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Advance Auto Parts NBA All-Star 2018 Sweepstakes

Mobil 1™ and Advance Auto Parts Speedperks with All-Star sweepstakes tagline

It’s game time for the NBA All-Star 2018 Sweepstakes.

Don’t miss your shot at the NBA All-Star experience of a lifetime. One lucky fan will win a trip to Los Angeles to experience the NBA All-Star 2018 game live and in person. Two hundred fifty runners-up will win an official NBA All-Star 2018 hat.

Promotion ends: Aug. 30, 2017
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NBA All-Star 2018 Sweepstakes ("Sweepstakes")

Participation constitutes entrant's full and unconditional agreement to, and acceptance of, these Official Rules. NBA All-Star 2018 Sweepstakes (the "Sweepstakes") begins at 12:00:01 AM Eastern Time ("ET") on June 1, 2017, and ends at 11:59:59 PM ET on August 30, 2017 ("Sweepstakes Period"). Sweepstakes is sponsored by ExxonMobil Oil Corporation, Exxon Mobil Corporation, 22777 Springwoods Village Parkway, Spring, TX 77389 (the "Sponsor").

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The Sweepstakes is subject to all applicable federal, state and local laws, rules and regulations. Void where prohibited or restricted by law, rule or regulation.

WAYS TO ENTER: During the Sweepstakes Period eligible participants may enter one of three (3) ways:

  1. Online: To enter via the internet, complete the electronic entry form during the Sweepstakes Period, and no later 11:59:59 PM ET on August 30, 2017, on Sponsor’s website located at, by providing your name, address, city, state, ZIP code, telephone number, email address and your Speed Perks number to receive one (1) entry into the random drawing. For purposes of this Sweepstakes, "receipt" of an internet entry occurs when Sponsor/Administrator’s server records the entry information resulting from the entrant selecting the "SUBMIT" button. Automated entries (including but not limited to entries submitted using any robot, script, macro, or other automated service) are not permitted and will be disqualified. Entrants may not enter using multiple email addresses. To receive a bonus entry, prior to hitting "SUBMIT" upload a proof of purchase showing a purchase of five (5) or more quarts of any Mobil™ product from Advance Auto Parts/CARQUEST. Limit one (1) online and one (1) bonus entry per person, email address or Speed Perks number during the entire Sweepstakes Period.
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LIMIT: Only three (3) entries into the Sweepstakes per person, per mailing address, mobile phone number, email address or Speed Perks number during the Sweepstakes Period, regardless of the method of entry. Multiple entries received during the Sweepstakes Period from any person, mobile phone number, household address or email address after the first entry received from that person, number, household address or email address will be void. Once submitted, an entry cannot be deleted, canceled or modified. All entries become the property of the Sponsor and will not be acknowledged. Sponsor reserves the right, in its sole discretion, to void any and all entries of an entrant that submits more than the one permitted entry, or who Sponsor believes has attempted to tamper with, influence, or impair the administration, security, fairness, or proper play of this Sweepstakes.

DRAWING AND ODDS OF WINNING: One (1) Grand Prize winner and two hundred fifty (250) Second Prize winners will be selected in a random drawing which will take place at the conclusion of the Sweepstakes Period on or about from among all eligible entries received. Drawing will be conducted on or about September 21, 2017, by Arrowhead Promotion & Fulfillment Co., Inc. (the “Administrator”), an independent judging organization whose decisions are final and binding in all matters relating to this Sweepstakes. Odds of winning will depend on the total number of eligible entries received. Decisions of judges are final.

PRIZES: One (1) Grand Prize – A 3-day/2-night trip for winner and one (1) guest to Los Angeles, California for NBA All-Star 2018 in Los Angeles, California between February 17-19, 2018 (exact dates to be determined by Sponsor). Trip includes round-trip coach air transportation from a major U.S. gateway airport nearest winner’s residence (as determined by Sponsor) to Los Angeles, California for winner and one (1) guest, two (2) nights’ hotel accommodations (one (1) standard room, double occupancy) (room rate and room tax only), two (2) tickets to State Farm All-Star Saturday Night and the NBA All-Star Game 2018, and $500 spending money awarded in the form of a gift card for the winner only. Approximate Retail Value ("ARV"): $5,000.00.

Actual value may vary based on point of departure and dates of travel. If winner resides within a 100-mile radius of trip destination, Sponsor reserves the right to provide ground transportation in lieu of air transportation and no compensation or substitution will be provided for difference in prize value. Car rental subject to winner holding a valid U.S. driver’s license and qualifying as an eligible driver under applicable car rental company policy and other rental car company terms and conditions. Car rental does not include gas, tolls, parking fees, collision waiver insurance, or any other related fees and is subject to car rental company terms and conditions. Travel arrangements must be made through Sponsor’s agent, on a carrier of Sponsor’s choice, at least thirty (30) days prior to travel. Winner and guest must travel together on the same itinerary and are responsible for obtaining all travel documents prior to travel. Flight schedules are subject to change without notice. Sponsor and Sweepstakes Entities are not liable for any expenses incurred as a consequence of flight cancellation/delay. Location of seats will be determined by Sponsor. Tickets are subject to certain terms and conditions specified thereon. Winner and guest agree to comply with all venue rules and regulations. Failure to do so may result in forfeiture of such portion of prize.

Travel guest must be eighteen (18) years of age or older (or 19 of a resident of Alabama or Nebraska) and must execute and return a liability/publicity release prior to issuance of travel documents. By participating and accepting Grand Prize, the Grand Prize winner represents that he/she understands that travel, whether by plane, automobile or other conveyance, or by foot, and in the air, contains some inherent element of risk of accident, illness, injury, loss or death, which may be caused by negligence, forces of nature, or other agencies, known or unknown. Winner and his/her guest represent that they recognize that such risks may be present at any time before, during and after the trip. Certain travel restrictions may apply.

Air transportation and hotel accommodations are subject to availability. All other costs and expenses associated with the acceptance and use of the prize not stated in the prize description above as being provided including, but not limited to, all federal, state and local taxes, luggage fees, meals, gratuities and tips will be the sole responsibility of the winner. In the event Sponsor is unable to provide tickets to the rodeo or any portion of the trip is cancelled for any reason, no compensation or substitution will be provided for that portion of the prize; however, the remainder of the prize package will be awarded and Sponsor will have no further obligation to winner. Winner must travel on dates specified by Sponsor in February 2018 or prize will be forfeited and Sponsor will have no further obligation to such winner. No substitution, transfer or cash equivalent is permitted except by Sponsor, which reserves the right to substitute the prize (or portion thereof) with one of comparable or greater value at its sole discretion. Prize is awarded “as is” with no warranty or guarantee, either express or implied by Sponsor. All prize details are at the sole discretion of Sponsor.

Two-hundred fifty (250) Second Prizes – An NBA All-Star 2018 Hat. ARV: $16.50 each.

Total Prize pool ARV is $9,125.00. Participant is only eligible to win one (1) prize during the entire Sweepstakes Period. Exact Prize details shall be determined in the sole and absolute discretion of Sponsor.

PRIZE CONDITIONS: Only the prize specified above will be awarded. Prizes are non-transferable, with no cash redemption or equivalent. All federal, state, and local income taxes, insurance, registrations and title fees connected to the prize are the sole responsibility of winner. All federal, state, and local regulations apply. All Winners agree to release the Sponsor and the Sweepstakes Entities from any and all liability whatsoever for any injuries, losses, or damages of any kind caused by entering the promotion or for damages of any kind caused by any prize or resulting from acceptance, possession, or use/misuse of prize awarded. No substitution or transfer of prize will be permitted.

CLAIMING PRIZE: As a condition of winning the prizes, Winners may be required to complete, sign and return an affidavit of eligibility and other applicable forms required by a taxing authority within ten (10) days of written prize notification, or prize/s will be forfeited and an alternative winner selected. All prize Winners will be contacted by an email sent to their email address that they used in their online or mail-in entry (in the sole discretion of Sponsor). If a potential prizewinner does not respond within ten (10) calendar days from the first notification attempt an alternate Winner may be selected. All taxes are the sole responsibility of each Winner. In the event that any potential Winner does not respond to any such Prize Notification within ten (10) days of the date of issuance or declines the Prize for any reason, a disqualification will result, the Prize will be forfeited and, at Sponsor's sole discretion and time permitting, an alternate potential Winner may be randomly selected from among all remaining eligible entries received for the drawing at issue.

PUBLICITY RELEASE: Except where prohibited by law, the Winner's acceptance of a Prize constitutes permission for the Sweepstakes Entities use Winner’s name, photograph, likeness, statements, biographical information, voice, and address (city and state of residence), photos, videos or film clips, and/or other visual likeness worldwide and in all forms of media, in perpetuity, for advertising and/or trade purposes and/or for any other purpose now or hereafter known without further compensation.

