Article Category: Cancer Research

Your Commitment to Producing Reproducible Research is Critical

Sign up for LI-COR’s “Reproducibility in Science Webinar” Series. The first webinar is on March 25, 2015.

Reproducibility is becoming a highly discussed issue in all research sciences. The ability for major research findings to be independently replicated after an initial experiment is essential to building upon foundational discoveries. When experiments are not conducted thoroughly or published articles lack sufficient details for replication, we lose the ability to move ahead with accurate science. This is a major problem for researchers today.

NIH QuoteThis problem will only begin to be addressed if institutions, universities, industry, and others alike take on the responsibility of producing scientific experiments and reporting scientific methods that can be replicated at a later date. Thus, the conventions of reproducible science are paramount to the future of biomedical research findings in particular.

Several areas are being scrutinized in the discussion on biomedical reproducibility. Including:

  • Thoroughness of experimental details in journal articles
  • Review of studies submitted to journals
  • Scientific fraud
  • Utilization of highly reproducible techniques

reproducibility initiative logo smallThoroughness in research is important, because without knowing all the details of a foundational experiment future scientists are unable to efficiently build upon that research. To increase thoroughness, the Reproducibility Initiative, headed by Elizabeth Iorns, is advising full disclosure of experimental procedures in published papers. The initiative aims to identify and reward high quality, published research that can be successfully reproduced by independent validation labs. The first step in this process is pinpointing a pool of research that is true and accurate —a task The Reproducibility Initiative has begun by investigating 50 of the most impactful cancer biology studies from 2010 – 2012.

In light of the growing concern regarding scientific reproducibility, the review processes for scientific journal submissions are seeing stringent changes as well. The plans to increase the reproducibility of published papers laid out by the National Institutes of Health (NIH) at the beginning of the year are just one example. In their plan the NIH instituted a training module for enhancing the transparency of cited methods, provided a checklist for routine evaluation of grant applications, and began to urge scientific journals to revise their current review practices. Since then, high-impact journals like Nature and Science have implemented precautionary statistical checklists intended to qualify submitted research papers before publishing them in their magazines.

Unfortunately, though, there are times reported science is proved to be inaccurate, and fraudulent papers claiming breakthrough research are retracted. These retractions can severely affect scientists who have based their careers on such published inaccuracies.

ireland flag smallIn response, Ireland has taken precautions against fraudulent publication. By the end of the year The Science Foundation Ireland will be funding auditors at leading universities. The auditors will look into best practices related to research, procedures “for reporting and investigating misconduct; whether management has followed those procedures in real cases; and whether any investigations have been carried out to a satisfactory standard.” The purpose of these audits is to encourage researchers to take protocols seriously and to put standards in place that will decrease the likelihood of scientific fraud occurring.

Another area of the reproducibility discussion highlights the need for highly consistent research techniques and instrumentation. The nature of complex research and varying protocols between labs can cause inherent fluctuating results from experiment to experiment. To help combat the variability, there is a need for improved and consistent training of researchers using Western blotting and other scientific techniques in their research, just as there is a need for the instruments researchers use to be of the highest quality and to generate reproducible results. Putting more emphasis on training researchers and utilizing the highest quality instruments will help to improve the reproducibility of the studies research labs are currently conducting.

Only time will tell if the scientific community will really begin to take the issues and repercussions of reproducible science seriously. While science is shifting it is important you stay ahead of the curve and close the gaps in your research confidently. Your commitment to producing reproducible research is critical to redressing the reputation of the scientific method from beginning research stages to the published piece.

Are your findings reproducible? Read more about how reproducibility is affecting the life sciences and where the future of Western blotting may be headed.

If you’d like to learn more about reliable instrumentation, check out LI-COR Imaging Systems, which offer a digital imaging solution that ensures reproducible results. See how LI-COR can help you improve your research.

Studying Colon Cancer? Use the C-DiGit® Scanner for Western Blots.

Cortactin (CTTN) is a substrate of Src tyrosine kinase involved in actin dynamics, and is overexpressed in several cancers. Phosphorylated cortactin (pTyr421) is required for cancer cell motility and invasion. This study demonstrates elevated expression of pTyr421-CTTN in primary colorectal tumors, with no change in mRNA levels. Curcumin (a natural compound derived from the spice turmeric) reduced association of CTTN with plasma membrane fractions in surface biotinylation, mass spectrometry, and Western blot experiments. Curcumin also decreased pTyr421-CTTN levels in certain cell lines.

