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7701 Krause Avenue
Oakland,CA 94605
Free food!
The Justice 4 Alan Blueford coalition is hosting a community BBQ in Arroyo Park on Saturday, April 13th. We hope you'll come out with friends and family to eat, play, and connect as we build the strength of our community.
Expect delicious home-cooked food, great music, powerful speakers, kid-friendly activities, a nurse's station – all donated to this community event in honor of Alan Blueford, a Skyline High School student who was murdered by OPD Officer Miguel Masso almost one year ago on May 6th, 2012.
Please join us in demanding the arrest of Officer Miguel Masso for the murder of Alan Blueford, and an end to racial profiling and violent, unaccountable police in Oakland.
Officer Masso must be fired because factual evidence exists that:
- he participated in an illegal 'Stop & Frisk,' precipitating Alan's death
- he did not activate his lapel camera as required by OPD protocols
- he executed Alan Blueford, firing three shots at someone who was unarmed
- he lied after the fact about the events of May 5th & May 6
- he should never have been hired in the first place because of his NYPD record
- he refused a prisoner medical treatment, as documented by NYPD Internal Affair
- he is not psychologically fit to be a police officer, as evidenced by his own statements in the District Attorney's report


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DHS News October 2011
October 2011 DHS Newsletter
As we experienced with the recent 5.8 magnitude earthquake that shook some buildings, the potential for a natural disaster to strike exists regardless of the season, the time or the place. With this in mind, DHS has posted disaster preparedness information, including a tip sheet provided by the Federal Emergency Management Agency (FEMA) titled "How to Communicate During a Disaster." The tips are intended to help people better prepare for major disasters, knowing that communications networks are more likely to be compromised or damaged. Such information was especially vital less than a month ago when the earthquake and Hurricane Irene hit the East Coast. With both events, there was significant disruption to mobile networks and severe network congestion, making it harder for people to reach loved ones and emergency services. Although Allegheny County was left unscathed in both incidences, it is impossible to determine when the next emergency disaster situation will affect our region. Some of the tips included in FEMA's "How to Communication During a Disaster" include:
View the full list of disaster communication tips
Know your SERP While the earthquake was a rare event for our region and no significant damage or injuries were reported, events like this should serve as a reminder to staff that Standard Emergency Response Plans (SERPs) for all of our buildings are available on the DHS Intranet at and that staff should be familiar with them. The objectives of the SERPs are to establish procedures for responding to fire emergencies, natural and man-made disasters and medical emergencies. They also outline methods for safe and orderly evacuation (or shelter-in-place requirements, if deemed necessary) of any part of our buildings. Please review the SERP for your building or other DHS buildings that you visit frequently at your earliest convenience.
DHS News Newsletter DHS News Archive

subset: commoncrawl


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The groups have purchased 400 carbon monoxide detectors to create both awareness and compliance with the Carbon Monoxide Poisoning Prevent Act. This July celebrates the second year of this program which requires that carbon monoxide detectors are installed in every "dwelling unit intended for human occupancy."
Studies show that less than 25 percent of the homes have carbon monoxide detectors.
It's hoped that 400 new detectors will be installed in Woodland homes by the end of the year.
People interested in receiving a Carbon Monoxide Detector for their home should contact the Yolo County Board of Realtors at 449 Elm Street; or call 666-4253.
Those assisting with the program include Meals on Wheels, Community Care Car, Senior Center, Yolo County Food Bank, and the Woodland Fire Department.


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With a covered up three-month-old baby Blue Ivy attached to her chest, Beyonce and Jay-Z emerged from a yacht in the Caribbean this weekend.
Look ma, I'm on a boat.
Blue Ivy
Baby's First Yacht Ride
4/9/2012 7:20 AM PDT BY Johnny Lopez
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Making pictures of the vast Taiga forest is not easy. You need a higher elevation to give an impression of this endless seeming landscape. Hannu Hautala – in his friendly and supporting manner – gave me the advice to follow the ridges close beside the Kitka River in the Oulanka National Park. It was a great day in the woods: Rainy, misty, silent, the scent of the wet forest and a single siberian jay following me for a while– just perfect!
Location: Finland


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PhotoFriday: Brother
Photo Friday sent out a Sister challenge around the new year, and I didn't really have any pictures of my own family that would fit the bill. I do have a half sister, but I haven't seen her in many years. When this week's challenge came up, I finally had some material to work with.
This picture was taken around Thanksgiving at the dinning room table. My precious little niece was rarely set down, and passed from one warm embrace to another. Following grandmothers, uncles and aunts finally it was turn for Rod, my brother, to hold his daughter. No flash was to be used, less it disturbed the baby, so this is shot wide open at F1.4 on my 50mm. I'm quite impressed with the warm dinning room lighting contrasting the cool window light.
One of my favorite parts of Photo Friday is improving my craft by looking over the note worthy photos from each week. The challenge from a fortnight ago had this photo submitted, and I can say I love absolutely everything about it. From the tack sharp wood grain to the noisy clouds along with the contrast, color and composition all make this an amazing photograph. I'd better keep on practicing!


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An inspiring lecturer turned Marcus Raichle's focus from music and history to science. Since then, he has pioneered the use of imaging to study how our brains function.
An inspiring lecturer turned Marcus Raichle's focus from music and history to science. Since then, he has pioneered the use of imaging to study how our brains function.
Vision researcher John Dowling has spent a lifetime studying the neural architecture of the retina. He is closing his laboratory after 53 years, opting to extend these studies as a postdoc.
A groundbreaker in the study of Listeria monocytogenes, Pascale Cossart continues to build her research tool kit to understand how to fight such intracellular human pathogens.
In lieu of a career in punk rock, James Pfaus opted to study the brain signals underlying sexual behavior and then see what happened when he manipulated them.
György Hajnóczky uncovers the chemical and physical strategies by which mitochondria communicate and function within a cell.
With much of his early career dictated by US Navy interests, Carl June drew inspiration from malaria, bone marrow transplantation, and HIV in his roundabout path to a breakthrough in cancer immunotherapy.
Joel Cohen uses the tools of mathematics to deconstruct questions of life.
By audaciously pursuing an abandoned area of research, Ana María Cuervo discovered how cells selectively break down their waste, and revealed the health consequences when that process malfunctions.
Thanks to a book, a war, and a big green caterpillar, John Hildebrand found himself mapping the exquisite and surprising wiring of the insect olfactory system.
Raising one evolutionary question after another, Brandon Gaut has harvested a crop of novel findings about how plant genomes evolve.


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found a new home!
Plenty of my friends are looking
for one too. Find a pet to adopt.
Find a pet to adopt |
My name is Nicco!
Facts about Nicco
- Breed: Corgi/Chihuahua Mix
- Color: Red/Golden/Orange/Chestnut
- Age: Adult
- Size: Med. 26-60 lbs (12-27 kg)
- Sex: Male
Nicco's Info...
I am already neutered, up to date with shots, good with kids, and good with dogs.
Come meet me and all my canine friends on Saturday from 10am-3pm at the Petsmart in Chandler, 855 N 54th St near Ray Rd and I-10. See you there!
This little boy was rescued from a very bad situation. He is sweet and playful and looking for a forever home. We aren't quite sure what he is....his previous owners thought shepherd/chihuahua, but we're thinking there is some corgi in him. One look and you'll wonder too. But he's a cutie! For more information or to request an application, please email email@example.com. Rescue a forever friend!
About our adoption process: For more information or to request an adoption application (required), please email the dog's foster listed above (no phone calls, please). There is no commitment by submitting the application - it just helps us determine if the pet you are interested in will be a good match for you. Next is a meet and greet at one of our adoption events - typically held on Saturdays at the Petsmart in Chandler, AZ (Ray Rd & I-10). Not every pet is at every event so please contact us first to find out when and where you can meet the pet you are interested in. If there are other dogs in your household, we suggest bringing them to ensure everyone gets along. The standard adoption fee for dogs and puppies is $140 (cash or check) which includes spay/neuter, current vaccinations, microchip and collar/tags.
Other Pets at Benji's Buddies Animal Rescue
Why Adoption is a Great Option
Nicco, a Chandler Corgi/Chihuahua dog was adopted! Here's how you can benefit by looking at adoption as an option to buying a Corgi/Chihuahua for sale in Chandler, or seeking a Corgi/Chihuahua dog from a breeder in Chandler.
- Rescues and shelter volunteers in Chandler already know the personality and tendencies of their pets. This means they can match you with the right pet. That will makes it much easier transition to owning a pet for yourself and your new pet.
- On average adoption fees are much less than you'd pay a Chandler breeder, or pet store. Plus most often rescue pets have already been to the veterinarian for a check-up, had a round of vaccinations, and are spayed or neutered. That is a lot of savings!
- You will become a hero, and give a needy dog or cat a loving home. In fact, there are even Corgi/Chihuahua breeders who help with Corgi/Chihuahua rescue. One puppy or kitten adopted in Chandler paves the way for another one to be saved.


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Maili Elementary Principal named as finalist in $25,000 School Leadership Award | Business
Disa Hauge, principal of Maili Elementary School was named a finalist in the 2012 Masayuki Tokioka Excellence in School Leadership Awards.
One Hawaii public school principal will receive $25,000 ($15,000 towards a school project of his or her choice and a $10,000 personal cash award). The recipient of this year's award will be announced at the Public Schools of Hawaii Foundation dinner on April 13, 2012 in Honolulu.
The award, which is named after Island Insurance founder Masayuki Tokioka, is underwritten by the Island Insurance Foundation, the philanthropic arm of Island Insurance Companies. 11 public school principals from across the state are finalists for this year's award which recognizes outstanding school principals across the state and their efforts to provide high-quality learning opportunities for their students.
Hauge has been an educator at Ma'ili Elementary for 18 years. The past 8 years as principal and prior to that as vice principal. Growing up in Waianae, Hauge understands the challenges that teachers at Ma'ili Elementary face including: children in poverty, homelessness, foster care and transiency. Hauge believes the will to succeed is not nearly as important as the will to prepare to succeed. Through her leadership, several innovative programs have been initiated that have resulted in nearly 70% of students in grades 4-6 at or above their grade level in reading.
"Since she was raised in Makaha and attended Makaha Elementary and Waianae Intermediate as a youngster, Principal Hauge has a clear understanding of the many challenges our student on the Waianae Coast face on a daily basis," said Suzie Lee, Vice Principal of Ma'ili Elementary. "She perseveres and does not allow any obstacles to hinder student achievement. She is a dedicated child advocate who envisions a brigh and successful future for all students."
The Island Insurance Foundation was established as the charitable arm of Island Insurance, the only locally-owned property and casualty insurance carrier to have a charitable entity solely dedicated to supporting the non-profit community. Island Insurance is Hawaii's largest locally owned and managed P&C insurance carrier with financial strength ratings of "A" by A.M. Best and Fitch Ratings and is the only Hawaii-Based company to be recognized as one of the Top 50 property & casualty insurance companies in the nation by the Ward Group for four consecutive years. For more information, visit its website at: www.islandinsurance.com.