LIMITATIONS OF LIABILITY: If for any reason this Sweepstakes is not capable of running as planned due to a virus, bugs, tampering, unauthorized intervention, fraud, technical failures, or any other causes beyond the control of the Sponsor which corrupt or affect the administration, security, fairness, integrity or proper conduct of this Sweepstakes, the Sponsor reserves the right at its sole discretion, to disqualify any individual who tampers with the entry process, and to cancel, terminate, modify or suspend the Sweepstakes in whole or in part, at any time, without notice and award the prizes using all non-suspect eligible entries received as of, or after (if applicable) this cancellation, termination, modification or suspension date, or in any manner that is fair and equitable. Under no circumstances will entrants be permitted to obtain awards for, and entrants hereby waive all rights to claim, punitive, indirect, incidental and consequential damages.

Sponsor assumes no responsibility for any error, omission, interruption, deletion, defect, delay in operation or transmission, communications line failure, theft or destruction or unauthorized access to, or alteration of, entries or text messages.

USE OF DATA: Sponsor will be collecting entrants’ personal data in accordance with the Sponsor’s Privacy Policy that can be reviewed at the following link: Please see the full Privacy Policy for details. Entrant’s personally identifiable information is not shared with third parties unless we give prior notice and choice. We do not rent, disclosed or share personal information or aggregated demographic information with our partners and advertisers. Though we may use an intermediary to conduct promotions, they may not use entrant’s personally identifiable information for any secondary purposes. By participating in the Sweepstakes, you hereby agree to Sponsor's collection and usage of your personal information and acknowledge that you have read and accepted Sponsor's privacy policy. Entrant’s information will be used for purposed of direct marketing, research, and monitoring or improving the use and satisfaction of ExxonMobil’s products.

TERMS & CONDITIONS: This Sweepstakes is subject to all applicable federal, state and local laws. By entering the Sweepstakes, where permitted by law, entrants agree to release, discharge, indemnify, and hold harmless the Sweepstakes Entities, and each of their respective directors, officers, employees, shareholders, and agents from any and all liability, loss, damage, or injuries to any person (including death) or property of any kind sustained in connection with their participation in the Sweepstakes or from their acceptance, receipt, possession, and/or use or misuse of the prize, or for any typographical or other error in the printing, offering, or announcement of the prize. Entrants further acknowledge that the Sweepstakes Entities have neither made, nor are in any manner responsible or liable for any warranty, representation, or guarantee express or implied, in fact or in law, relating to the prize, including, but not limited to, its quality or fitness for a particular purpose.

Any person attempting to defraud or in any way tamper with the Sweepstakes mechanics and/or implementation will be prosecuted to the fullest extent of the law. By participating, entrants agree to be bound by these Official Rules and the decisions of the Sweepstakes Administrator, and waive any right to claim ambiguity in the Sweepstakes or these Official Rules. The Administrator reserves the right to disqualify any entrant that Administrator determines, in its sole discretion, violates these Official Rules. This Sweepstakes is void where prohibited. By accepting a prize, Winners agree to allow the Sweepstakes Entities the right and license to use winner’s likeness, voice and/or address (city/state) for promotional purposes without any additional compensation or prior approval, except where prohibited.

DISPUTES: In the event of a dispute as to the identity of a Winner based on an email address or a phone text entry, the winning entry will be decided in favor of the authorized account holder of the email address or the authorized account holder of the phone number used at time of entry. "Authorized account holder" is defined as the natural person who is assigned to an email address by an internet access provider, online service provider, or a phone number by a phone service provider or other organization (e.g., business, educational, institution, etc.) that is responsible for administering the phone number chosen and/or the domain responsible for the email address chosen.

By entering the Sweepstakes, entrants agree that 1.) Any and all disputes, claims and causes of action arising out of or connected with the Sweepstakes, or any prizes awarded, shall be resolved individually, without resort to any form of class action; 2.) Any and all claims, judgments and awards shall be limited to actual out-of-pocket costs incurred, including costs associated with entering the Sweepstakes but in no event attorneys' fees; and 3.) Under no circumstances will any entrant be permitted to obtain any award for, and entrant hereby waives all rights to claim, punitive, incidental or consequential damages and any and all rights to have damages multiplied or otherwise increased and any other damages, other than for actual out-of-pocket expenses, and any and all rights to have damages multiplied or otherwise increased.

All issues and questions concerning the construction, validity, interpretation and enforceability of these Official Rules, or the rights and obligations of the entrants and Sponsor or the Sweepstakes Entities in connection with the Sweepstakes, shall be governed by, and construed in accordance with United States law. Any legal proceedings arising out of this Sweepstakes or relating to these Official Rules shall be instituted only in the federal or state courts of Texas, and the parties consent to jurisdiction therein with respect to any legal proceedings or disputes of whatever nature arising under or relating to these Official Rules. The invalidity or unenforceability of any provision of these Official Rules shall not affect the validity or enforceability of any other provision. In the event that any provision is determined to be invalid or otherwise unenforceable or illegal, these Official Rules shall otherwise remain in effect and be construed in accordance with their terms as if the invalid or illegal provision were not contained herein. The value of the prize set forth above represents Sponsor’s good faith determinations of the ARV thereof and such determinations are final and binding and cannot be appealed. If the actual value of any prize turns out to be less than the stated ARV, the difference will not be awarded in cash.

SWEEPSTAKES RULES: These Official Rules will be posted on Sponsor’s website at during the Sweepstakes Period.

WINNERS LIST REQUESTS: To request confirmation of the name and address of the winners, please send a self-addressed, stamped business-size envelope by September 30, 2017, to: NBA All-Star 2018 Sweepstakes, c/o Arrowhead Promotion & Fulfillment Co. Inc., PO Box 2354-W, Grand Rapids, MN 55745-2354.

SPONSOR: ExxonMobil Oil Corporation, 22777 Springwoods Village Parkway, Spring, TX 77389.

ADMINISTRATOR: The administrator of this Sweepstakes is Arrowhead Promotion & Fulfillment Co., Inc., 1105 SE 8th Street, Grand Rapids, MN 55744.

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Open Access

Elevated autocrine EDIL3 protects hepatocellular carcinoma from anoikis through RGD-mediated integrin activation

  • Ming-Xuan Feng1,
  • Ming-Ze Ma2,
  • Ying Fu2,
  • Jun Li2,
  • Tao Wang1,
  • Feng Xue1,
  • Jian-Jun Zhang1,
  • Wen-Xin Qin2,
  • Jian-Ren Gu2,
  • Zhi-Gang Zhang2Email author and
  • Qiang Xia1Email author
Molecular Cancer201413:226

Received: 10 May 2014

Accepted: 28 September 2014

Published: 1 October 2014

A remolded microenvironment in hepatocellular carcinoma (HCC) caused by abnormally expressed matricellular proteins could promote HCC progression. The cell-matrix interactions mediated by integrins play an important role in tumor microenvironment. Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), an extracellular matrix (ECM) protein with angiogenic and anti-inflammatory effects, is abnormally highly expressed in HCC. Here we aim to analyze its expression in liver and HCC tissues, investigate the underlined mechanisms accounted for HCC progression.

EDIL3 expression level is examined in normal liver, cirrhotic liver and HCC at both mRNA and protein level. The association between EDIL3 and clinical outcomes is analyzed. The pattern of EDIL3 expression and location is examined using Immunofluorescence and ELISA. Overexpression or knock-down of EDIL3 in a panel of cell lines are subjected to assays related to proliferation, invasion, and anoikis to investigate the mechanisms of this matrix protein in HCC progression. Recombinant EDIL3 treatment is applied to confirm the results.

Compared with normal liver and cirrhotic liver, EDIL3 is elevated in HCC. High level of EDIL3 protein is much more commonly in patients with larger tumor or portal vein tumor thrombus (PVTT) formation, associated with poor prognosis. EDIL3 is abundantly expressed in HCC cells and secreted by cancer cells. In vitro and in vivo studies indicate that EDIL3, probably in an autocrine manner, inhibits anoikis and promotes anchorage-independent growth of HCC cells. Further mechanistic studies suggest integrin ligation by EDIL3 and thus that the sustained activation of the FAK-Src-AKT signal is responsible for the anoikis resistance and anchorage independence. Both the administration of cilengitide, a RGD-containing integrin antagonist, and silencing of integrin αV, an important RGD-binding integrin, results in the blockade of anoikis-resistance induced by EDIL3.

Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of detached HCC cells and may involve in cancer cell spreading. We also highlight the importance of interaction between EDIL3 and integrin αV and suggest disrupting the ligation of EDIL3 to integrins via RGD-blocking in selected patients may bear potential therapeutic value.