Western blot analysis of cortactin, actin and GAPDH proteins

Figure 1. Western blot analysis of cortactin, actin and GAPDH proteins from DMSO and curcumin treated cell fractions of HCT116 cells. Total cell lysates were used to represent total protein input. Cytosolic and cytoskeletal proteins were extracted using Cell Fractionation kit (Cell Signaling, MA) and quantification of the blots are summarized in graphs. The images were scanned using C-Digit and quantified using Image Studio Digits (LI-COR Biosciences, NE). The data are expressed as a ratio to total protein (mean ± SD). * p<0.05 DMSO vs. curcumin; Student’s T-test. All images are representative of three independent experiments.

Quantitative chemiluminescent Westerns (using the LI-COR® C-DiGit Blot Scanner and SuperSignal® West Pico substrate) showed that curcumin treatment reduced CTTN levels in cytoskeletal fractions, and increased cytoplasmic localization. In Western blotting and immunofluorescent microscopy studies, curcumin induced dephosphorylation of cortactin by activation of the PTPN1 protein tyrosine phosphatase. Western blotting demonstrated that biotinylated curcumin directly binds to PTPN1, and that curcumin blocks the interaction between CTTN and p120 catenin. Curcumin inhibits cell migration in colon cancer cells overexpressing CTTN, and it holds promise as a colon cancer therapeutic.


pTyr421 cortactin is overexpressed in colon cancer and is dephosphorylated by curcumin: involvement of non-receptor type 1 protein tyrosine phosphatase (PTPN1)
VM Radhakrishnan, P Kojs, G Young, R Ramalingam, B Jagadish, EA Mash, JD Martinez, FK Ghishan, PR Kiela
University of Arizona Health Sciences Center, Tucson, Arizona; Arizona Cancer Center, Tucson, AZ, USA
PLoS ONE 9(1): e85796 (2014). 10.1371/journal.pone.0085796

Journal Articles Citing Use of Odyssey® or Pearl® Imaging Systems and Near-Infrared Fluorescence

The following are 4 journal references citing the use of either Odyssey or Pearl Imaging Systems.

Affibody-DyLight Conjugates for in vivoAssessment of HER2 Expression by Near-Infrared Optical Imaging.

Zielinski R, M Hassan, I Lyakhov, D Needle, V Chernomordik, A Garcia-Glaessner, Y Ardeshirpour, J Capala and A Gandjbakhche
Radiation Oncology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA
PLoS ONE 7(7): e41016 (2012). doi:10.1371/journal.pone.0041016

The HER2/neu gene is overexpressed in ~20% of invasive breast carcinomas. in vivo assessment of HER2 levels would aid development of HER2-targeted therapies and perhaps assist in selection of appropriate treatment strategies. This study describes HER2-specific probes for in vivo monitoring of receptor levels by near-infrared (NIR) optical imaging. Affibody molecules were labeled with DyLight750 dye, and affinity and specificity were confirmed in vitro. in vivo, Affibody-DyLight probes accumulated in HER2-positive breast cancer xenografts, but not in HER2-negative xenografts.

Fluorescent images were acquired at different time intervals after probe injection.
Fluorescent images were acquired at different time intervals after probe injection. Mouse bearing BT-474 xenograft tumor was injected with 10 µg HER2-Affibody-DyLight750 conjugate. Images were acquired every second for 1 minute with Pearl Impulse Imager (LI-COR Biosciences). doi:10.1371/journal.pone.0041016.s004

Animals were imaged with a custom NIR fluorescence-lifetime imaging system. The Pearl® Impulse Imager (LI-COR Biosciences) was used to monitor real-time accumulation of the Affibody probe in HER2-positive tumors during very early time points. Probe was injected during image acquisition, and images were captured every second for 1 minute. Probe accumulation in the kidney first, followed by tumor accumulation. Tumor fluorescence could still be detected 5 days after probe injection. This Affibody conjugate is useful for preclinical monitoring of HER2 status, and may have clinical utility.

Disruption of Kv1.3 Channel Forward Vesicular Trafficking by Hypoxia in Human T Lymphocytes

AA Chimote, Z Kuras, and L Conforti
Departments of Internal Medicine and Molecular & Cellular Physiology, University of Cincinnati, Cincinnati, Ohio
Journal of Biological Chemistry 287(3): 2055-67 (2012) DOI 10.1074/jbc.M111.274209

In solid tumors, hypoxia decreases immune surveillance. Kv1.3 channels on T lymphocytes are down-regulated by an unknown mechanism, inhibiting T cell function. The authors hypothesize that changes in membrane trafficking cause reduced expression of Kv1.3 at the cell surface. On-Cell Western cell based assays (Odyssey® Imager, LI-COR Biosciences) were extensively used to measure cell surface expression of Kv1.3.