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Ah, the countryside of central Italy! It makes me think of golden sunsets, scenic vineyards and homemade tomato sauce! (So maaaybe I've seen too many cliché TV movies...) This outfit reminds me of those things (okay, minus the sauce part—but hey, if pasta's on the dinner menu, the print will camouflage spills) and glamorous Dolce & Gabbana campaign-y Italian women in general. So, if you get to squeeze in some sightseeing on the side, mix the pieces with whatever else you packed—people will think you're a local!
French ladies are famous for their understated sense of style, so don't go overboard on wild patterns or statement makeup. It's better to stick with a simple, solid sheath dress (black is always good, but navy is much more Parisian, non?) and have your fun with accessories.
You may never get another opportunity to wear a crazy hat at a wedding and completely fit in (over-the-top headwear is the norm at formal British events), so go for it! The bigger, the better! To keep your whole ensemble from looking too conspicuous, however, do like the Middleton sisters and balance things out with a simple lace dress and nude pumps.
More on Luckymag.com:
- Five Easy and Extraordinary Outfits For Your Next Beach Wedding
- 17 Cocktail Dresses Tailor-Made for the Summer Wedding Circuit
- What to Wear to an Evening Wedding
- You Fancy, Huh? 20 Dresses You Can Actually Wear to a White Tie Wedding
- 25 Dreamy Dresses Perfect for Your Next Black Tie Wedding
- 17 Cocktail Dresses Tailor-Made for the Summer Wedding Circuit


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Medicine Alumni Homecoming Weekend 2014
The UI Carver College of Medicine invites you to join our 2014 Homecoming celebration! We hope you'll attend all the events to reconnect with former classmates and faculty emeriti, tour our Medical Education and Research Facility, and enjoy a weekend on the beautiful University of Iowa campus. To view the full schedule of Homecoming events and to find hotel information, click here.
Please complete your registration below by Friday, Sept. 19. For questions, please contact email@example.com or 319-335-8886.
This event is supported by the UI Alumni Association.
Ticket information: The Medicine reunion block of football tickets has ended. To purchase tickets, please contact the Athletic Ticket Office at 1-800 IA-HAWKS (1-800 424-2957).


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Test your knowledge of the weird and wonderful world of the round-headed manc, star of 'An Idiot Abroad' and 'The Ricky Gervais Show', the one and only Karl Pilkington!
In a mysterious and frankly implausible event, everything Shakespeare ever wrote has been destroyed. Prove Karl wrong and re-write the 'Complete Works Of Shakespeare' by answering 15 consecutive questions correctly and winning an infinite amount of monkeys with typewriters.
Use your lifelines wisely:
Fifteh Fifteh: Remove two wrong answers
Ask Suzanne: When Karl doesn't know something, he asks his girlfriend. Now you can too.
Ask some monkeys: Get the monkeys to vote on what they think is the correct answer.
Who Wants To Be A Pilkington?
* Note: This application is not endorsed by Karl Pilkington or his management in any way *


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Science and technology studies bring inter- and transdisciplinary approaches to the social dimensions and effects of scientific and technological practices and institutions. Major orientations in STS look at science, technology, and medicine through the lenses of history, philosophy, sociology, and public policy. In addition, to subject guides in those fields, researchers should also explore social and humanistic content in the library's many subject guides for physical and life sciences, engineering, and medicine. Virginia Tech Department of Science and Technology in Society
Getting started with academic research | Overview of science and technology studies | Recommended databases
The History of Science, Technology, and Medicine database indexes citations and abstracts of journal articles, conference proceedings, books, dissertations, maps, and other materials on the history of science, medicine, and technology. 1600s-present.
Sociological Abstracts indexes abstracts and citations from journal articles, book reviews, books, conference papers, and theses and dissertations in the field of sociology, social and behavioral sciences, and related topics. You can limit to peer-reviewed sources. 1952-present.
The Philosopher's Index indexes citations and abstracts from journal articles, books, chapters/essays, book reviews in the fields of philosophy, aesthetics, metaphysics, social philosophy, and axiology. You can limit to peer-reviewed sources. 1940-present
ASSIA is an index of citations and abstracts to journal articles in the social sciences and education, economics, and health fields. You can limit to peer-reviewed sources. 1987-present.
Engineering Village is the search interface to three engineering databases: Compendex, Inspec, and NTIS. Compendex indexes citations and abstracts from over 5,000 engineering journals, conference papers, books, patents, dissertations, and technical reports. Inspec indexes citations and abstracts from journal articles, conference proceedings, books, and dissertations in engineering and physical sciences. NTIS indexes citations and abstracts of National Technical Information Service reports and audiovisual materials from sources such as NASA and the departments of Energy and Defense. 1884-present.