Epidermal growth factor-like repeats and discoidin I-like domains 3Hepatocellular carcinomaAnoikis resistanceIntegrin activation

Hepatocellular carcinoma (HCC) is a lethal disease with high mortality due to the high rate of postoperative recurrence and metastasis [1]. Both genetic and epigenetic alterations [2] within the HCC and a remolded microenvironment [3, 4] surrounding HCC affect the biological behaviors of HCC and thus result in different outcomes of HCC patients. Extracellular matrix (ECM) proteins are the main non-cellular components of the tumor microenvironment; many of them can interact with the tumor cell through direct binding to specific receptors or by modulating the activation of other cytokines [5, 6]. Some reports have suggested that microenvironmental changes caused by abnormally expressed matricellular proteins may modulate HCC progression by affecting cell growth, migration, invasion, anoikis and metastasis; thus, targeting ECM proteins may have therapeutic value [3, 710].

Epidermal Growth Factor-like repeats and Discoidin I-Like Domains 3 (EDIL3), also known as DEL-1, is a secreted ECM protein that was firstly characterized in vascular morphogenesis [11]. Structurally, EDIL3 contains 3 EGF-like repeats and 2 discoidin-like repeats, with an RGD motif located in the second EGF-like repeat [11]. Three-dimensional crystal structures reveal a unique RGD finger, which is believed to be advantageous for its ligation with integrins over other RGD-containing ECM proteins [12]. EDIL3 has been intensively studied in vascularization, exhibiting strong angiogenic or vascularizing function through integrin αvβ3 modulation [13, 14]. Moreover, this protein plays a role in modulating immunocyte adhesion through interactions with leukocyte-specific integrins [15]. The role of EDIL3 in cancer is also revealed in several malignancies, albeit in observational level. For example in pancreatic carcinoma, EDIL3 is one of the SHH-dependent stromal factors that predict poor prognosis [16]; another study focusing on carcinogenesis of ulcerative colitis-associated colorectal cancer suggests EDIL3 may add some power in this process [17]; interestingly, EDIL3 could also be detected in exosomes of bladder cancer cells and facilitate cancer progression trough EGFR signal [18]. In the field of HCC, high-throughput genomic data suggests elevated EDIL3 in HCC compared with adjacent liver [19], and a clinical analysis reveals that EDIL3 might affect the prognosis of HCC [20]. However, no study has addressed how EDIL3 is involved in HCC development and progression.

Anoikis is a special apoptotic process resulting from the loss of or inappropriate cell adhesion. Upon detaching from the primary lesion, the lack of cell-ECM adhesion fails to maintain the pro-survival signals within cancer cells, leading to decreased anti-apoptotic signals, thus activating anoikis [21]. Gaining anoikis resistance is a prerequisite for intra-hepatic spreading and extra-hepatic metastasis of HCC. In addition to adaptive alterations, such as switch in integrin expression patterns or the hyperactivation of receptor tyrosine kinases, the abnormal microenvironment also helps the cancer evading anoikis [22]. Integrins are the key mediators of the cell-ECM interaction, sending pro-survival signals in the presence of ECM ligands, e.g., collagens and laminins, and inducing apoptosis in their unligated status [23]. EDIL3 is an important ligand for αV-coupled integrins via RGD recognition and has been shown to be able to bind these groups of integrins [24] and inhibit anoikis in the endothelium [25].

In the present study, we focus on the role of EDIL3 in HCC and demonstrate that EDIL3 is highly expressed in HCC patients. Moreover, the accumulation of tumor-derived EDIL3 in the microenvironment promotes anoikis resistance and anchorage independent growth advantage through the activation of integrin signal pathways.

The EDIL3 level is significantly elevated in HCC tissue and is associated with adverse biological behaviors

To observe the expression change in HCC, we first tested the EDIL3 level in 5 normal livers (NL), 10 cirrhotic livers (CL), and 49 HCCs by qRT-PCR and western blot. The result revealed that EDIL3 was mildly expressed in NLs and CLs but was significantly elevated in HCC both at the mRNA and protein level (Figure 1 A, B, Additional file 1: Figure S1A). IHC stain of another array of samples including 3 NLs, 10HCCs and 6 PVTTs confirmed the high expression of EDIL3 in HCC compared with NL and CL (Figure 1C). Notably, co-stain of EDIL3 and endothelium marker CD31 in HCC samples showed EDIL3 is not only stained with endothelium marker, but also widely and diffusively located in cancer cells clusters (Figure 1C, D), suggesting that cancer cells becomes an important sources of EDIL3 besides endothelium.
Figure 1

EDIL3 is significantly up-regulated in HCC and PVTT compared with normal liver (NL) and cirrhotic livers (CL) and is closely related to the prognosis of HCC. A, The transcriptional level of EDIL3 is measured via qRT-PCR in 5 NLs, 10 CLs and 49 HCCs. The relative mRNA level is normalized to β-actin and is presented as Δ–ΔCq. The mRNA of EDIL3 in HCC is significantly higher than both NL and CL; B, The protein level of EDIL3 in NL, CL, HCC and PVTT was examined by western blot with β-actin as a loading control. The EDIL3/β-actin densitometry is performed and shown as density value below. EDIL3 is mildly expressed In NL and CL while significantly elevated in HCC and PVTT; C, Representative pictures demonstrating EDIL3 staining in different liver samples including NL, CL, HCC, PVTT and microscopic thrombi by IHC. Scale bars, 50 μm or 150 μm; Arrow heads indicate that high EDIL3 expression is largely localized to cancer cells. D, Confocal microscopic observation of immunofluorescence staining of CD31 (green), an endothelium marker, and EDIL3 (red) in HCC samples show EDIL3 is not only localized with endothelium, but also widely and diffusively located in cancer cells clusters. White arrows indicates the endothelium; Grey arrows indicates the cancer cell cluster. E, Kaplan-Meier analysis of overall survival between EDIL3-negative or moderately positive patients and highly positive patients shows a significant survival advantage in EDIL3 low express group. **: P < 0.01.

We then explored the clinical value of EDIL3 in HCC by analyzing the protein level in another independent 202 HCC samples gathered on tissue microarrays (TMA). We observed approximately 72% (57/202) of patients were EDIL3-positive, and high levels of EDIL3 positivity was significantly associated with adverse clinicopathological parameters of HCC, including tumor size, PVTT formation, and TNM stage (Table 1). Moreover, Kaplan-Meier survival analysis in the 192 patients with follow-up demonstrated a higher postoperative recurrence risk and poor survival in patients with high EDIL3 expression. Because high EDIL3 expression was much more common in patients with PVTT, we examined EDIL3 protein levels in 6 PVTT samples. The result revealed that EDIL3 stain was very strong in all of the PVTT samples (6/6) as well as microscopic tumor thrombus (Figure 1C), and this was validated by western blot (Figure 1B). Taken together, these results reveal the high level of EDIL3 in HCC and suggest its association with adverse clinicopathological parameters and poor prognosis.
Table 1

Correlation between EDIL3 and key clinicopathological parameters

EDIL3 (n = 202)

P value

Age (years)

Tumor size

≤5 cm

>5 cm

TNM stage

The bold numbers represent the P-values with significant differences.

EDIL3 is abundantly expressed in HCC cell lines and exhibits unique expression pattern

We examined EDIL3 expression in 7 HCC cell lines and 2 non-HCC cell lines. The mRNA level of EDIL3 varied among all of the cell lines, with the HCC cell lines Huh-7 and CSQT-2 exhibiting much higher mRNA levels of EDIL3 (Figure 2A), while THLE-3 and Lo-2, which derived from normal hepatocyte, also showed mild transcription. Surprisingly, inconsistent with mRNA, EDIL3 protein was detected at almost the same level in the cell lysates of all the cell lines (Figure 2A) and the Immunofluorescence confirmed the existence of EDIL3 in cytoplasm of all 9 cell lines (Figure 2C and Additional file 1: Figure S1). Indeed, we also observed this inconsistency in other cancer cell lines (Additional file 2: Figure S2). Because EDIL3 is an ECM protein, we examined the EDIL3 that was secreted in conditioned medium (CM). All cell lines were subjected to standardized condition (see methods for details), and the secreted proteins in the CM was analyzed by Western Blot and ELISA. Not surprisingly, the Huh-7 and CSQT-2 secreted the most EDIL3 into the CM, whereas there was only detectable EDIL3 in the CM of other cell lines (Figure 2A, B). These observations in cell lines reveal a unique autocrine expression pattern of EDIL3, and the high level of EDIL3 in CSQT-2, a PVTT derived cell line, is consistent with the observation in patient’s samples above.
Figure 2

EDIL3 exhibits a unique expression pattern in cell lines. A, detailed analysis of EDIL3 expression in mRNA level, cell lysates and CMs of 7 HCC and 2 non-HCC cell lines demonstrated an approximate correlation between mRNA and secreted EDIL3 in CMs, whereas EDIL3 in cell lysates exhibited almost same intensity. Notably, Huh-7 and CSQT-2 exhibited a much higher level of secreted EDIL3. B, ELISA assay testing the EDIL3 in CMs of 9 cell lines validates the secreted EDIL3 level is approximately correlated with mRNA level. C, Immunofluorescence staining of EDIL3, F-actin and DAPI in confocal microscope shows EDIL3 is localized within cells and at almost the same intensity in all the 9 cell lines under test, despite their varied mRNA level.