Chronic hypoxia decreased cell surface expression of Kv1.3 in Jurkat cells. Inhibition of protein synthesis, degradation, or endocytosis did not block this effect. However, inhibition of forward trafficking in the trans-Golgi with brefeldin A (BFA) prevented hypoxia-induced reduction of Kv1.3 cell surface expression. Confocal microscopy confirmed retention of Kv1.3 in the trans-Golgi. Quantitative fluorescent Westerns (Odyssey Imager) demonstrated that expression of AP-1, which is required for clathrin-coated vesicle formation, is downregulated by hypoxia. These data indicate that chronic hypoxia disrupts clathrin-mediated forward trafficking of Kv1.3, thereby reducing immune surveillance by T cells.

Sequential Application of Anticancer Drugs Enhances Cell Death by Rewiring Apoptotic Signaling Networks

M Lee, A Ye, A Gardino, A Hheijink, P Sorger, G MacBeath, and M Yaffe
Dept of Biology, David H. Koch Institute for Integrative Cancer Research, Cambridge, Massachusetts, USA.
Cell 149:780-794 (2012). doi: 10.1016/j.cell.2012.03.031

Historically, standard treatments for human malignancies have been single drug therapies that cause DNA damage. Systems-based approaches and network analysis are now being used to examine how signaling can be re-wired by drug treatments that target dynamic network states. This study suggests that the timing and order of administration of certain drug combinations increases treatment effectiveness. Lee et al. pre-treated cells with epidermal growth factor receptor (EGFR) inhibitors, prior to DNA-damaging chemotherapy drugs.

Pre-treatment with erlotinib (an EGFR inhibitor) sensitized triple-negative breast cancers (TNBCs) to the DNA damage agent doxorubicin, and cell death increased by nearly 500%. Sensitization occurred only if the drugs were given sequentially. Transcriptional, proteomic, and computational analysis of signaling networks showed that dynamic network re-wiring was responsible for sensitization. Quantitative Westerns (Odyssey Imager; high-density, 48-sample blots) were used to monitor systems-level signaling dynamics. Erlotinib treatment made cells more susceptible to DNA damage by reactivating an apoptotic pathway that had been suppressed.

Investigation of Ovarian Cancer Associated Sialylation Changes in N-linked Glycopeptides by Quantitative Proteomics

V Shetty, J Hafner, P Shah, Z Nickens, and R Philip
Immunotope, Inc., Doylestown, Pennsylvania, USA
Clinical Proteomics 9:10 (2012) doi:10.1186/1559-0275-9-10.

CA125 is currently used as a biomarker for ovarian cancer, but is ineffective for detection of early stage disease. Previous research indicates that the level of sialic acid in total serum of ovarian cancer patients is elevated. Based on that idea, the authors suggest using N-linked sialyated glycopeptides as potential targets for early stage ovarian cancer biomarker discovery.

Shetty et al. used Lectin-directed Tandem Lableing (LTL) and iTRAQ quantitative proteomics to investigate N-linked sialyated glycopeptides, and identified 10 that were up-regulated in serum from ovarian cancer patients. Quantitative Western blot analysis of lectin-enriched glycoproteins (Odyssey Imager) was used to confirm the proteomic analysis. In ovarian cancer, increased sialylation of haptoglobin, PON1, and Zinc-alpha-2-glycoprotein was observed. Cancer-specific sialylation of glycopeptides may be a target for biomarker discovery.

Check out some of our Publications Lists for:

Optical Probe for Prostate Tumor Imaging – IRDye® 800CW YC-27

Optical Probes Icon

IRDye 800CW YC-27 (available through LI-COR Custom Services) is a near-infrared dye-labeled imaging agent specifically designed to target prostate specific membrane antigen (PSMA), also known as folate hydrolase I or glutamate carboxypeptidase II.

This small molecule can be used as an optical imaging agent for in vitro (such as In-Cell Western™ Assays), in vivo, whole organ, and tissue section analysis, allowing the same probe to be used in all steps of the biomarker discovery process.

Example of tumor imaging with IRDye 800CW YC-27.
Figure 1. Example of tumor imaging with IRDye 800CW YC-27. Nude mouse bearing 22Rv1 xenograft tumor on the right hip (white arrow) received IRDye 800CW YC-27 (0.5 nmole) 24 hours prior to imaging on the Pearl® Small Animal Imaging System. Orange arrows point to residual kidney clearance of optical imaging agent.