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Kristina E. Hoot1, Masako Oka2, Gangwen Han2, Erwin Bottinger3, Qinghong Zhang4 and Xiao-Jing Wang1,2
1Departments of Cell and Developmental Biology and Otolaryngology, Oregon Health and Science University, Portland, Oregon, USA. 2Department of Pathology, University of Colorado Denver, Aurora, Colorado, USA. 3Charles R. Bronfman Institute for Personalized Medicine, and Department of Medicine, Mount Sinai School of Medicine, New York, New York, USA. 4Department of Dermatology, University of Colorado Denver, Aurora, Colorado, USA.
Address correspondence to: Xiao-Jing Wang, Department of Pathology, Building RC1-N, Room P18-5128, Mail Stop 8104, University of Colorado, Denver, Aurora, Colorado 80045-0508, USA. Phone: 303.724.3001; Fax: 303.724.3712; E-mail: firstname.lastname@example.com.
Authorship note: Kristina E. Hoot and Masako Oka contributed equally to this work.
First published September 13, 2010Submitted: April 12, 2010; Accepted: July 28, 2010.
TGF-β signaling can promote tumor formation and development or suppress it, depending on the cellular context and tumor stage. A potential target of this dual effect of TGF-β is HGF, as TGF-β can inhibit or promote its expression, although the mechanisms underlying this are largely unknown. In the present study, we found that mice with keratinocyte-specific deletion of the TGF-β signaling mediator Smad2 (referred to herein as K5.Smad2–/– mice), which have increased susceptibility to squamous cell carcinomas (SCCs), exhibited angiogenesis associated with epithelial overexpression of HGF and endothelial activation of the HGF receptor c-Met. Application of a c-Met inhibitor abrogated angiogenesis, suggesting that HGF overexpression plays a major role in angiogenesis associated with epithelial Smad2 loss. On the Hgf promoter, Smad2 was mainly associated with transcriptional corepressors, whereas Smad4 was mainly associated with the transcriptional coactivator CREB-binding protein (CBP/p300). Smad2 loss caused increased binding of Smad4 and CBP/p300 to the Hgf promoter. Consistent with this, knocking down Smad2 in human keratinocytes caused increased levels of HGF, which were abrogated by concomitant knockdown of Smad3 and Smad4. Importantly, the incidence of HGF-positive human SCC was high in cases with Smad2 loss and lower when Smad4 was also lost. We therefore conclude that Smad2 loss causes HGF upregulation via loss of Smad2-mediated transcriptional repression and enhanced Smad3/4-mediated transactivation. Since Smad2 is often downregulated in human SCCs, our data suggest a therapeutic strategy of blocking HGF/c-Met activation for Smad2-deficient SCCs.
TGF-β signaling can be tumor promoting or tumor suppressing depending on the cellular context and tumor stage (1), and its signaling mediators, Smads, are involved in these dual effects. TGF-β ligand binds to heteromers of TGF-β type I and type II receptors (TGF-βRI and TGF-βRII) to induce TGF-βRI–mediated phosphorylation of Smad2 and Smad3. Phosphorylated Smad2 (p-Smad2) and p-Smad3 bind the common Smad, Smad4, and the heteromeric complexes translocate into the nucleus to regulate transcription of TGF-β target genes (2). Smad3 binds to the Smad-binding element (SBE) of a target gene, resulting in binding of Smad2 and/or Smad4 to the same SBE to transactivate or repress target genes (3). Smads can recruit transcriptional activators, such as CBP/p300, to induce gene expression (2). Smad2 can recruit transcriptional corepressors such as TGF-β–induced factor homeobox protein (TGIF), which has been shown to bind histone deacetylases (HDACs) leading to gene silencing (4). TGIF binds to Smad2 and Smad3 in competition with CBP/p300, so the ratio of CBP/p300 to TGIF in the promoter of a target gene depends on the relative levels of these 2 proteins in each cell type (4), and the same may be true for other transcriptional coregulators. Increasing numbers of studies now show competition between Smad2 and Smad3 or Smad4 for transcriptional regulation (5–7).
In human squamous cell carcinomas (SCCs), Smad2 and Smad4 are frequently downregulated through loss of 1 genetic allele or at the mRNA level (7, 8), whereas Smad3 is retained (7). Consistently, loss of even 1 allele of Smad2 in mice is sufficient to increase susceptibility to skin carcinogenesis (7, 9) due, in part, to increased Snail-mediated epithelial-to-mesenchymal transition (EMT) (7). Μice lacking epithelial Smad4 develop spontaneous SCCs in the skin and oral cavity (8, 10, 11), indicative of a dominant tumor suppressor function for Smad4. Although Smad3-null keratinocytes transduced with a v-ras oncogene exhibited increased malignancy when grafted to immunocompromised mouse skin (12), Smad3-knockout mice are resistant to skin chemical carcinogenesis (9, 13) due to abrogation of TGF-β1–mediated inflammation and gene expression critical for tumor promotion (13). Therefore, Smad3 appears to mediate both tumor suppression and promotion effects of TGF-β.
One of the potential TGF-β target genes involved in dual effects of TGF-β on cancer is HGF. HGF acts as a ligand for the receptor tyrosine kinase, c-Met. HGF and its receptor are often overexpressed in cancer (14). Activation of the c-Met receptor can lead to proliferation, antiapoptotic survival, invasion, migration in tumor epithelia, and angiogenesis in tumor stroma (14). In the latter case, HGF has been shown to be an independent, potent angiogenic factor through stimulation of endothelial cell growth, migration, scatter, and elongation (14, 15). Previous studies have shown TGF-β can promote angiogenesis and tumor invasion via stimulation of HGF expression (16, 17). Conversely, TGF-β has also been shown to inhibit HGF transcription, potentially through binding a TGF-β inhibitory element located approximately 400 bp upstream of the HGF transcription start site (18, 19), and abrogation of this effect leads to cancer development (20). The molecular mechanisms and microenvironments controlling the positive or negative effects of TGF-β signaling on HGF expression are largely unknown.
In the current study, we found that epithelial Smad2 loss caused increased angiogenesis associated with HGF overexpression and endothelial c-Met activation. Further analysis revealed a repressive role for Smad2 but an activating role for Smad4 in HGF transcription. Our findings provide important biomarkers for targeted therapy for cancer with Smad2 loss.
Epithelial Smad2 loss caused increased angiogenesis. Mice with epidermal-specific Smad2 deletion induced at 6 weeks of age were exposed to a 2-stage chemical carcinogenesis protocol as we previously reported (7). We have previously found that keratinocyte-specific deletion of Smad2 leads to increased susceptibility to skin carcinogenesis (7). We analyzed angiogenesis in SCCs from the above experiments from 19 mice with keratinocyte-specific deletion of the TGF-β signaling mediator Smad2 (referred to herein as K5.Smad2–/– mice) and 24 WT mice. CD31 staining revealed that K5.Smad2–/– mice demonstrated 3 times the vessel area of control mice (25.9% ± 4.4% vs. 7.7% ± 2.9%) (Figure 1, A and B).
Increased angiogenesis in K5.Smad2–/– SCCs and neonatal skin. (A) Immunofluorescence of K5.Smad2–/– SCCs and skin for CD31 (green) showed increased vessel area compared with SCCs derived from WT mice. Keratin 14 (K14, red) was used as a counterstain. Scale bar: 100 μm. (B) Quantification of percentage of vessel-covered stromal area in K5.Smad2–/– SCCs as determined by quantitation of immunofluorescence images. (C) Quantification of percentage of vessel-covered stromal area in K5.Smad2–/– neonatal skin as determined by quantitation of immunofluorescence images. *P < 0.05. Data are expressed as mean ± SEM.
To assess whether increased angiogenesis in K5.Smad2–/– SCCs was due to epithelial Smad2 loss or due to the secondary effects of carcinogenesis, we examined angiogenesis in the skin and oral cavity of K5.Smad2–/– and WT mice. Smad2 was deleted in the epidermis at birth or in oral epithelia of 5-week-old mice by RU486 application topically or in oral cavity of K5.CrePR1/Smad2f/f bigenic mice as we previously described (7, 8). On days 3–5, K5.Smad2–/– skin and WT skin treated with RU486 were excised for CD31 staining. K5.Smad2–/– neonatal skin contained approximately 4 times the stromal area covered in vessels compared with WT neonates (8.4% ± 2.1% vs. 2.7% ± 0.7%) (Figure 1, A and C). Similar results were also seen in oral tissues (not shown). These results indicate Smad2 loss in keratinocytes was sufficient to increase angiogenesis in the underlying stroma.
Activated HGF signaling contributed to angiogenesis associated with epithelial Smad2 loss. Since TGF-β is a known positive mediator of angiogenesis via endothelial TGF-βR Alk1–mediated Smad1/Smad5 activation (21), we assessed whether K5.Smad2–/– SCCs had increased endothelial TGF-β signaling. As previously reported, K5.Smad2–/– SCCs do not have increased TGF-β1 ligand when compared with WT SCCs (7). Consistently, K5.Smad2–/– SCCs and skin did not show increased staining of endothelial pSmad1/5/8 compared with WT (Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI43304DS1). These data suggest that angiogenesis in K5.Smad2–/– SCCs is not a direct effect of TGF-β signaling in tumor stroma.
Since K5.Smad2–/– SCCs had increased angiogenesis independent of TGF-β–mediated angiogenesis, we screened potential angiogenesis regulators associated with epithelial Smad2 loss, using an angiogenesis microarray from Superarray (OMM-033). Among the angiogenesis factors included in the array (e.g., VEGF, Flt, sFLT, MMPs, MAPKs, FGFs, IL-8, PDGFs, and TNF-α), only HGF showed a significant increase in K5.Smad2–/– SCCs compared with WT SCCs (Supplemental Figure 2). Increased HGF was primarily located in tumor epithelial cells as visualized by immunohistochemistry (IHC) staining (Supplemental Figure 3). To determine whether increased HGF ligand in K5.Smad2–/– tumors activated its signaling, we examined phosphorylation (activation) status of the HGF receptor, c-Met (p–c-Met). Immunofluorescence staining showed that K5.Smad2–/– tumors had increased p–c-Met in both tumor epithelia and endothelia compared with stage-matched WT tumors (Supplemental Figure 3). Accordingly, downstream mediators of p–c-Met, e.g., p-Akt and eNOS, were activated in both tumor epithelia and endothelia (Supplemental Figure 3). As seen in tumor samples, K5.Smad2–/– neonatal skin had markedly increased HGF compared with WT skin (Figure 2, A and B). IHC showed that HGF staining was strongest in the epidermis, followed by the superficial dermis in K5.Smad2–/– skin (Figure 2A). This staining pattern suggests keratinocyte-produced HGF acts in a paracrine nature. However, increased p–c-Met and its downstream targets p-AKT and eNOS were primarily seen in endothelial cells (Figure 2A), presumably due to a much higher level of c-Met in normal endothelial cells than keratinocytes. These results suggest that HGF upregulation in epithelial cells and its paracrine effect on c-Met activation in endothelial cells is an early effect of epithelial Smad2 loss, whereas activation of c-Met in epithelial cells is secondary to carcinogenesis, presumably due to increased c-Met levels in tumor epithelia compared with normal keratinocytes. We therefore focused on analyzing the direct effect of epithelial Smad2 loss on HGF-induced angiogenesis.
Increased HGF with activation of its downstream mediators in endothelial cells in K5.Smad2–/– skin. (A) IHC shows increased HGF staining in K5.Smad2–/– epidermis and dermis compared with WT skin (first row). Immunofluorescence (IF) shows K5.Smad2–/– skin had increased activated endothelial HGF receptor, p–c-Met (green, second row, CD31 counterstain in red), compared with WT skin. HGF signaling pathway activation demonstrated by increased endothelial p-AKT (green, third row, CD31 counterstain in red) and IHC for eNOS (fourth row) in K5.Smad2–/– skin. Arrows indicate endothelial eNOS. Scale bar: 100 μm. (B) qRT-PCR demonstrating an increased HGF mRNA expression level in K5.Smad2–/– skin compared with WT skin (fold). HGF level in WT skin was arbitrarily assigned as 1. 5 skin samples from each group were used. *P < 0.05. Data are expressed as mean ± SEM.
To determine whether HGF upregulation plays a major role in angiogenesis associated with epithelial Smad2 loss, we treated Smad2-deficient skin or oral cavity with the c-Met inhibitor PHA665752 (Tocris). Adult K5.CrePR1/Smad2f/f mice together with WT littermates were treated with RU486 topically in the skin or oral cavity for 5 days to induce Smad2 deletion (Smad2–/–) in the epidermis or oral mucosa. Since adult mouse skin has a low level of angiogenesis, we topically treated mice with tetradecanol-phorbol-13-acetate (TPA), which induces acute inflammation and angiogenesis. Subsequently, PHA665752 was topically applied to the TPA-treated mouse skin daily for 3 days. WT skin treated with the c-Met inhibitor did not exhibit a significant reduction in vessel density compared with untreated WT skin, indicating that endogenous HGF/c-Met signaling does not significantly contribute to TPA-induced angiogenesis (Figure 3A). However, the vessel density in Smad2–/– skin treated with the c-Met inhibitor was reduced to a level comparable to WT controls (Figure 3A). To determine whether c-Met inhibition also attenuates naturally occurring angiogenesis in tissues with epithelial Smad2 loss, we applied the c-Met inhibitor orally (dissolved in sesame oil) for 3 days. Examination of angiogenesis in oral tissue revealed similar effects of c-Met inhibition on epithelial Smad2 loss–associated angiogenesis in oral tissue (Figure 3B and Figure 4). Immunofluorescence staining shows that the c-Met inhibitor did not alter the number of p-Smad2–positive cells in the skin (not shown) or oral mucosa (Figure 4), but significantly reduced p–c-Met–positive cells in vessels of K5.Smad2–/– stroma (Figure 4). These results suggest that HGF-mediated c-Met activation in endothelial cells is a major factor contributing to angiogenesis associated with epithelial Smad2 loss.
Abrogation of angiogenesis by c-Met inhibition in K5.Smad2–/– skin or oral cavity. (A) Quantitation of angiogenesis demonstrates abrogation of angiogenesis by a c-Met inhibitor PHA665752 in TPA-treated K5.Smad2–/– skin. (B) Quantitation of angiogenesis demonstrates abrogation of angiogenesis by a c-Met inhibitor PHA665752 in K5.Smad2–/– tongue mucosa. **P < 0.001. Data are expressed as mean ± SEM.
c-Met inhibition abrogates angiogenesis associated with epithelial Smad2 loss. (A) IF staining of CD31 (green) shows increased angiogenesis in K5.Smad2–/– tongue mucosa, which was abrogated by a c-Met inhibitor PHA66572. K14 (red) was used for counterstain. (B) IF staining shows that PHA66572 did not affect Smad2 activation, as evidenced by staining of p-Smad2 (green). Note that p-Smad2 was specifically ablated in tongue epithelial cells but not in K5.Smad2–/– stroma. K14 (red) was used for counterstain. (C) Double IF staining of p–c-Met (green) and CD31 (red) shows that the vessels in K5.Smad2–/– tongue had increased p–c-Met staining (yellow or orange), which was abrogated by PHA66572 treatment. Bright green staining surrounding the vessels represents autofluorescence of the muscles in the tongue. Scale bar: 100 μm.
Smad2-mediated transcriptional repression of HGF in keratinocytes. We have previously shown that Smad2 and Smad4 are frequently downregulated while Smad3 is largely retained in human SCCs (7). To determine whether Smad4 loss has an effect similar to Smad2 loss on HGF overexpression, we examined HGF levels in the skin with keratinocyte-specific deletion of Smad4 (7, 8). No difference in levels of HGF mRNA (Supplemental Figure 4) and protein (not shown) were found in K5.Smad4–/– skin, suggesting that Smad4 has little effect on HGF regulation in normal keratinocytes.
Previous reports have shown that HGF is either positively or negatively regulated TGF-β (16, 18–20). To determine whether Smad2 loss–associated HGF overexpression is linked to Smad-dependent TGF-β1 effects on HGF regulation, we examined HGF levels after knocking down Smad2, Smad3, or Smad4 in human HaCaT keratinocytes using individual siRNAs specific for Smad2, -3, or -4 validated in our previous study, which knocked down each Smad without affecting other Smads (7). We verified knockdown efficiencies at the mRNA and protein levels (Supplemental Figure 5). Similar to previous reports that TGF-β1 suppresses HGF expression in normal cells (18–20), 2 ng/ml TGF-β1 treatment of mock-transfected HaCaT keratinocytes for 2 hours caused a significant reduction in HGF expression (Supplemental Figure 6). Similar to the finding in K5.Smad2–/– skin, knocking down Smad2 resulted in an approximately 4- to 5-fold increase in HGF expression with or without TGF-β1 treatment in comparison with corresponding controls (i.e., TGF-β1–untreated and –treated HaCaT cells, respectively) (Supplemental Figure 6). In contrast, knocking down Smad3 or Smad4 had no significant effect on HGF levels with or without TGF-β1 treatment (Supplemental Figure 6). These data suggest that Smad2 loss attenuated endogenous or exogenous TGF-β1–mediated HGF inhibition.
The specific link between Smad2 loss and HGF overexpression found in our study prompted us to evaluate whether Smad2 directly inhibits HGF transcription. From the TRANSFAC database, we identified a SBE –466 bp upstream of the mouse HGF transcriptional start site (TSS), a region previously shown to be repressed by TGF-β (18–19). We performed in vivo ChIP assays in neonatal mouse skin using validated Smad antibodies as we previously described (7) and confirmed that Smad2, -3, and -4 all bound to this site (Figure 5A). To evaluate whether this SBE site is critical for Smad regulation of the HGF promoter activity, we created luciferase reporter constructs with –500 bp HGF promoter sequence with and without an SBE mutation. HaCaT human keratinocytes were transfected with the HGF luciferase constructs, control renilla construct, and individual siRNAs for Smad2, -3, or -4. Consistent with the above data, knockdown of Smad2 resulted in a 5-fold increase in luciferase activity, which was abrogated by mutation of the SBE (Figure 5B). In contrast, knocking down Smad3 or Smad4 did not affect luciferase activity with either WT SBE or mutant SBE (Figure 5B). These results suggest that Smad2 binding to this SBE is critical for its repressive effect on HGF transcription; hence, Smad2 loss caused HGF overexpression.
Smad2 binding to the SBE of the HGF promoter is critical for transcriptional regulation of HGF. (A) ChIP assay showing Smad2, Smad3, and Smad4 all bound to the SBE located 466 bp upstream of the transcription start site of the HGF promoter. IgG (rabbit) was used as a negative control. (B) siRNA knockdown of Smad2 leads to 5-fold increase in HGF promoter activity, which is abrogated by mutation of the SBE. Knockdown of Smad3 and Smad4 do not increase HGF promoter activity. **P < 0.001. Data are expressed as mean ± SEM.
We then performed ChIP assays to identify potential corepressors recruited by Smad2 at the –466 bp SBE site. As expected, in K5.Smad2–/– skin, Smad2 binding was lost (Figure 6A). Conversely, in K5.Smad4–/– skin, Smad2 binding was increased by 8-fold at the SBE compared with WT skin (Figure 6A). We then performed ChIP assays for corepressors TGIF, CtBP1, and HDACs-1, -2, and -3, which have been shown to be recruited by Smad2 to other transcriptional targets (2–3). In K5.Smad2–/– skin, TGIF binding to the HGF promoter was significantly reduced whereas CtBP1 and HDAC3 binding was not significantly reduced in comparison with WT skin (Figure 6B), suggesting that TGIF binding to the HGF promoter is primarily recruited by Smad2, whereas CtBP1 and HDAC3 may also be recruited by the remaining Smad4. However, along with increased Smad2 binding in K5.Smad4–/– skin, TGIF and CtBP1 exhibited modest increases in binding to the SBE of the HGF promoter, whereas HDAC3 binding to this site increased by 45-fold compared with WT skin (Figure 6B). These data indicate Smad2 has a stronger activity than Smad4 to recruit the transcriptional corepressors, particularly HDAC3 to the SBE of the HGF promoter. To confirm that Smad2 directly bound in a complex with HDAC3 on the HGF promoter, we performed a dual-IP ChIP using WT neonatal mouse skin. We immunoprecipitated chromatin using an antibody specific to either Smad2 or Smad4, followed by precipitation of chromatin-Smad complexes using an antibody specific to HDAC3. HDAC3 bound to Smad2 10-fold more than to Smad4 (Figure 6C).
Smad2-mediated repression of HGF transcription. (A) HGF promoter occupancy by Smad2 in WT skin, K5.Smad2–/– skin, and K5.Smad4–/– skin. Smad2–/– skin has reduced Smad2 binding with remainder attributable to nonepithelial tissues. Smad4–/– skin showed an 8-fold increase in Smad2 binding. (B) Transcriptional repressors TGIF, CtBP1, and HDAC3 bound to the HGF promoter with greater affinity in K5.Smad4–/– and reduced affinity in K5.Smad2–/– skin compared with WT skin by comparative ChIP. (C) Dual-IP ChIP was performed by first immunoprecipitating Smad2 and Smad4 on the HGF promoter in WT skin, then performing a second IP to HDAC3. *P < 0.05; **P < 0.01. Data are expressed as mean ± SEM.
Increased Smad4-mediated transactivation of HGF contributed greatly to HGF overexpression in Smad2–/– keratinocytes. We have previously shown that Smad2 loss in keratinocytes causes increased Smad4 binding and transactivation of the Snail promoter without changes to the level of Smad3 or Smad4 protein expression (7). To assess whether a similar mechanism also contributes to increased HGF overexpression in Smad2-deficient cells, i.e., a compensatory activation of Smad4-mediated HGF transcription, we knocked down Smad2, -3, or -4 individually or in combination in human HaCaT keratinocytes and assayed for expression levels of endogenous HGF. Similar to the HGF promoter reporter assay (Figure 5B), siRNA knockdown of Smad2 leads to increased HGF mRNA levels, whereas knocking down either Smad3 or Smad4 alone did not significantly affect HGF expression levels (Figure 7A). However, knockdown of Smad3 and Smad4 together resulted in reduced HGF expression compared with control (Figure 7A). In addition, concomitant knockdown of Smad3 or Smad4 with Smad2 abrogated Smad2 loss–associated HGF overexpression (Figure 7A). These results suggest that endogenous Smad3 and Smad4 together activate normal HGF expression, which could contribute to HGF overexpression in Smad2-depleted cells.
Smad4-mediated transactivation of HGF transcription. (A) Knockdown of Smad2 dramatically upregulates HGF expression, which is abrogated by concomitant knockdown of either Smad3 or Smad4. Knockdown of Smad3 and Smad4 leads to reduced HGF expression compared with mock treatment. *P < 0.05 compared with mock treatment. ‡P < 0.05 compared with Smad2 siRNA. (B) HGF promoter occupancy by Smad3 and Smad4 in WT skin, K5.Smad2–/– skin, and K5.Smad4–/– skin. As expected, Smad4 binding is reduced in Smad4–/– skin. Smad2–/– skin showed an increase in Smad3 and Smad4 binding. *P < 0.05 compared with WT skin. (C) Transcriptional activators CBP/p300 and RNA Pol II bound with greater affinity in K5.Smad2–/– skin, correlating with Smad2 loss–associated HGF upregulation. CBP/p300 binding was modestly increased in Smad4–/– skin, which showed an increase in Smad2 binding. RNA Pol II was reduced in Smad4–/– skin. *P < 0.05; **P < 0.01 compared with WT skin. (D) Dual-IP ChIP was performed by immunoprecipitating Smad2 and Smad4 on the HGF promoter in WT skin, then performing a second IP to CBP/p300. Transcriptional activator CBP/p300 bound to Smad4 with 75-fold greater affinity than to Smad2. *P < 0.05. Data are expressed as mean ± SEM.