Autocrine EDIL3 promotes anoikis resistance and anchorage-independent growth in HCC cells in vitro

Anoikis resistance and invasion are two important features of the HCC cells that are prone to forming PVTT or metastasis. We continued to investigate the effect of EDIL3 on tumor cell invasion and anoikis. A eukaryotic expression system suitable for secreted protein synthesis was used to generate recombinant EDIL3 with high fidelity (Additional file 3: Figure S3). SMMC-7721 and MHCC-97H, two cell lines with low basal levels of autocrine EDIL3, were chosen to undergo functional assays in the presence of EDIL3. EDIL3 did not affect invasion or proliferation (Additional file 2: Figure S2B). However, using WST-8 and Caspase3/7 activity assay, we found EDIL3 significantly lowered the anoikis rate. EDIL3 treatment reduced the Caspase3/7 activity and increased the viability in cancer suspended in poly-hema coated dishes for long to 72 h (Figure 3A-D). In soft agar clone formation, EDIL3 also significantly promoted the anchorage-independent growth in SMMC-7721 and MHCC-97H cells (Figure 3E). These effects above are both dose-dependent, with the 50 nM safely reaching significance.
Figure 3

Treatment of EDIL3 contributes to anoikis resistance and anchorage-independent growth advantage in HCC. A, Different concentration of recombinant EDIL3 is used to treat SMMC-7721 suspended in poly-hema coated dishes. It sustains the viability of SMMC-7721 as demonstrated by caspase3WSt-8 assay in a time and dose dependent manner compared with control protein. B, Recombinant EDIL3 ameliorate the anoikis compared with control protein in SMMC-7721 as demonstrated by caspase3/7 intensity assay in a time and dose dependent manner. C-D, MHCC-97H is subjected to the assays same as SMMC-7721 and obtain consistent result. E, Administration of recombinant EDIL3 increases the anchorage-independent growth of SMMC-7721 and MHCC-97H cells in soft agar compared with control protein in a dose dependent manner. The assays last for 28 days. *: P < 0.05; **: P < 0.01.

Furthermore, we transfected SMMC-7721 and MHCC-LM3 with overexpression vectors. Elevated mRNA of EDIL3 did not increase intracellular EDIL3 levels in western blot; however, we did detect an elevation of autocrine EDIL3 by ELISA and western blot of CMs (Figure 4A). SMMC-7721 overexpressing EDIL3 exhibited a significant advantage of anoikis resistance and anchorage-independence compared with control cells (Figure 4B-E) without affecting the proliferation and invasion (Additional file 2: Figure S2C). Importantly, the addition of EDIL3 in control cells partly mimicked, albeit to a lower degree, these effect (Figure 4B-E). We obtained similar results in MHHC-LM3, a cell line that also exhibited a low basal level of autocrine EDIL3 (Additional file 4: Figure S4A-E).
Figure 4

Overexpression of EDIL3 in SMMC-7721 cells significantly increases the anoikis resistance and anchorage independence. A, Overexpression of EDIL3 is validated by real-time PCR, western blot and ELISA assay, confirming that the increase in transcription leads to a higher autocrine EDIL3. B, EDIL3-overexpressing SMMC-7721 forms more clones in soft agar compared with control cells, which can be partly mimicked by recombinant EDIL3 (50 nM). C, After being suspended for up to 72 h, the overexpressing group demonstrates a high WST-8 value compare to the control cells at 48 h and 72 h, and administration of recombinant EDIL3(50 nM) in control cells partly mimics the effect induced by EDIL3 overexpression. D, Caspase3/7 intensity assay validate that the difference in cell viability between the two groups is caused by lower apoptosis induction. E, Annexin V/PI stain by FACS is performed to validate the apoptosis result. EDIL3 overexpression lowers both the early phase and later phase apoptosis percentage and recombinant protein partly mimics this effect. *: P < 0.05; **: P < 0.01.

We tested whether knocking down EDIL3 would cause the opposite effects. Using two shRNA sequences, we significantly suppressed the mRNA level of EDIL3, leading to a decrease in secreted EDIL3 in Huh-7 cells but no changes in intracellular EDIL3 in control cells (Additional file 5: Figure S5A). In agreement with the result from overexpression experiments, the decrease in EDIL3 in CM increased the caspase3/7 activity, lowered the WST-8 value and inhibited the colonies formed in soft agar, while adding recombinant EDIL3 partly rescued these inhibitory effects (Additional file 5: Figure S5B-D).

Taken together, these results indicate that EDIL3, especially secreted by cancer cells in our current study, is effective in protecting them from anoikis after suspension, thus supporting anchorage independent growth.

EDIL3 promotes HCC tumorigenesis in vivo

To further elucidate the functional role of anoikis resistance conferred by EDIL3 in vivo, subcutaneous tumor formation assays with EDIL3-overexpressing and control SMMC-7721 were performed in nude mice. The results clearly indicate that the size of tumor formed in SMMC-7721 with EDIL3-overexpression is significantly larger than the control cells in the 2–4 week after subcutaneous implantation (Figure 5A, C). IHC staining and qRT-PCR confirmed the overexpression of EDIL3 in cancer cells (Figure 5B, C) and revealed that tumors with high EDIL3 exhibited a much lower apoptosis rate by TUNEL assay, whereas PCNA, a proliferative marker, was dominantly positive in EDIL3-overexpressing tumors but only sporadically positive in control tumors (Figure 5D). These results demonstrate that EDIL3 supports subcutaneous tumor formation.
Figure 5

EDIL3 promotes HCC tumorigenesis in vivo. A total of 1.0 × 106 of EDIL3-overexpressing or control SMMC-7721 cells are subcutaneously implanted into the right flank of 5 nude mice of each groups. The mice were observed and tumors formed are measured every week and resected after 6 weeks. A, After 6 weeks, EDIL3-overexpressing group shows relatively larger tumors compare with control group. B, qRT-PCR of 5 tumors in each group demonstrates a significant elevation in mRNA level. C, Tumor growth curve reveals a shorter latency in the EDIL3-overexpressing group (2 weeks) vs. the control group (4 weeks) and tumors in the EDIL3-overexpressing group are significantly larger than control group from 2nd week to 4th week. D, IHC stain in the same region of the tumor confirms the overexpression of EDIL3 and suggests more active proliferation (PCNA) and lower apoptosis (tunel) upon EDIL3 overexpression. **: P < 0.01.

Activation of FAK-Src-AKT signaling by EDIL3 ligation is associated with anoikis resistance

Because EDIL3 is a known ligand of integrins, we first examined whether HCC cell lines express receptors for EDIL3, such as integrin αV and α5. All of the cell lines used in this study expressed these integrins (Additional file 3: Figure S3B). We then examined the alteration of integrin-mediated signal pathways that were key to cell survival upon autocrine EDIL3. Indeed, we observed that EDIL3 activates signals downstream of integrins. During the 48 h of suspension, the pro-survival FAK-Src-AKT signal gradually faded away, suggesting the anoikis triggered by detachment. However, the FAK-Src-AKT signal was significantly sustained in EDIL3-overexpressing cells but tapered much more quickly in control cells (Figure 6A). The administration of recombinant EDIL3 also elevated these signal pathways, although to a lesser degree than overexpression (data not shown). However, pretreating cells with RDG-blocker cilengitide reversed the activation of FAK-Src-AKT (Figure 6B).
Figure 6

Sustained activation of FAK-Src by EDIL3 through RGD recognition. A, Western blot and densimetric analysis suggests EDIL3 overexpression sustains the signal intensity of FAK-Src and results in higher AKT phosphorylation within suspended SMMC-7721 cells over 48 hours. The elevated 397p-FAK, 416p-Src and 473p-AKT axis in overexpressing cells compare with control cells exists at most of the time points. B, Cilengitide (10 μM) reversed the activation of the FAK-Src-AKT axis induced by EDIL3. The signal intensity was examined after 24 h of suspension.

