor pool
- Fitness facilities
- Spa tub
- 24-hour business center
- 24-hour front desk
- Dry cleaning/laundry service
- Laundry facilities
- Number of buildings/towers - 1
- Elevator/lift
- Accessible bathroom
- In-room accessibility
- Roll-in shower
In the room
- In-room climate control (air conditioning)
- Air conditioning
- Coffee/tea maker
- Iron/ironing board
- Free toiletries
- Hair dryer
- Premium TV channels
- Free WiFi
- Free local calls
- Refrigerator
- Microwave
- Daily housekeeping
- In-room safe
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Sleep Inn North, Phoenix's small print
Mandatory fees
Deposit: USD 50 per stay
Pet deposit: USD 50 per stay
Optional extras
Rollaway beds are available for USD 15 per night
Pets are allowed for an extra charge of USD 15 per pet, per night
We have included all charges provided to us by this hotel. However, charges can vary, for example, based on length of stay or the room you book.
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