We then determined whether Smad3 and Smad4 binding to the HGF promoter was increased in K5.Smad2–/– skin. ChIP assays showed that Smad3 binding was modestly increased (~3.5-fold) and Smad4 binding was dramatically increased (~48 fold) to the SBE of the HGF promoter in K5.Smad2–/– skin (Figure 7B). Since we have shown that Smad2 loss does not cause compensatory Smad4 overexpression (7), increased Smad4 binding to the HGF promoter in Smad2-deficient cells could be target specific instead of global Smad4 activation. To further evaluate whether increased Smad4 binding in K5.Smad2–/– cells changed the recruitment of transcriptional coactivators on the SBE site of the HGF promoter, we performed ChIP assays for CBP/p300, a previously known coactivator of Smad4 (2–3) and RNA polymerase II (RNA Pol II). Consistent with increased Smad4 binding in K5.Smad2–/– skin, CBP/p300 showed increased binding to the SBE of the HGF promoter over 200-fold compared with WT skin (Figure 7D). Additionally, RNA Pol II binding to the SBE of the HGF promoter was increased by 2.5-fold in K5.Smad2–/– skin (Figure 7D). In contrast, in K5.Smad4–/– skin that has increased Smad2 binding (Figure 6A), CBP/p300 binding was only increased 10-fold, whereas RNA Pol II binding was actually reduced by 75% compared with WT skin (Figure 7D), which is consistent with increased binding of Smad2 and corepressors in the HGF promoter (Figure 6). Further, we compared recruitment of CBP/p300 by Smad2 and Smad4 to the HGF promoter, using a dual-IP ChIP assay in WT mouse skin with an antibody specific for Smad2 or Smad4, followed by immunoprecipitation of Smad/DNA complexes with the CBP/p300 antibody. We found 75-fold more CBP/p300 bound to Smad4 than to Smad2 on the HGF promoter (Figure 7E). Taken together, these data show that Smad4 primarily recruits a transcriptional coactivator to the HGF promoter and transactivates HGF; hence, a significant increase in Smad4/CBP binding to the HGF promoter in Smad2-deficient cells contributes greatly to HGF overexpression.
High incidence of HGF overexpression in human SCCs with Smad2 loss. HGF is normally expressed in mesenchymal cells but is often overexpressed in epithelial tissues of developing cancers (22). To determine whether the mechanisms of Smad2 loss–associated HGF overexpression found in our analyses contribute to HGF overexpression in human SCCs, we performed IHC staining of HGF on human SCC tissue arrays containing 74 skin SCCs and 113 head and neck SCCs (HNSCCs) (Biomax). Similar to our previous reports (7, 8), approximately 60%–70% of SCCs lost either or both Smad2 and Smad4 (not shown). HGF was not detectable in normal tissues (Figure 8), but was detected in 60% (45/75) of skin SCCs and in 45% (38/84) of HNSCCs. Consistent with our findings in animal models and in in vitro analyses, among skin SCCs that lacked Smad2 protein but retained Smad3/Smad4 protein, HGF was detectable in most of the SCCs (Table 1 and Figure 8). HGF-positive cases were reduced in Smad2-negative cases when they also lost Smad4 protein (Figure 8) and were further reduced in SCCs when Smad3 was also lost (Table 1). These data further support that Smad2 loss together with Smad3/Smad4-mediated transactivation contributed to HGF overexpression in at least some human SCC cases. HGF overexpression in cases of all 3 Smads positive or all 3 Smads negative for Smad2, Smad3, and Smad4 could represent Smad-independent mechanisms of HGF regulation. For instance, hypoxia-induced factors and increased MMP activity, which are commonly associated with cancer, largely contribute to HGF induction (22).
HGF expression correlates with Smad2 loss but Smad4 retention in human SCCs. IHC staining showing that normal oral mucosa is HGF negative but positive for both Smad2 and Smad4. In SCCs from head and neck (here) or skin (not shown), epithelial HGF staining correlated with the Smad2-negative, Smad4-positive staining pattern. In SCC without strong staining of both Smad2 and Smad4, HGF staining in epithelia was weak. Scale bar: 100 μm.
HGF-positive cases correlated with Smad2-negative/Smad4-positive status
HGF overexpression plays a critical role in angiogenesis associated with epithelial Smad2 loss. We have previously shown that Smad2 loss in keratinocytes caused an early onset EMT, which contributes to increased susceptibility to skin cancer formation and malignant progression (7). In the current study, we observed increased angiogenesis in K5.Smad2–/– tissues and tumors. Since Smad2 was only deleted in keratinocytes, increased angiogenesis in the stroma suggests that angiogenic regulators secreted from keratinocytes were altered by Smad2 deletion. As loss of a TGF-β signaling component in keratinocytes often causes compensatory TGF-β1 overexpression (8, 23), we determined whether increased angiogenesis in K5.Smad2–/– tissues was due to increased TGF-β1 that could directly induce angiogenesis (21) or due to increased VEGF, which can be activated by Smad3 and is seen after Smad4 is knocked out in keratinocytes (24) or in breast cancer cells after knocking down Smad2 (25). However, we observed neither increased TGF-β1 nor increased VEGF production in nonneoplastic K5.Smad2–/– tissues or SCCs compared with WT samples, possibly due to a lack of further enhancement of Smad3 activation seen in Smad4–/– keratinocytes (8), which directly transactivates VEGF (26, 27). These results highlight the context-specific nature of Smad transcriptional regulation. Using an unbiased screening, we identified that Smad2 loss induces overexpression of HGF. In nonneoplastic K5.Smad2–/– tissues, we did not observe consequent activation of HGF receptor c-Met in epithelial cells, presumably due to a low level of c-Met in normal epithelial cells. However, at this stage, overexpressed HGF is sufficient to activate c-Met in endothelial cells. Further, treating K5.Smad2–/– mice with a c-Met inhibitor completely abrogated increased angiogenesis to a baseline level seen in normal tissues, suggesting that HGF overexpression is a major contributor to angiogenesis associated with epithelial Smad2 loss. This finding has an important implication for a therapeutic strategy targeting SCCs. We have shown that loss of 1 Smad2 allele, which contributes to at least a 50% reduction of Smad2 protein, occurs in approximately 40% of human SCCs and that overall Smad2 protein loss occurs in approximately 70% of human SCCs (7). Our current study suggests that Smad2 loss is an important factor contributing to HGF overexpression in human SCCs. Since Smad2 is haploid insufficient, i.e., 50% of Smad2 loss is sufficient to increase skin cancer susceptibility (7), it would be difficult to restore genetically lost Smad2 to a normal level when treating SCC patients. Therefore, if we can block Smad2 loss–mediated angiogenesis using a c-Met inhibitor, Smad2 loss–associated malignant progression may be attenuated or delayed. As seen in our current study, since HGF is barely detectable in normal tissue, the c-Met inhibitor did not significantly affect normal angiogenesis, which could be beneficial as a targeted therapy. Nevertheless, since cancer-associated angiogenesis involves multiple pathways and often harbors oncogene addiction (28), it remains to be determined whether blocking HGF-mediated angiogenesis can significantly slow down or starve tumor cells in Smad2-deficient SCCs.
HGF transcription is negatively regulated by Smad2 but positively regulated by Smad4. TGF-β can stimulate HGF production (16, 17, 29) but can also represses HGF (30, 31). As summarized in Figure 9, our current study revealed an important mechanism underlying this context-specific effect of TGF-β signaling on HGF transcriptional regulation, which largely depends on the ratio of Smad2 and Smad4 in cells. In normal keratinocytes, Smad2, -3, and -4 all bind to the –466 bp SBE of the HGF promoter (Figure 5). In this complex, Smad2 primarily recruits transcriptional corepressors (e.g., TGIF, CtBP1, and HDAC3; Figure 6), whereas Smad4 primarily recruits transcriptional coactivator CBP/p300 (Figure 7). Since normal keratinocytes produce very low levels of TGF-β, the recruitment of either corepressors or coactivators are expected to be at low levels. Together with the balance between the recruitment of corepressors and coactivators, virtually no HGF can be detected in normal keratinocytes. Because Smad3 has the strongest DNA binding, loss of Smad2 only modestly increased Smad3 binding, whereas loss of Smad4 did not significantly affect Smad3 binding to the HGF promoter. Smad4 loss in normal keratinocytes had no significant effect on baseline HGF expression, despite increased binding of Smad2 and corepressors, i.e., TGIF, CtBP1, and HDAC3. Two possibilities could contribute to this result. First, consistent with a previous report that Smad2 can recruit both corepressors and coactivator CBP/p300 (4), increased CBP/p300 binding to the HGF promoter was also seen in K5.Smad4–/– skin, which was presumably a result of increased Smad2 binding. Second, the remaining Smad3 could still transactivate HGF, as knocking down both Smad3 and Smad4 exhibited reduced baseline HGF levels. Therefore, the balance between Smad2/Smad3-mediated transactivation and an increase in Smad2-mediated repression kept HGF levels largely unaffected in Smad4–/– keratinocytes. In contrast to Smad4 loss, Smad2 loss had a dramatic effect on HGF levels. Smad2 loss caused a dramatic increase in Smad4 binding to the HGF promoter. The shift from Smad2 binding to Smad4 binding also caused a significant increase in the recruitment of transcriptional coactivator CBP/p300. Therefore, the significant increase in CBP/p300 binding apparently dominates transactivation of HGF. Supporting this notion, ablating Smad2 in keratinocytes resulted in an increased HGF expression, which was abrogated when Smad4 was also ablated. The correlation between HGF expression and Smad2-negative/Smad4-positive status in human SCCs is also consistent with our molecular analysis and our findings from K5.Smad2–/– and K5.Smad4–/– mouse models. In addition to the direct transcriptional regulation of HGF by Smad2 and Smad4, it has been shown that mesenchymal cells have HGF-dependent angiogenesis (30–34). Since Smad2–/– keratinocytes undergo EMT (7), this may also allow a mesenchymal transcriptional environment that promotes HGF transcription.
Schematic of Smad transcriptional regulation of HGF. WT skin has Smad2, -3, and -4 bound to the HGF promoter 466 bp upstream of the TSS, with the various Smads having recruited transcriptional activators (CBP/p300) and repressors (TGIF, CtBP1, and HDAC3). When Smad4 is lost, increased Smad2 is bound, recruiting transcriptional repressors and preventing HGF expression. In contrast, when Smad2 is lost, increased Smad3 and Smad4 bind to the HGF promoter, recruiting transcriptional activators and promoting HGF expression.
In summary, we report that Smad2 normally represses HGF expression, and its loss caused HGF overexpression associated with loss of this repression and, perhaps even more, with increased Smad4 transactivation of HGF. Increased HGF contributes greatly to Smad2 loss–associated angiogenesis, which can be abrogated by inhibition of c-Met. Our study indicates that although Smad4 is a potent tumor suppressor, Smad2 loss–associated increase in Smad4 binding to the HGF promoter beyond a physiological level facilitates angiogenesis, which could contribute to tumor promotion. Our study necessitates future analysis of whether HGF-mediated angiogenesis contributes to tumor formation and malignant progression in tissues with epithelial Smad2 loss and therefore can be pharmacologically targeted for SCC treatment.
All studies were reviewed and approved by IACUC and IRB at Oregon Health and Science University and by IACUC and IRB at University of Colorado Denver.
Generation of inducible and keratinocyte-specific Smad2- and Smad4-knockout mice. Inducible K5.Smad2–/– and K5.Smad4–/– mice were generated as we previously reported (7, 8). Cre-mediated Smad2 or Smad4 deletion in keratinocytes was achieved with topical application of RU486 (20 μg in 100 μl ethanol in the skin or 40 μg in 50 μl in sesame oil in oral mucosa) once a day for 3–5 days at time points specified in the Results section. Smad2 or Smad4 gene deletion was detected by PCR on DNA extracted from RU486-treated skin, using deletion-specific primers (35, 36).
RNA extraction and quantitative RT-PCR. Total RNA was isolated from mouse skin and tumors using RNAzol B (Tel-Test), as previously described (37), and further purified using a QIAGEN RNeasy Mini Kit (QIAGEN). Quantitative RT-PCR (qRT-PCR) was performed as we have previously described (38). HGF levels were determined using Power SYBR Green Master Mix (Applied Biosystems) and custom primers (for mouse: 1F-GAACTGCAAGCATGATGTGG, 1R-GATGCTGGAAATAGGGCAGAA; for human: 1F- AAAGGACTTCCATTCACTTGC, 1R-CGCTCTCCCTTACTCAAGCTA). A GAPDH RNA probe was used as an internal control. Three to 9 samples from each genotype of mice were used for qRT-PCR. In analyzing the relative expression levels of individual genes, the average expression level from WT samples (unless otherwise specified) of each particular gene being analyzed was set as "1" arbitrary unit.
Tissue histology and IHC. Dissected mouse tissue and tumor samples were fixed in 10% neutral-buffered formalin at 4°C overnight, embedded in paraffin, and sectioned to 6-μm thickness. Human tissue arrays of skin SCCs and head and neck SCCs (without personal information) were purchased from Biomax. IHC was performed on paraffin sections of mouse and human SCC samples, as we have previously described (39), using primary antibodies against HGF (1:10; R&D Systems), p–c-Met (1:50; Cell Signaling), p-Smad2 (1:100; Cell Signaling), Smad2 (1:100; Zymed), Smad4 (1:100; Upstate), and eNOS (1:50; Abcam). Sections were counterstained with hematoxylin. A double-blind evaluation for IHC was performed by 2 investigators using methods we have previously described (39).
Double-stain immunofluorescence. Each section was incubated overnight at 4°C with a primary antibody together with either a guinea pig antiserum against mouse K14 (1:400; Fitzgerald), which highlights the epithelial compartment of the skin or oral mucosa (37), or CD31 (1:200; BD Biosciences) which highlights endothelium. The primary antibodies included p-Smad1/5/8 (1:50; Cell Signaling), p-Smad2 (1:100, Cell Signaling), p-AKT (1:100; Cell Signaling), and p–c-Met (1:50; Cell Signaling). Frozen sections were fixed for 2 minutes in methanol. An Alexa Fluor 488–conjugated (green) secondary antibody against the species of the primary antibody (1:400; Molecular Probes) and Alexa Fluor 594–conjugated (red) anti-guinea pig secondary antibody (1:400; Molecular Probes) for K14 counterstain or Alexa Fluor 594–conjugated (red) anti-rat secondary antibody (1:400; Molecular Probes) for CD31 was used to visualize the staining. Sections were visualized under a Nikon Eclipse E600W fluorescence microscope. Images were acquired using MetaMorph (Universal Imaging Corp.) and processed using Adobe Photoshop 6.0. Quantitation of angiogenesis was done using ImageJ software (NIH) and expressed as percentage of stroma occupied by vessels ± SEM.
c-Met inhibitor treatment. Adult K5.CrePR1/Smad2f/f mice together with their WT littermates were topically treated with RU486 for 5 days to induce Smad2 deletion (Smad2–/–) in the skin. These mice were then topically treated with 5 μg of tetradecanol-phorbol-13-acetate (TPA), which induces acute inflammation and angiogenesis in the skin. Subsequently, a c-Met inhibitor, PHA665752 (Tocris, dissolved in DMSO and then diluted in 100% ethanol), was topically applied to the skin of the above mice, 16.5 μg /mouse, daily for 3 days, beginning on the same day of TPA application. In a separate experiment, Smad2 was deleted by oral RU486 treatment for 5 days, followed by a c-Met inhibitor PHA665752 (dissolved in sesame oil) daily for 3 days to assess its effect on naturally occurring angiogenesis in oral mucosa.
Quantitative chromatin immunoprecipitation. At least 4 mouse backskins from each group of WT, K5.Smad2–/–, and K5.Smad4–/– mice were homogenized on ice in 5 ml of 1% formalin and incubated at room temperature for 30 minutes after adding an additional 5 ml of 1% formalin to each tube. Each sample was then diluted in 1 ml of 10× Glycine Stop Solution (Active Motif), incubated at room temperature for 5 minutes, and then centrifuged at 250 g for 10 minutes at 4°C. The resulting pellet was used for ChIP Enzymatic Digestion following the manufacturer's protocol (Active Motif). Antibodies, 3 μg each, to Smad2 (Zymed), Smad3 (Upstate), Smad4 (Upstate), RNA Pol II (Upstate), CBP/p300 (Upstate), TGIF (Santa Cruz Biotechnology Inc.), CtBP1 (Upstate), and HDAC3 (Abcam) were used to immunoprecipitate the sheared chromatin complexes. Rabbit IgG (3 μg; Santa Cruz Biotechnology Inc.) or mouse IgG (3 μg; Upstate) was employed as a negative control for antibody specificity. DNA obtained from eluted beads was used for quantitative PCR using Power SYBR Green Master Mix (Applied Biosystems). Primers encompassing the SBE sites of the HGF promoter (FP, AGTCCAACGGGTCTCAAGTG; RP, CCAAACCACTGCAAAAGGAT) were used for PCR. Positive binding was defined as antibody binding greater than 10-fold of the IgG-negative control. PCR encompassing the coding region was used as a negative control for specificity of SBE binding. ΔCt values were obtained by normalizing IP Ct values to input values for each group. ΔΔCt values were obtained by comparing the ΔCt values of K5.Smad2–/– and K5.Smad4–/– skin to WT skin. Values are expressed as fold change based on ΔΔCt values.
Dual-IP ChIP. At least 4 mouse backskins from WT mice were homogenized on ice in 5 ml of 1% formalin in 10 mM dimethyl adipimidate 2HCl. After homogenizing, another 5 ml of cross-linking solution was added and samples were incubated at 37°C for 10 minutes and then at room temperature for 20 minutes. Each sample was then diluted in 1 ml of 10× Glycine Stop Solution (Active Motif), incubated at room temperature for 5 minutes, and then centrifuged at 250 g for 10 minutes at 4°C. The resulting pellet was used for ChIP Enzymatic Digestion following the manufacturer's protocol (Active Motif). Antibodies, 3 μg each, to Smad2 (Zymed) and Smad4 (Upstate) were used for the initial immunoprecipitation of the sheared chromatin complexes overnight at 4°C. Rabbit IgG (3 μg, Santa Cruz Biotechnology Inc.) was used as a negative control for antibody specificity. Complexes were eluted from Protein G Beads using 50 μl of the provided elution buffer (Active Motif). Antibodies, 3 μg each, to CBP/p300 (Upstate) and HDAC3 (Abcam) were used for the second immunoprecipitation, performed overnight at 4°C. Rabbit IgG (3 μg, Santa Cruz Biotechnology Inc.) was used as a negative control for antibody specificity. Elution and cross-link reversal was performed as per manufacturer's instructions (Active Motif). DNA obtained from eluted beads was used for quantitative PCR using Power SYBR Green Master Mix (Applied Biosystems). Primers encompassing the SBE sites of the HGF promoter (above) were used for PCR. Positive binding was defined as antibody binding greater than 10-fold of the IgG-negative control. ΔCt values were obtained by normalizing IP Ct values to values for chromatin having been immunoprecipitated only once for Smad2 or Smad4 respectively. ΔΔCt values were obtained by comparing the ΔCt values of Smad4-initial-IP to the Smad2-initial-IP, i.e., Smad4(1st IP)-HDAC3(2nd IP) versus Smad2(1st IP)-HDAC3(2nd IP). Values are expressed as fold change based on ΔΔCt values.
HaCaT keratinocyte culture and siRNA knockdown. HaCaT keratinocytes were cultured in high-glucose DMEM with 10% FBS and penn-strep antibiotics. Twenty-four hours prior to siRNA transfection, cells were switched to low-glucose DMEM with 0.2% FBS and penn-strep antibiotics. Cells were transfected with siRNAs for human Smad2, Smad3, or Smad4: Smad2: UUCUCAAGCUCAUCUAACCGUCCUG, Invitrogen (7), or GGAGUGCGCUUAUACUACAtt, Applied Biosystems (Supplemental Figure 5); Smad3: CCUGCUGGAUUGAGCUACACCUGAA, Invitrogen (7), or GUCUACCAGUUGACCCGAAtt, Applied Biosystems (Supplemental Figure 5); Smad4: GGUGAUGUUUGGGUCAGGUGCCUUA, Invitrogen (7), GGACAUUCAAUUCAAACCAtt, Applied Biosystems (Supplemental Figure 5). XtremeGene siRNA Transfection Reagent (Roche) was used in 6-well plates at a final concentration of 50 pmol siRNA/μl in Opti-MEM media (Gibco; Invitrogen). Four hours after transfection, media was switched to high-glucose DMEM. Cells were harvested at 72 hours after siRNA transfection for RNA extraction using QIAshredder and RNeasy kits (QIAGEN). For HaCaT cells treated with TGF-β1, a final concentration of 2 ng/ml TGF-β1 was added to culture medium the last 2 hours prior to harvesting the cells.
Luciferase constructs. Normal C57BL/6 mouse genomic DNA was used to amplify the HGF promoter region using the following set of primers using the Easy-A cloning kit (Promega): FP, CTCCCTCCCTGAAGACTGTG; RP, CTCAGCCCAATCGCTAAGTC. The gel-extracted promoter was ligated to the pGEM T-Easy vector (Promega) as per the manufacturer's instructions. The vector was sequence verified. Using XhoI- and HindIII-labeled forward and reverse primers from above, we amplified the promoter. Using XhoI and HindIII (New England Biosystems) restriction enzymes, we cut the promoter fragment and cloned it into pGL4.26 Luciferase Vector (Promega). A Stratagene QuikChange Kit was used to mutate the SBE from CTGT to TTGA as we previously described (24).
HGF-pGL4.26, pGL4.74 (Promega), and siRNAs were transfected into 50% confluent HaCaT cells in a 24-well plate using XtremeGene siRNA Transfection Reagent (Roche). Cells were harvested for luciferase using the Dual Luciferase Assay reagents (Promega) as per the manufacturer's instructions. Luciferase reporter assays were done using the 96-well plate GloMax dual-injector luminometer (Promega).
Protein extraction and ELISA. Protein extraction was performed by homogenizing tissue in Complete Lysis Buffer M (Roche). Total protein was determined using detergent-compatible Bradford Assay reagents (Bio-Rad). Quantikine ELISA kit for VEGF (R&D Systems) was used to determine the concentration of VEGF, as per the manufacturer's instructions.
Statistics. Significant differences between the values obtained in each assay on samples from various genotypes were determined using Student's t test and expressed as mean ± SEM, with the exception of evaluation of human SCCs, where a 2-tailed Fisher's exact test was used. P values considered significant (P < 0.05) are denoted in each figure or table.
View Supplemental data
This work was supported by NIH grants CA87849, CA79998, and DE15953 to X.J. Wang; and R01CA115468 to Q.H. Zhang. K. Hoot is a recipient of NIH training grant HD46420. M. Oka is a recipient of the fellowship from the Uehara Memorial Life Science Foundation, Japan. The authors thank Pamela Garl for proofreading this manuscript.
Conflict of interest: The authors have declared that no conflict of interest exists.
Citation for this article:J Clin Invest. 2010;120(10):3606–3616. doi:10.1172/JCI43304.