Disrupting integrin ligation restores anoikis susceptibility to HCC

Based on the results above, we continued to examine weather disrupting the EDIL3/ integrins interaction could affect the anti-anoikis effect of EDIL3. In first effort, Cilengitide, an RGD-containing integrinαV antagonist, almost abrogated the anti-anoikis conferred by EDIL3 overexpression by lowering the WST-8 value (and increasing casepase3/7) to the control cells. However, Cilengitide did not result in an obvious pro-apoptotic effect on control cells, which have low EDIL3 expression (Figure 7A). In another effort, silencing of integrin αV by RNAi also led to a loss of protection of EDIL3. Surprisingly, the relative caspase3/7 intensity declined significantly with the down-regulation of integrin αV (Figure 7B); indeed, we observed similar results in an integrin α5 knock down assay (data not shown). These results suggest that RGD binding of integrinαV or integrinα5 is crucial for EDIL3-induced anti-anoikis and that integrins alone or when unligated might activate apoptosis in detached cancer cells.
Figure 7

Disrupting integrin-EDIL3 ligation deprives HCC of anoikis resistance induced by EDIL3 in SMMC-7721 and MHCC-LM3. A, Cilengitide (10 μM) reduces the WST-8 value and increased casepase3/7 of the EDIL3 overexpressing group back to levels of control group, suggesting a blockage of EDIL3’s effect. There was not any effect observed in the control group in WST-8 and caspase3/7 assay. Both the two cell lines show consistent results. B, when integrin αV was knocked down to a very low level in both cell lines by two siRNAs (Si2 and Si3), the alteration in WST-8 and casepase3/7 value of EDIL3-overexpressing cells compared with control cells disappear. Interestingly, the WST-8 value and casepase3/7 intensity results suggest the overall anoikis significantly declined upon integrin αV silencing in both siRNA knock-down groups compare with si-control group. **: P < 0.01.

This study provides a clear expression pattern of one ECM protein, EDIL3, in clinical samples and highlights the role of autocrine EDIL3 in the integrin-mediated interaction between HCC and ECM, which resulted in an anti-anoikis and anchorage-independent growth advantage. Our results also suggest a potential therapeutic strategy for personalized medicine by targeting ECM-integrin interactions for cancer cells in a subgroup of HCC patients.

In tissue level, EDIL3 is dominantly expressed and secreted by the endothelium, acting as a regulator of vascularization, immunocyte-endothelium adhesion and platelet microparticle clearance [14, 15, 26], or by a subset of macrophages to mediate engulfment [27]. Our data reveal that EDIL3 expression is turned on in adult human normal liver (cells) and is amplified during malignant transformation, which is different from mouse data, wherein EDIL3 is undetectable in the liver [15]. The cause of the up-regulation of EDIL3 in HCC is not clear; however, there is evidence suggesting that VEGF induces EDIL3 expression in malignant cells [28] and some inflammatory cytokines in endothelium [25]. Therefore it is possible that EDIL3 is elevated in response to cytokines in HCC. However, other events, such as HBV incorporation or epigenetic modifications, may also affect EDIL3 expression. Meaningfully, by analyzing an adequately sized sample, we demonstrated that when EDIL3 protein is accumulated to a high level, it is associated with larger tumor size and more PVTT formation, as well as greater relapse risk and shorter overall survival, which is consistent with a previous study, although the previous report only reached a positive result in overall survival [20], most likely due to the sample size and difference in patients selection.

In cell lines, however, EDIL3 demonstrates a unique expression. By examining multiple HCC cell lines and normal hepatocyte derived cell line, we find EDIL3 is expressed by both the normal and malignant cell lines, suggesting EDIL3 may not be directly linked to the relative tumorigenicity of HCC cell lines. Interestingly, we reveal that intracellular EDIL3 protein is observed at almost the same level despite the large differences in transcription. This inconsistency in transcription and translation was explained by the difference in secreted EDIL3. Moreover, either overexpressing or knocking-down the transcription level of EDIL3 only affected the secreted EDIL3. Based on these observations in cell lines and human samples, we postulate that EDIL3 is maintained in a standby status by many types of cells in quiescent (or normal) conditions and is secreted upon elevated transcription in physiologic or pathophysiological conditions; notably, the secretion is probably key to its biological functions. However, it will also be interesting to see the effects of EDIL3 knock-out in malignancies, and the potential regulation of EDIL3 secretion.

EDIL3 is well documented as an integrin ligand, modulating a wide range of biological processes that require integrins, such as integrin αV in angiogenesis and integrin αL in the neutrophil adhesion cascade [13, 29]. Because integrins such as integrin αV are also abnormally expressed in HCC [30], we postulated that EDIL3 may affect HCC cells through integrin ligation when it is secreted and anchored on the cell membrane, or deposited in the ECM. By overexpressing EDIL3 or recombinant EDIL3 treatment, we observed that secreted EDIL3 reduced the likelihood of anoikis in cancer cells and promoted anchorage-independent growth, both of which are indispensable for HCC spreading and PVTT formation, whereas do not affect proliferation or invasion. Indeed, all of the fresh PVTT samples in our study exhibited a very high level of EDIL3 protein, so it is highly possible that tumor cells bring this protective protein when leaving primary lesion, thus assisting PVTT formation. Similar strategies to overcome anoikis have been reported in other types of cancer, in which another ECM protein, collagen IV, is expressed [31].

The anoikis is a complex apoptosis process resulted from cell detachment from ECM or inappropriate cell-cell adhesion. The lack of ECM contact or the engagement with inappropriate ECM leads to the activation of anoikis from death receptors (extrinsic pathway) or mitochondria (intrinsic pathway) [32]. Integrins on the cell surface are crucial for anoikis modulation by modulating both the extrinsic and intrinsic pathway, passing opposite signals in their ligated or unligated status [21, 23, 33] or even independent of their ligation status [34]. The 397FAK-416Src-473AKT axis is a well-documented pro-survival pathway in anoikis resistance [32, 3537]. Our results suggest that this axis is sustained when cancer cells are located in an environment rich with EDIL3, whereas phosphorylation of all three effectors was partly inhibited when RGD binding sites were blocked by cilengitide, suggesting that RGD recognition is critical for this pro-survival signal. Another interesting finding is that the suppression of integrin αV (or α5) expression led to a strong anoikis resistance, suggesting that unligated integrin may trigger anoikis, which is supported by other studies [38, 39]. However, intriguingly enough, integrin αV or α5 expression by cancer cells is required for invasion and spreading [4042]. This seemly contradictory role of integrins reflects the importance of a suitable interaction between cancer cells and the microenvironment, or ECM, at the suitable stage of tumor progression; in this study, an EDIL3-integrin alliance may be exploited by detached HCC cells to escape anoikis when cells detach from the primary lesion, which resulted into a higher success rate of metastasis.

Finally, a variety or integrin inhibitors has entered clinical trial of different types of cancers [33, 43]. However, the outcomes have fallen short of expectations, revealing the complexity of integrin function as discussed above. Therefore, refined patient selection is required. Cilengitide was initially documented as an angiogenesis and invasion inhibitor [44]. However, our preliminary study suggests a potential role of cilengitide in inhibiting anoikis resistance in EDIL3-overexpressing HCC, thus pointing to a new potential indication of integrin inhibitors in HCC and providing a reference for patient selection.

Our study suggests that high levels of autocrine EDIL3 may contribute to a receptive microenvironment for the survival of HCC cells and therefore assist cancer cell progression, which lead to adverse clinical results. Although other explanations may be concealed in our current effort, the anti-anoikis conferred by high level of EDIL3 is an important mechanism for through which EDIL3 provide protection for HCC cells. Disrupting the ligation of EDIL3 to integrins via RGD-blockers or inhibitors in selected patients with high EDIL3 may bear potential therapeutic value.

Methods and materials

Cell culture

The HCC cell line Sk-Hep1 was purchased from the American Type Cell Culture Collection (ATCC), HuH-7 was purchased from RIKEN Resource Centre, Japan, SMMC-7721 was purchased from the Cell Bank of the Chinese Academy of Sciences Cell Bank of Type Culture Collection. The metastatic HCC cell lines MHCC-97 L, MHCC-97H and MHCC-LM3 were obtained from the Liver Cancer Institute, Zhongshan Hospital, Fudan University. The HCC cell line CSQT-2 derived from a portal vein tumor thrombus (PVTT) has been described elsewhere [45]. Human liver epithelial cell line THLE-3 was purchased from ATCC;. All cell lines except THLE-3 were cultivated in DMEM (Dulbecco’s modified Eagle medium) supplemented with 10% (v/v) fetal calf serum at 37°C in a humidified incubator under 5% CO2 condition. THLE-3 was cultivated in BEGM (Lonza) with the addition of BEGM bullet kit according to the instruction on ACTT.