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Reviewed Garden Vegetable Soup
Nov 1, 2012 in Food Network Community Toolbox on FoodNetwork.com
"I followed the recipe to the T and found it lacking flavor- even after letting the flavors meddle in the fridge overnight. I decided to add some butter beans and kale, then thyme, celery seed, dried oregano, and dried basil. I'm always looking for go-to soup recipes--- this one did not make the cut. I won't be making this again, and I don't recomm""


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IN THE WINNER'S CIRCLE with JESSICA KORDA
2014 Pure Silk-Bahamas LPGA Classic
Ocean Club Golf Course
January 26, 2014
Born: Bradenton, Fla.
Lives: Bradenton, Fla.
Birthdate: February 27, 1993 – currently 20 years, 10 months, and 30 days
Qualified for LPGA Tour: Finished runner-up at the 2011 LPGA Final Qualifying Tournament
Turned Professional: December 2010
- LPGA: 2nd career victory. Previous wins: 2012 ISPS Handa Women's Australian Open
- CAREER: With the $195,000 first-place check, Jessica moves to 236th on the LPGA Career Money List with $795,511
- SEASON: Jessica has earned $195,000 this season and is first on the 2014 LPGA Official Money List
- Previous best finish was T32 at the 2013 Pure Silk Bahamas LPGA Classic
- With her win, Jessica is projected to move from No. 40 to No. 26 in the Rolex Women's World Golf Rankings
- 21 events, 21 cuts made, $593,389 (25)
- 6 top-10 finishes including a season-best tie for second at the Mobile Bay LPGA Classic
- Competed on her first U.S. Solheim Cup Team and posted a 1-2-1 record
- Earns 500 points toward the Race to the CME Globe
- Earns 30 points toward the Rolex Player of the Year race and currently ranks first
- Earns 40 points toward the U.S. Solheim Cup Team and moves into fourth place with 59 points
Topics: In the winners circle, Pure Silk Bahamas LPGA Classic, Korda, Jessica


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I am going there this summer. My family has property in Jeffrey's Bay, right by the beach. How is it there? I don't know what to expect I have no idea how it looks, I haven't been there ever, and the last tim it was seen was at least 20 years ago. |
Follow-Up Postings:
- Posted by frankstives Z10 (My Page) on Sun, Mar 21, 04 at 5:31
I went there last October, mainly in Cape area ,West as far as Nysna and then up the West Coast to Springbok We went Flydrive, good roads with virtually no cars navigation no problem Safety again no problem, we did however avoid parts of Capetown late at night, everyone was very friendly We visited Kirsenbosch, Stellenbosch, Harold Porter and Little Karoo BG. also Karas Farm and The Hester Malin Garden in The Geogap Nature Reserve all worth a visit Lots of other small Botanic places to numerous to mention Hope this helps Frank |
You mean, nobody has seen the property in 20 years?! Are you sure it is still there?? ;-) |
Here is a link that might be useful: Jeffrey's Bay
- Posted by Phoenicophorium z 8/9/11 W/Z/CA (email@example.com) on Mon, Mar 22, 04 at 15:15
Nobody I that I know of. We bought it and then moved to the United States. Don't know whats become of it. WHat types of weeds are from that area? |
Hi I am sure you are going to love it. I live in Johannesburg but I have been to "J-Bay" as it is known. Frank is right. It is fairly safe but you must not make yourself a target. No matter where you are in cities or in rural areas, always be aware of what's going on around you. Crime is everywhere. If you are just careful as you would be in any major city, you should not have a problem. Do not stop your car in deserted areas. Try not looking like tourists, ie with cameras, etc. Do not openly display expensive items like cell phones, rings, etc. I hope I haven't scared you but do not fall into the trap of thinking you are out in the country, away from big cities so you can relax completely. While you are in that area visit Mountain Zebra National Park and Addo Elephant National Park. Both are beautiful. Visit www.parks-sa.co.za for more info. |
- Posted by Phoenicophorium z 8/9/11 W/Z/CA (firstname.lastname@example.com) on Tue, Mar 23, 04 at 19:07
Thank you that will be useful! Coincidently, oday at school, someone brought a gun and ran around with it poitning it at people. (newport Highschool) It was a very dramatic day today, but everything turned out alright, no one was hurt, except for the kid, who is now arrested. Yesterday many people were making fun of him, and he is very individual. |
What types of weeds are from that area? None! The weeds all come mostly from the Americas, but also Europe or Australia! ;-) But seriously, now. If you are interested in succulent plants, the Eastern Cape is a paradise! Many wonderful wild areas around Port Elisabeth to explore. Even a Nature Reserve in the middle of Town where you can see Haworthia cymbiformis growing in big chunks on the cliffs. Go east to Grahamstown and Alicedale (where, incidentally, is also the Shamwari Game Reserve). Or west to Uniondale and Willowmore. Or north to Graaf-Reinet which is very well worth a visit, too. Enjoy your holiday and have a lot of fun! |
We just got back from 2 months in the Western Cape, mainly the Knysna -Plettenberg area, but also saw most of the Cape, including parts of the Little Karoo, and went as far east as Jeffrey's Bay. That part of the Eastern Cape is very windy and kind of barren, but succulent plants do well there, especially aloes. The weeds really are from other continents, and you don't find them all over the place or smothering gardens the way you do in North America. Try to get to the Western Cape for the fynbos, seeing proteas in flower, and sugarbirds on them, is a great experience; the botanical gardens mentioned are a must, and Harold Porter, near Betty's Bay is fabulous (while in that area, you should also see the penguins) and as special in its way as Kirstenbosch in Cape Town. Ttsitstikamma National Park (not too far from Jeffrey's) is exquisite, and shouldn't be missed. South Africans really are very friendly; we met with no crime, and didn't have to take any more precautions than at home - just use common sense. We visited townships and informal settlements, and while the poverty is very sad, we found people friendly. By the way, the only places we've ever been mugged and pickpocketed were in Europe! |
My wife and I spent the first two weeks of Feb. in South Africa, mostly the Western Cape, but also east somewhat on the garden route. Other than a few aggressive child panhandlers, we found Capetown very safe, although we did not go out at night. We took the local minitaxis and buses and had no problems. Everyone was very friendly. I hear Jo'berg can be problematic, tho. Like others on this board, I would recommend you keep your wits about you at all times. In Capetown, tho, I saw tourists digging in their wallets and flashing cameras all the time in public. If you get a chance, Kirstenbosch is fantastic, and the Little Karoo and Stellenbosch University gardens also are wonderful. We drove quite a bit and once I got used to driving on the left side and to South Africans' driving habits (very fast), it actually was fun. The roads are in better shape than here in the U.S. |
- Posted by RobertEire (My Page) on Mon, Mar 29, 04 at 4:04
Be aware that the area you are going to is a summer rainfall region, and you are heading there in their winter, so it will be mostly dry. Also be aware that they are in the midst of a drought that shows no signs of letting up, so aside from the Knysna and Tsitsikamma forests things are likely to look a bit barren. If you find yourself in Cape Town for any length of time, there is a municipal nature reserve in Durbanville (just before the racecourse) that is well worth a visit. It is a small but very rich collection of native fynbos and renosterbos. |
- Posted by SowthEfrikan z7b/NE TXexas (My Page) on Thu, Apr 1, 04 at 12:48
National Geographic is featuring SA in it's new issue, specifically South Africa. The journalist sounds like a bit of a woosie, he was gaaing aan about it being his most dangerous assignment ever and needing a bodyguard. Shame hey. |
- Posted by RobertEire (My Page) on Fri, Apr 2, 04 at 10:05
SowthEfrikan, there is a documentary soon on BBC from another bitter Englishman doing his best to down South Africa. You know what they say - never let the truth get in the way of a good story... |
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James Dando
James Dando is an avid writer and consumer of current affairs.
He is educated in the arts and, like other members of Gen Y, he has traipsed the world, never alighting anywhere for long. He's motorcycled through West Africa, chaperoned celebrity tours of London and assembled time clocks in Annandale.
He currently makes a living in Sydney, sorting excited children into the Hogwarts House of their choice.