Sample information

All samples were collected in Department of Liver Surgery, Renji Hospital, Shanghai Jiaotong University School of Medicine. Fresh samples, including tumor tissues and PVTTs, were obtained from HCC patients during tumor resection. Normal livers and cirrhotic livers were collected from healthy liver donors and cirrhosis patients, respectively, during liver transplantation. Approximately 202 HCC samples were collected from 2004 to 2010 and were constructed into tissue microarray (TMA). The median age of this cohort of patients was 50 years (range 17–73 years). The majority of patients are HBV-positive (187/202). The follow-up was ended in December 2012, and the median period was 33 months (range 2–90 months). All human samples were obtained with informed consent, and protocols were approved by the ethical review committee of the World Health Organization Collaborating Center for Research in Human Production (authorized by the Shanghai Municipal Government).

Quantitative real-time PCR

Total RNA was extracted using Trizol reagent (Takara) and reverse transcribed using PrimeScript qRT-PCR kit (Takara) according to the protocol. Quantitative real-time PCR analyses were performed with SYBR Premix Ex Taq (Takara) on a 7300 Real-time PCR system (Applied Biosystems Inc.), and the primer for EDIL3 was as follows: forward, GTGAACTGTCGGGTTGTTCTGAG; and reverse, 5′-GGTTCCCAAGTGAACATGTCCAT-3′. The primers for ACTB were as follows: forward, 5′-TCACCCACACTGTGCCCATCTACGA-3′; and reverse, 5′-CAGCGGAACCGCTCATTGCCAATGG-3′. The relative expression of EDIL3 was analyzed by the comparative cycle threshold method (ΔΔCt method), which was normalized to ACTB.

Protein collection, Western blot and antibodies

The HCC samples were handled with T-Per tissue protein extraction reagent (Thermo Scientific) according to the manufacturer’s protocol. Total cell protein was obtained by IP lysis buffer (Beyotime) for further assays. The secreted proteins in conditional media were collected by ethanol precipitation. Briefly, when cells grew to approximately 80% confluence, normal media were replaced by serum-free media. After 24 h, the media were collected, and 95% ethanol was added and kept overnight. The precipitated proteins were collected with SDS loading buffer and underwent standard western blot immediately. Western blot was performed using SDS-PAGE gel for proteins separation and nitrocellulose membrane for proteins blotting. The total volume of protein used in WB is 50 μg for tissue sample assays, and 30-60 μg in cell line assays. The signal was detected by the Odyssey infrared system (LI-COR).

The antibodies used were the following: EDIL3 (ProteinTech), ITGAV (Abcam), p-FAK397 (Cell Signal Technology), FAK (Abcam), p-Src416 (Epitomics), Src (Cell Signal Technology), p-AKT473 and AKT (Cell Signal Technology), StrepII (Abcam), GAPDH (ProteinTech) and β-actin (Abcam).

Immunohistochemistry stain and analysis

A total of 202 samples of HCC on 2 TMA slices, 3 NLs, 10 HCCs and 6 PVTTs are subjected to IHC. Paraffin-embedded sections 5 μm thick and TMA were stained according to standard Immunohistochemistry (IHC) protocols. Heat-mediated antigen retrieval in pH 6.0 citric acid was performed. The antibodies used here were EDIL3 (ProteinTech). The scoring of EDIL3 was performed according to the ratio and intensity of positive-staining cells: 0-5% scored 0; 6-35% scored 1; 36-70% scored 2; and more than 70% scored 3. The final score was designated as negative (score 0), positive (score 1 or 2) and high positive (score 3). These scores were determined independently by two experienced pathologists in a blinded manner.

Enzyme-linked immunosorbent assay

HCC cell lines were planted on 10 cm petri dish until 90% confluence, then were incubated in 3 ml serum free media for 48 hour. At the end of the incubation period, the conditional media were harvested and stored at -80C until use. The secreted EDIL3 in media were quantified using ELISA kits according to the manufacturer’s instructions (CUSABio).

Immunofluorescence staining

In cell assay, all the HCC cell lines under tests were seeded on cover slides in 24-well plates and incubated overnight. For F-actin staining, cells were incubated with phalloidin-FITC (Sigma-Aldrich) for 75 minutes at room temperature. For EDIL3 staining, cells were incubated with primary antibodies against EDIL3 (Abnova) for 75 minutes at room temperature, followed by an Alexa Fluor 594-conjugated secondary antibody. In tissue staining, two samples embedded in paraffin were subjected to heat-mediated antigen retrieval in pH 6.0 citric acid, and blocked by 10% BSA. The primary antibodies used are EDIL3 (Abnova) and CD31 (Abcam) with Alexa Fluor 594-conjugated secondary antibody against EDIL3 and Fluor 488 against CD31. Immunofluorescence signals were captured using confocal micro-scopy (LSM 510, METALaser Scanning Microscope, Zeiss).

Recombinant EDIL3 expression, purification and characterization

Human EDIL3 (NM_005711 Origene) with the signal peptide truncated were cloned into the episomal expression vector pCEP-Pu-Strep II-tag (N-terminal) in-frame with the sequence of the BM-40 (SPARC/osteonectin) signal peptide downstream of the CMV promoter, which has been described elsewhere [46]. Briefly, the expression vector was transfected into human embryonic kidney 293/Epstein-Barr nuclear antigen cells (EBNA-293) by Xetrem (Roche). The cells were screened with puromycin, and the surviving cells were cultured on a large scale. The culture medium was collected and centrifuged, and the supernatant was subjected to the StrepTactin sepharose column (IBA) for purification. The collected proteins were quantified and validated by Coomassie Brilliant Blue (CBB) staining and western blot. The empty vector was applied as control proteins.

Establishment of stable overexpression or knock-down cell lines

For the overexpression of EDIL3 in cell lines, the full-length cDNA of EDIL3 (NM_005711 Origene) was subcloned into pCDH-CMV-MCS-EF1-Puro vector (System Biosciences). Lenti-virus was packaged in 293 T cells using Lipofectamine2000 (Invitrogen), and the virus DNA was transduced into cell lines. For knock-down of EDIL3, short hairpin RNA (shRNA) sequences (Sh1:5′-CCGGCCCAAGTTTGTCGAAGACATTCTCGAGAATGTCTTCGACAAACTTGGGTTTTTG- 3′; Sh2: 5′-CCGGGGAGGTTGCATCAGATGAAGACTCGAGTCTTCATCTGATGCAACCTCCTTTTTG- 3′) targeting EDIL3 were cloned into pLKO.1-puro vectors (Roche). ShRNA-containing plasmids were packaged into lenti-viruses and transduced into target cell lines as above. The efficiency of over-expression or knock-down was tested by qRT-PCR and western blot.

Anoikis assay

Anoikis was induced by culturing cells in poly-HEMA coated plates as described by others [47]. Briefly, poly-HEMA were prepared as a 10 mg/ml solution in ethanol, which covered completely the petri-dish or plates, then dried and repeated once. Cells in serum-free medium were seeded into the coated plates with or without adding the recombinant EDIL3 at the corresponding concentration. To avoid survival effects caused by the clumping of cells, 0.5% methyl cellulose (Sigma-Aldrich) was added into the medium. At the designated time points, the suspended cells were collected and subjected to cell viability assays by WST-8 kit (Dojindo), apoptosis assays by Caspase3/7 Glo kit (Promega), and Annexin V/PI staining on FACS (BD) according to the protocols from the respective manufacturers.

Anchorage-independent growth assay

Colony formation in soft agar was tested to assay anchorage-independent growth. Stable overexpressing EDIL3 cell lines SMMC-7721 and MHCC-LM3 or their control cells were suspended in the upper layer consisting of culture medium with 1% FBS and 0.35% agar, which is above a basal layer of 0.6% agarose in 6-well plates in a triplicate manner. The cell density was 2,500 cells per well. The soft agars were fed twice a week with 0.3 ml of culture medium. In recombinant EDIL3 treatment assay, the soft agars were fed every 2 days with serum-free culture medium containing EDIL3 or empty vector. Colonies were stained with 0.05% crystal violet, and all the visible colonies were counted by microscopy after 21–28 days.

Tumorigenesis in vivo

A total of 1.0 × 106 of lenti-vector or lenti-EDIL3 SMMC-7721 cells were implanted subcutaneously into the right flank of 5 BALB/c (nu/nu) mice in each group. Tumor sizes were measured once a week, and mice were sacrificed for the analysis of tumor burden after 6 weeks. All procedures were performed in accordance with The Animal Care and Use Committee of Jiaotong University. The resected mice tumors were fixed in methanol and embedded in paraffin before being subjected to IHC using anti-EDIL3 (ProteinTech), anti-PCNA (Abcam) and anti-TUNEL (Millipore).