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ISLAMABAD - The arrest of an imam on suspicion of planting evidence to frame a Christian girl accused of defaming Islam is sparking divisions in Pakistan, with some residents see the detention as a plot against the religious leader.
This is a conspiracy against him (Imam), and he is being penalized for raising this issue, Malik Banarus, a member of the Ameena Mosque, told OnIslam.net on Monday, September 3.
We all stand alongside him.
Pakistan Muslims Defend Blasphemy Girl
Twitter Blasphemy Freedom of Expression?Why Should Blasphemy Be Punishable at All?
Imam Khalid Jadoon was arrested on Sunday on suspicion of putting two burnt pages of the Noble Qur'an in the page of Christian girl Ramsha.
Jadoon was ordered to remand in custody on Monday till September 16, after Hafiz Zubair, a mosque Muezzin, testified in court that the imam had put the burnt pages in Ramsha's bags to underpin the blasphemy charge against her.
Two more eyewitnesses also backed Zubair's testimony, a claim denied by the imam.
But residents cast doubts on the testimonies.
Hafiz Zubair is not even an n area resident, Banarus said. But even that, he forcibly pokes his nose into mosque affairs.
Malik Shaukat, a member of the mosque committee, also denies the charges against the imam.
Hafiz Zubair harbors bitterness for Imam Jadoon, he told OnIslam.net.
He (Zubair) a few months ago conspired against the former imam of this mosque, who had been leading prayers here for last 22 years, on the pretext of wrongly accentuating Qur'anic verses.
There are witnesses who saw her (Ramsha) throwing the plastic bags, carrying half-burnt pages of the Holy Qur'an on a pile of trash in the locality, he said, arguing that the mother of the Christian girl not only admitted the mistake committed by her daughter, but also sought apology.
We did not take law into the hand despite the fact the area residents were enraged. We have taken the matter to the court, and we expect justice.
Rimsha, which is reported to be suffering from down syndrome, was arrested last month under Pakistan's blasphemy law over holding burnt pages which had Islamic text and Qur'anic verses on them.
But the arrest has prompted outcry from Western governments, the Vatican and rights groups, who have complained that blasphemy law are often abused to settle personal scores.
A conviction for blasphemy is punishable by death in Pakistan.
But the imam's arrest triggered a wait-and-see position from Pakistan's religious parties.
It is premature to say anything definitive in this regard, Syed Munawwar Hasan, Chief of Jammat-e-Islami, one of the two mainstream religious parties in Pakistan, told OnIslam.net.
Let the court do its job. It is not a right time to give any reaction at this stage.
He opines that any interference in judicial rulings will be counterproductive.
The most important thing in this matter is that the culprit should be taken to task, and innocent should be released.
Allama Tahir Ashrafi, the chairman of Ulema Council, called for the release of the Christian girl.
The arrest of the imam and statements (of witnesses) against him have proved that the girl is not involved in desecration of the Holy Qur'an. Therefore, she should be released immediately, Ashrafi told OnIslam.net.
She (Ramsha) is the daughter of Pakistan. I demand the government to release and provide her full security.
If the government cannot provide her security, then Ulema Council is ready to perform this duty, he said.
Ayaz Ameer, a veteran columnist and a member of the National Assembly of the right-wing Pakistan Muslim League (N), said he personally had investigated the incident and found that the half-burnt pages were not from the Qur'an, but from Noorani Qaeda, a primer for children learning Arabic alphabets.
Pakistan's blasphemy laws came under the spotlight in 2010 after a Christian woman was sentenced to death in a case stemming from a village dispute.
In January 2011, Punjab governor Salman Taseer was killed by his bodyguard over his criticism of the blasphemy law.
In addition to Taseer death, Pakistan's Minister of Minorities Shahbaz Bhatti, the only Christian member of the government, was assassinated later on March 4, 2011.
According to Pakistan's blasphemy law, insulting any Prophet in Pakistan, a country where 95 percent of the population is Muslim, is a crime punishable with death or life imprisonment.
The law, commonly known as 295-C, was introduced in early 1980s by late President General Zia-ul-Haq.
Since then, some 700 cases of blasphemy have been registered, half of which are against Muslims.But rights groups say the law is often exploited to settle personal scores.
Reproduced with permission from OnIslam.net


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Saturday, April 12, 2014
Please complete the registration form below. Just a couple of things to remember... the VUU Open House is for Fall 2014 accepted students. If you can not locate your student ID# please contact the Office of Enrollment Management, 804-342-3570, to check on your application status. If you have applied and not received a decision, please put #0000 for you student ID and you will have a decision mailed to you before Open House.