RGD-blocking assay and knock-down of ITGAV

Cyclic RGD-containing peptide cilengitide (Selleckchem) or control peptide RGE was used to block the ligation of EDIL3 to integrins on the surface of HCC cells at a final concentration of 10 μM. Briefly, the cells was suspended in Poly-HEMA coated plates and exposed to the peptides about 3 hours after suspension. Then, the cells were subjected to the assays as described above. SiRNA targeting ITGAV (Si2: 5′-GCCAGCCAAUUGAAUUUGATT- 3′; Si3: 5′-CCCAGUUGUAUCUCACAAATT - 3) was used to knock down the protein level of this integrin and was confirmed by western blot. After 72 h of transfection, the cells were subjected to the assays as above.

Statistical analysis

Statistical analyses were performed with SPSS 16.0 software. The association between EDIL3 expression and clinicopathological parameters was analyzed by Pearson Chi-Square test. Overall survival and relapse risk was calculated by the Kaplan-Meier method and compared by the log-rank test. Statistical significance was determined by two-tailed Student’s t test for any differences observed (P < 0.05).

The study was supported by the Training Program for Superb Academic Leaders in Shanghai Health System (No. XBR2011029); the Special Fund for Building of Leading Talent Teams in Shanghai; Doctoral Innovation Fund Projects of Shanghai Jiao Tong University School of Medicine (BXJ201222); National Key Sci-Tech Special Projects of Infectious Diseases (2013ZX10002-007-006) and National Science Foundation of China (81472678; 81101600; 81201624; 81372368). We are grateful to Drs Xiao-Jin Liu, Xiao-Mei Yang, Ya-Hui Wang, Guang-Dong Yang and Shu-Heng Jiang for their assistance with the experiments. We also received constructive comments on the manuscript from other members in our lab.

Authors’ Affiliations

Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiao Tong University
State Key Laboratory of Oncogenes and Related Genes, Shanghai Cancer Institute, Renji Hospital, Shanghai Jiao Tong University School of Medicine

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© Feng et al.; licensee BioMed Central Ltd. 2014

This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

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text:
How Ponzi Schemes Work

Ponzi Scheme Basics
People often compare a Ponzi scheme to building a house of cards. It must collapse eventually.
People often compare a Ponzi scheme to building a house of cards. It must collapse eventually.

Though it landed him in jail, Charles Ponzi's infamous scam spawned many imitators. The get-rich-quick scheme has proved too alluring for other scoundrels to pass up. However, these imitators need not use the front of international reply coupons to make it work.

The basic framework of a Ponzi scheme can be applied and reapplied in countless contexts. The scheme revolves around the process of paying old investors with the money you get from new investors. The central method remains the same. All one has to do is hook a few investors who are willing to get in early on a once-in-a-lifetime business venture. The details of the investment don't matter too much. What suckers people in is the promise of fantastic returns on investments.

After the schemer has convinced a handful of investors to fork over money, those funds can bankroll a nice car -- or, if the schemer is truly sneaky, he or she can use it to rent office space and buy some fancy furniture. These props will help con the next round of investors. Now, he or she is ready to find more investors. This time, the schemer takes a slice off the top for himself or herself and uses the rest to pay off the first rung of investors with some initial returns. (Some liken this to robbing Peter to pay Paul, but it's not quite the same, as we'll see.)

Eventually, the second rung of investors will need its payout. This is a simple matter of wash, rinse and repeat: The money from a newly recruited third rung of investors can pay off the second rung and deliver more returns to the first rung.

But as the cycle goes on, it gets more complicated. Earlier rungs of investors will get suspicious if they don't continue to see returns. New investors will have to be paid back their initial investment, and the schemer will have to appease them with regular returns. This means that new investors will have to be added to the Ponzi scheme continuously in order to pay all the previous rungs. The schemer is under an enormous amount of pressure to keep adding investors, and one person can only do so much. (This is why the most successful schemes typically involve accomplices, but this merely delays the inevitable.) The scheme will eventually become unsustainable. The upside-down house of cards the schemer has built will finally collapse.

More to Explore


text:
Sainsbury's Bank Fixed 1.34% until 31/12/2019

Sainsbury's Bank Fixed 1.34% until 31/12/2019
Overpayments allowed Portable Initial rate Reverts to Max LTV
Whether the deal allows you to pay more than you have to each month so you can pay your mortgage off more quickly and save on interest.
Whether the deal can be transferred to a new property if you move so you don’t have to pay charges for paying the mortgage off early.
The interest rate you'll pay during the initial deal period.
The interest rate you'll pay once the initial deal period ends.
The maximum amount you can borrow as a percentage of the property value.
Yes Yes 1.34% 3.99% 60.0%

Assumed borrowing of £125,000 over 25 years, representative 3.7% APRC. Total amount payable: £190,980.96. Includes interest of £65,235.96, Valuation fee of £0, Booking fee of £0 and Administration fee of £745. Repayments: 23 monthly repayments of £490.58 at 1.34% (fixed), then 277 monthly repayments of £646.04 at 3.99% (variable).

Sainsbury's Bank customer satisfaction scorecard:

How do we work this out?
Clarity of statements Flexibility of payments Transparency of charges Customer service Overall customer score
n/a n/a n/a n/a

Not rated

n/a = sample size is too small.

We have not been able to rate this provider as our customer satisfaction survey didn't generate a big enough sample of its customers but we aim to rate as many providers as possible

Maximum age 76
Minimum age N/A
Location Any
Restricted location note N/A
Existing customer note N/A
Eligibility notes N/A

Mortgage product details

Mortgage types Purchase, First time buyer
Deal type Fixed
Tracker type N/A
Payment options Repayment

Mortgage product features

Overpayments allowed Yes
Overpayment limits note Up to 10% of the outstanding balance in a 12 month anniversary period
Offset mortgage No
Help to Buy mortgage No
Shared ownership mortgage No
Shared equity mortgage No
New-build mortgage No
Self-build mortgage No
Portable mortgage Yes

Product fees

Arrangement fee £745
Arrangement fee can be added to loan Yes
Booking fee £0
Telegraphic transfer fee £25
Insurance fee £0
Other fees £0

Rate information

Minimum borrowing amount £40,000
Maximum borrowing amount £1,000,000
Minimum term (years) 5
Maximum term (years) 40
Initial deal end date 31.12.19
Initial deal length (months) 24
APRC 3.66%
Initial rate 1.34%
Revert-to rate 3.99%
Maximum LTV 60.0%

Other charges

Exit fees £100
Valuation admin fee £0
Valuation fee From £191 for property value above £100,000
Early repayment note 2% then 1% until end of fixed period

Standard benefits

Valuation admin fee excluded from any refund offer No
Maximum free valuation £0
Cash back amount £0
Further notes about benefits N/A

Remortgage benefits

Cash back on legal fees No
Free legal work No
Valuation offer Payable

Purchase mortgage benefits

Cash back on legal fees No
Free legal work No
Valuation offer Free


text:
VA Dold’s newest release Stefan book tour and excerpt

El is a beautiful, successful, plus sized woman suffering from a debilitating humiliation that has left her hating all handsome, wealthy men exactly like Stefan Le Beau. Unfortunately for Le Beau, she’s known him since she was sixteen and was totally snubbed by him. To her, he’s a hound dog and a man-whore.
Stefan is a playboy to the extreme with one hard and fast rule: date a woman only once, take her to bed, and be gone before morning.
Until El. Stefan’s dream of finding his mate comes true when he bids two hundred thousand dollars to win a date with El at Simon’s charity ball. Money well spent in his opinion. Now, if she would only talk to him. Or look at him. Or touch him, or…like him.
Can Stefan convince El he’s a reformed man? Can El learn to trust a man who is the epitome of what she avoids and could shatter her heart?
It will require drastic, strategic measures from the entire family to make this mating happen.

va dold
BIO: V.A.Dold is the bestselling author of the Le Beau Brothers series, New Orleans wolf shifter novels. A graduate of Saint Cloud University, she majored in marketing. Prior to becoming a full time writer, she was a publicist to the authors, owning Innovative Online Book Tours and ARC Author & Reader Con’s (ARC NOLA) (ARC Phoenix). Still is. The companies mesh so well together, much like PB&J.  Her idea of absolute heaven is a day in the French Quarter filled with nothing but her computer, her coffee mug and the Brothers, of course. A Minnesota native with her heart lost to Louisiana, she has a penchant for titillating tales featuring sexy men and strong women. When she’s not writing, she’s probably taking in a movie, reading or traveling. Her earliest reading memories are from grade school. She had a major fixation with horses, and the Black Stallion was a favorite. Then junior high came along and teenage hormones kicked in. It became all about the Harlequin Romances. She has been hooked on romances ever since.
Stefan: Le Beau Brothers: New Orleans Billionaire Wolf Shifters with plus sized BBW for mates (Le Beau Series Book 3)

VADOLD FB Page banner

And now the excerpt!