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I had this free pattern and decided to use up some of my fat quarter stash to make my DH a quilt. Twin size amd just stitched in the ditch quilting.
Follow-Up Postings:
- Posted by solstice98 (My Page) on Tue, Mar 3, 09 at 22:08
It's beautiful! I really like the dark, rich tones of your scraps. It's a great contrast with the white. Kate |
Your placements and colors really are stunning! What a nice job. Lots of lovely jewel tones in your scraps. Mine seem to be all dull tans. Great work! |
- Posted by geezerfolks (My Page) on Wed, Mar 4, 09 at 8:40
Jayne, That is gorgeous and I really like the star effect around the richness of the center! Hmmmmmmm....... Thank you for sharing! Sharon |
What a great quilt. I can see now I will be making a bunch of HSTs for camp and putting together tops. Thanks for sharing the great quilt. beverly |
- Posted by bonnie_nmi (My Page) on Wed, Mar 4, 09 at 9:57
Lovely! The colors are wonderful and the pattern looks more complicated than it actually is - my kind of project! Bonnie |
- Posted by pirate_girl (My Page) on Wed, Mar 4, 09 at 12:05
Hey Nanaj, An interesting lesson in simplicity & how it can be strikingly beautiful. The contrast is great, lends it a real crisp feeling; the ditch quilting makes it pop, really excellent, hope DH LOVED it & may it keep him toasty warm! |
- Posted by toolgranny (My Page) on Wed, Mar 4, 09 at 12:27
I love your colors. Adding white really makes it. |
It's beautiful-- I love scrappy. |
- Posted by annabelle1941 (My Page) on Wed, Mar 4, 09 at 16:47
Just love it - colours are wonderful, well done Ann |
That is very pretty! The white really sets it off. Great job Jayne! Cathy |
What are HST's? There are alot of abbreviations, some I can figure out, some I can't. Just curious, |
HST's are half square triangles. Just a point, the white in the quilt is muslin. Jayne |
- Posted by sewbeeit42 (My Page) on Thu, May 7, 09 at 14:35
I want to make this quilt, but have lost the name of the place from where it came. Can you help me so I can get the pattern as I have been desperately search for it? My e-mail is email@example.com Thank you in advance for your help. Carolyn |
- Posted by teresa_nc7 (My Page) on Thu, May 7, 09 at 18:32
A beautiful scrappy quilt! Your DH will love it! Teresa |
- Posted by redpenny (firstname.lastname@example.com) on Thu, May 7, 09 at 19:22
I love scrappy.The white really sets it off. Red |
I was surprised to see the Cumberland back up. Always fun to read the comments. Carolyn, The pattern was a freeby that came in the mail. I had several copies but found only one tonight. I will e-mail the instructions later. It was a fun and easy quilt to use up some of my stash on. Jayne |
- Posted by sewbeeit42 (My Page) on Sun, May 10, 09 at 21:24
Jayne, Thank you for helping me out with this quilt pattern. My small quilt group wants to make it and some of the members need to have exact, printed directions. I was sure I had them only to discover I hadn't. My goose would have been cooked had I not found it, but you are saving the day. |
Glad to be of help. Wasn't sure they would print out large enough. It is a fun pattern to do and I hope your quilt group has as much fun as I did. Jayne |
- Posted by sewbeeit42 (My Page) on Mon, May 11, 09 at 17:03
I have continually checked my e-mail including the spam box but nothing has come through yet. Please advise me where to look as I am new to this group and may not be looking in the right place. I would e-mail you directly, Jayne, but don't know your address. Carolyn firstname.lastname@example.com |
Carolyn, I sent it the other day but I just checked and I may have used the wrong address, I will try again. Jayne |
- Posted by biwako_of_abi (My Page) on Tue, May 12, 09 at 0:36
I have never seen the Cumberland pattern before. It's a beautiful quilt and intriguing because, in spite of its simplicity, the more you look at it, the more facets you discover. Lovely use of color! |
- Posted by barbara_in_pa (My Page) on Tue, May 12, 09 at 6:48
I never heard of this pattern before and really love it... it is a wonderful way to use up your left overs. So simple, and really beautiful. I need to make this one. Barbara |
I must have missed this post the first time around. I'm glad it is back to the top! It is a beautiful quilt. Lola |
Where can I get this pattern?? It is soooo pretty. |
Where can I get this pattern?? It is soooo pretty. |
It was a "freebe" that came in the mail. E-mail me your address and I will send it to you. |
- Posted by littlehelen (email@example.com) on Tue, Jul 23, 13 at 20:53
Nanajayne...can I have a copy of the freebie too? Your quilt is lovely and I'd love to give this a go. I tried to email you, but did not find a link .?? V. |
Address: email@example.com. I only have one copy left but I will do the best I can. Glad you both enjoy it. Clinton has for some time. lol |
- Posted by geezerfolks (My Page) on Wed, Jul 24, 13 at 12:02
Am I confused.....again.....or is this more than HST's placed strategically? (Have I said anything about how I hate not being able to see clearly or that my eye doesn't tell my brain what I'm actually looking at?!) SharonG/FL |
I wonder if you really need a pattern for the individual blocks. Maybe just to set them together, but looking at Jayne's quilt, I think they are just set side by side And you can decide on whatever size you want those HSTs to be. Theresa |
This post was edited by nannykins on Wed, Jul 24, 13 at 13:15
It is a very simple quilt. As Theresa said the blocks are all the scrappy same, placed in rows going in the same direction. It is bordered with dark HST. HST's were made from 6 3/8 sq. and the blocks are 11" |
Looks great. I used to be shy of using white, but after participating in the block exchange for a while and seeing the quilts many of you make, white's become my new best friend! It's a great "manly" quilt. LindaB/CA |
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- Nuggets Ink - http://blogs.denverpost.com/nuggets -
Nuggets' Karl gets a new Hart
Posted By Christopher Dempsey On March 4, 2009 @ 5:25 pm In General Nuggets,NBA | No Comments
For about 10-15 minutes Wednesday afternoon, Nuggets coach George Karl sat in chairs along a sideline in the team's Pepsi Center practice gym and explained to his newest guard, Jason Hart, what will be expected of him.
The first time these two had a similar conversation was nine years ago, when Hart was a rookie point guard, drafted by Milwaukee, which was coached by Karl.
"When I was with George the first time it was tough," Hart said, with a smile. "I was a rookie. He doesn't like rookies."
And now?
"I'm not a rookie now," he said. "Anytime you're not a rookie in George's eyes you're a little bit better. But it's good. I'm just happy to be a part of what he's got going on here."
And that is simply to be ready when called.
Don't expect Hart to cut into anyone's minutes in the backcourt. Unless an injury causes either starting point guard Chauncey Billups or reserve Anthony Carter to go down, Hart's biggest contributions will be in practice, helping the regulars get ready for a tough stretch run and the playoffs.
"He told me that basically this is Chauncey and Anthony's show," Hart said.
Hart and Nuggets forward Chris Andersen share a dubious bond. They were the first two players ever to be called up from the NBA Development League to the NBA. Andersen was the first player to receive a call-up. Hart was the second. The Spurs signed him from the Idaho Stampede on Dec. 19, 2001.
This year Hart was with the Clippers, where he played in 28 games, starting two, with averages of 2.3 points and 1.5 assists. The Clippers waived him on Feb. 27, allowing him the opportunity to sign with another team.
"It didn't work out for me (with the Clippers)," Hart said. "The opportunity was just bad from the start. I'm just happy to be back with a winning team, and whatever I'm asked to do, if I'm even called, I'll be ready."
Chris Dempsey: 303-954-1279 or email@example.com
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The other day my husband had arranged to play golf with a friend. When he called the friend to finalize the meeting time the friend said, "I can't. I need to stay by the phone." The only phone the friend has is a mobile one. Meaning movable.
We got a good laugh about it, but it made me think about my love/not-so-much-love relationship with my cellular phone. I love it for being convenient and for helping me to not miss calls. I also understand that it is a part of operating in my world and that I need it to compete and communicate. But I don't love that I feel beholden to check it all the time. And to respond. I think that it has lessened a thinking or cooling down period that we used to have.
The other thing that annoys me — but which I find myself doing, too — is when I get a call and I cannot pick it up and the person does not leave a message. Wait!? Who was it?! Ed McMahon perhaps. Am I RICH? In the past I have called back only to find it was a wrong number, or my phone rings and it is someone saying, "You called me" — but I had made the call to the wrong number.
In the days of yore, before the existence of even the humble answering machine… the days when one truly could have given the excuse of having to stay by the phone, we might have happily gotten an infinite number of calls that did not go through. And you know what? We all survived.
Be a little more lax around your phone. You just might find that things can wait and you can be more relaxed. And for goodness sake, if you want to get out of something, think of a better excuse than, "I need to stay by the phone!"


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Aug 30, 2013 4:45 PM by ASSOCIATED PRESS
SAN DIEGO (AP) - Some women who identified themselves as targets of San Diego Mayor Bob Filner's sexual advances are holding a mock celebration to mark the mayor's last day in office.
Attorney Gloria Allred was flanked by her clients Friday as she displayed what she called parting gifts for Filner.
Allred showed off a handheld mirror that she said Filner can look at when asking himself who's to blame for his resignation.
She also displayed a card that says she looks forward to seeing him at a deposition for a client who accuses him of harassment.
A wallet-sized card contained California's legal definition of sexual harassment.
Filner is keeping a low profile on his final day in office.
(Copyright 2013 The Associated Press. All rights reserved. This material may not be published, broadcast, rewritten or redistributed.)
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Jean-Étienne Liotard Swiss, Switzerland, about 1760 Graphite and black, brown, and red pastels, with stumping and red gouache (recto); colored pastels with stumping (verso) 8 11/16 x 7 3/16 in. 2004.75
For his portrait of Charles-Benjamin de Langes de Montmirail, the Baron de Lubières, Jean-Étienne Liotard drew on both sides of a thin sheet of cream-colored paper. On the verso surface, the artist delicately applied red and black pastels to achieve richer and more subtle degrees of tone and relief on the finely detailed recto. This novel technique is found in several of Liotard's portraits of the period and derives from the tradition of toning miniatures on ivory on the verso. A wealthy and erudite amateur, the Baron de Lubières was part of the writer Voltaire's circle in Geneva and contributed several articles to Diderot's Encyclopédie. Replete with fur muff, the fashionably attired sitter is clearly a gentleman of his time. Despite the attention to dress, Liotard depicted his sitter without adornment. The Baron de Lubières is in profile against a plain background with little depth. Nothing is allowed to overshadow the close likeness of the sitter achieved by Liotard. And it is this close likeness, above all else, that is intended to speak of the Baron de Lubières as a man of singular refinement and distinction.