Fortyfive minutes later, the auction was finally over. She could finally go home. Her feet were swollen and something was doing a tap dance on the inside of her skull. The Tylenol she had taken fifteen minutes ago hadn’t kicked in yet.

She considered her options as she rubbed the edge of her sole, should I chance taking these shoes off? Crap, I might not get them back on. Then what? Go bare foot in this gown?

That wouldn’t be embarrassing, not at all.

El stood and limped to the door leading to the ballroom. She groaned as she looked at the crowd she would have to walk through to get to her truck.

This was going to be two hundred thousand times worse than a walk of shame.

She giggled at her bad joke. Yeah, she was losing it.

What’s so funny?”

The deep timber of his drawl had no business sending her pulse racing. She waited for a panic attack to kick in, but her body remained calm.

Nothing.” She squared off with Stefan. “Were you dropped on your head as a child?”

The smell of fresh leather surrounded her. She took a deep breath wondering once again where it was coming from.

He frowned. “No.”

She cocked her head and arched a brow at him.

Is there a history of insanity in your family?”

No.” He scowled. “What are you getting at?”

No one in his right mind would bid two hundred thousand dollars. That leaves one conclusion, you’re insane.”

Oh, I’m insane all right. Insane for you.”

Right. Go fishing with those lines somewhere else, this woman isn’t biting.”

Oh, cher,” he laid his hand across his heart, “you wound me.”

Shut up, Le Beau.” She tried to push past him but he didn’t budge. “Please move out of the way.”

From the ballroom, the band began to play a romantic waltz. Her father’s favorite songthe one he always insisted she dance with him to. How odd.

He presented his hand to her. “I do believe they’re playing our song.”

She looked at his hand suspiciously. “What are you doing now?”

I’m asking the single woman I won at the auction to dance. That is my right as the winner.” He grinned.

She stepped back and crossed her arms. “You’re kidding, right?”

I’ve never been more serious, I assure you. Now, may I have this dance?” His hand remained extended as he waited for her response.

She eyed the hand like it might bite. “Fine. I suppose I have to.”

Stefan led her to the dance floor and took her into his arms. She was perfect. His wolf howled in triumph. Now if he could only get her to actually like him.

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Blog Archives

The War on Cash Finds Its General

Kenneth Rogoff would sharply disagree with Peale, a character in the 1915 novel ‘It Pays to Advertise’, who said that the most beautiful word in the English language is “cash.” For Rogoff, a distinguished monetary economist (and chess grandmaster) who

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Posted in america, current events, freedom, government, liberty, life, news, random, real news, truth, uncategorized
Merry Christmas from Mountain Republic!
Just look at us. Everything is backwards, everything is upside down. Doctors destroy health, lawyers destroy justice, psychiatrists destroy minds, scientists destroy truth, major media destroys information, religions destroy spirituality and governments destroy freedom.

Michael Ellner

The control of information is something the elite always does, particularly in a despotic form of government. Information, knowledge, is power. If you can control information, you can control people.

Tom Clancy

If the freedom of speech is taken away, then dumb and silent we may be led, like sheep to the slaughter.

George Washington

I'm Gonna Kick Your Ass!
You learn that you either are going to have a police state where you don't have any freedom left, or you're going to build a world that doesn't create terrorists, and that means a whole different way of getting along.

John Shelby Spong

Stand Up to the TSA

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If you want to offend somebody, just tell him the truth.

Friedrich Nietzsche

       Thousands of Deadly Islamic Terror Attacks Since 9/11

Dr. Judy Wood - Evidence of Breakthrough Energy on 9/11

9/11 Firefighter Blows WTC 7 Cover-Up Wide Open

Evidence That Won't Go Away! - September 11, 2001

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text:
ByLily Masco, writer at
"It's kind of fun to do the impossible." -Walt Disney
Lily Masco

Our favorite ogre is back! As if we knew that many. NBC Universal chief Steve Burke revealed that the idea of a Shrek revival is in the works, providing ample opportunity to extend the franchise to different platforms.

The Shrek franchise, based on William Steig's picture book Shrek!, has been DreamWorks Animation's biggest moneymaker since its launch in 2001. Shrek is also the 12th highest-grossing franchise and the highest-grossing animated franchise of all time. Shrek will only continue to grow with each layer of brand stretching that is added to its collection (like an onion).

Thus far, the franchise has led to the creation of four official movies, in addition to holiday specials, shorts, a musical adaptation, and a video game. Shrek even led to the spin-off Puss in Boots (2011) featuring Antonio Banderas, which appears as both a film and a TV series.

There is an upcoming movie sequel, Puss in Boots 2: Nine Lives & 40 Thieves, which was removed from the release schedule in January 2015 following corporate restructuring. Another factor that delayed its release is DreamWorks Animation's new policy to release two films a year. It is unclear as to whether this Shrek reboot will have an effect on the Puss in Boots sequel.

During an investor gathering at the Sixth Annual TMT Symposium, Burke admitted:

"Comcast hopes to breathe new life into Shrek and crank out as many as four animated movies a year following its acquisition of DreamWorks Animation."

Burke told the Guggenheim Partners that the aim is to exploit the fictional characters in order to make a profit by brand extension. In other words, they could increase their profit by turning the movie's low single-digit returns into a "different kind of business."

Examples Of Shrek Merchandise
Examples Of Shrek Merchandise

Besides creating more adaptations and sequels, examples include producing licensed merchandise revolving around the iconic protagonists. This opens a world of endless opportunities, as pretty much everything can be branded by the franchise: video games, toys, bicycles, clothing, costumes, home decor, kitchen supplies, bedding, electronics, school supplies, food, drinks, and so on.

Another option that is guaranteed to make more money is the emergence of Shrek-themed amusement parks such as Shrek's Adventure, which opened in London (next to the London Eye) last summer. They could continue to expand by opening new branches across the globe, particularly in Beijing, China, where they already have a presence.

DreamWorks Animation (DWA) has been enduring some noticeable transitions since Comcast recently struck a deal to purchase the company for the hefty price of $3.8 billion. This new collaboration will give them an extra push in their rivalry with Disney, which acquired Pixar for $7.4 billion back in 2006.

Burke hopes that the deal will make a turn for profit in the near future, saying that the acquisition advances Comcast's "consumer products agenda by five years." The pressure is on for DWA to release phenomenal films now that the company has $250 million worth of overhead costs and is only releasing two movies per year.

Christopher Meledandri/LA Times
Christopher Meledandri/LA Times

Film producer Christopher Meledandri, the founder and CEO of Illumination Entertainment, will have creative control over the project. His goal is to help come up with the best tactic to not only resurrect Shrek, but to "take a lot of the existing DreamWorks franchises and add value as we create new franchises.”

If this is the case, then we can definitely get excited for the Shrek reboot, along with all future possibilities. Many DWA franchises are already extraordinarily successful, with legends such as Ice Age, Toy Story, Kung Fu Panda, and Despicable Me.

What would you like to see in the new Shrek reboot?

Sources: Deadline, ComicBook

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Can You Guess Which Horror Movie These Crazy, Brutal Deaths Are From?

By ⋅ Posted on ⋅ Last edit on

So many ways to die...can you remember which horror movie these deaths are from?

  1. Q: How d'ya kill him? A: Oh, I just jammed a blender into his head.

    • The Unborn
    • The Strangers
    • Halloween: Resurrection
    • You're Next
  2. Which movie shows this lovely nun getting murdered by a hammer-wielding child?

    • Case 39
    • The Orphanage
    • The Omen: 666
    • Orphan
  3. Life Rule: try not to fall into a pile of bear traps...

    • You're Next
    • Seed of Chucky
    • Saw VI
    • The Collector
  4. A drill drops into the jacuzzi...gory electrification ensues!

    • Valentine
    • My Bloody Valentine
    • Final Destination 3
    • Scream 2
  5. The body in the laundromat?

    • My Bloody Valentine
    • Cherry Falls
    • Leprechaun III
    • Friday 13th Part III
  6. Steamed to death by a coffee machine?

    • Leprechaun II
    • Troll II
    • Critters II
    • Chucky II
  7. A resourceful killer uses whatever's at hand: in this case, a bottle of alcohol.

    • American Psycho
    • Hatchet
    • Sorority Row
    • I Still Know What You Did Last Summer
  8. A series of unfortunate events involving a fridge magnet, spaghetti and a ladder...?

    • Final Destination 2
    • Final Destination 4
    • Final Destination 5
    • Final Destination 3
  9. Death in a folding bed, like the world's grossest calzone...

    • Freddy vs. Jason
    • Friday 13th VII: The New Blood
    • Jason Goes To Hell: The Final Friday
    • Friday 13th: The Final Chapter
  10. So, the guy gets hung from a tree and dropped onto the car windshield....

    • Scream 3
    • Urban Legend
    • One Missed Call
    • Drive-Thru
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