PSMA is a type II glycoprotein that is over-expressed in prostate cancer including metastatic disease. PSMA is also expressed on the tumor vascular endothelium of virtually all solid carcinomas and sarcomas but not on normal vascular endothelium. This expression suggests a potential mechanism for specific targeting of tumor-associated neovasculature. IRDye 800CW YC-27 (urea-based small molecule; MW 1743) has been characterized for in vitro and in vivo use with a number of tumor cell lines which include LNCaP, 22Rv1, PC3M-LN4 (prostate carcinomas), PC3-PIP (PC3 cells transfected with PSMA) and PC3-flu (PSMA-). These characteristics make it ideal for preclinical evaluation of PSMA-expressing tissue such as prostate tumors.

For information on BrightSite™ Small Animal Imaging Agents labeled with IRDye near-infrared fluorescent dyes, visit our LI-COR BIO website.

Would you like to label your own compounds with with NIR fluorescent dyes? Try one of our IRDye Protein Labeling Kits.

In-Cell Western™ Assay Webinar – Applications Review

In-Cell Western Assays - Fluorescent ImmunoassaysFor those of you that like to watch videos and listen to information, here is a great webinar on In-Cell Western Assays.

In this In-Cell Western webinar, the basics of ICW assays are covered and these applications:

For more information on these plate-based fluorescent immunoassays, go to the In-Cell Western assay application page. There are also several sample protocols and information on how to set up, optimize, and analyze ICW assays.

In-Cell Western™ Assay Application: Response of COS-7 Cells to Hydroxyurea

Application: Detecting phospho-p53 in COS cells in response to Hydroxyurea

Example of In-Cell Western Assay: Effects of Hydroxyurea on phospho-p53 on COS-7 cells

In this In-Cell Western assay application, the response of COS-7 cells to increasing doses of hydroxyurea was measured by a specific antibody (Anti-phospho-p53 from Cell Signaling Technology, P/N 9286) that detects phosphorylated-p53 (Ser16). Total ERK1 was used for normalization. The image represents a 96-well two-color In-Cell Western with the 700 and 800 nm channels detecting phosphorylated-p53 (Ser16) and total ERK1, respectively. Background wells were incubated with secondary antibody but no primary antibody. IRDye® 680RD secondary antibodies were used for detection in the 700nm channel and IRDye 800CW secondary antibodies were usd for detection in the 800nm channel.

Dose response graph of % induction of p53 phosphorylation with hydroxyurea in COS-7 cells

The graph represents the average of four sets of quantitative data, demonstrating the percent induction of phosphorylated-p53 (Ser16). Plate-based assays such as this can be imaged on the Odyssey® CLx or Odyssey Sa Infrared Imaging System.

For more uses of In-Cell Westerns Assays, visit our website.

Are You Studying Phosphorylation or Quantitative Cell Signaling Analysis? How About for IC50 Determinations?

In-Cell Western Assays - Fluorescent Immunoassays

In a previous post, I talked about how In-Cell Western™ assays could be used when studying apoptosis. So, you may be asking yourself, for what other applications can quantitative cell signaling analysis be used? GREAT QUESTION!!

Well, In-Cell ELISAs (as these immunofluorescent assays are also called) have been used successfully in studying protein phosphorylation. Whether you are looking at the effects of drug compounds on signaling pathways, or the timing/kinetics of signal transduction, or trying to determine the IC50 of compounds, In-Cell Western assays are a valuable tool.

Here are two examples of data from IC50 and EC50 determination experiments.
Use of labeling for In-Cell Western Assay normalization.
Figure 1. Use of cell labeling for In-Cell Western normalization. A) HeLa cells were treated with increasing amounts of rapamycin in a 384-well format. Fixed cells were stained with phospho-rpS6 antibody and NHS-ester reactive dye (for cell number). Dose dependent inhibition of phospho-rpS6-staining yielded an IC50 of 224 pM (n=4). B) Raw microplate image. For details, see Hoffman, GR et al. Assay Drug Dev Tech 8(2):186-99 (2010).

Dose titration of Wnt3a treatment of mouse L-cells.  An In-Cell Western Assay Application.
Figure 2. Dose titration of Wnt3a treatment of mouse L-cells. Half-maximal activation (EC50) of cellular beta-catenin levels occurs at 33 ng/ml ligand. Hannoush, RN. PLoS One. 3(10):e3498 (2008). Creative Commons license 2.5.

To help you get started in designing your experiment, here is a complete sample protocol for measuring IC50 of the inhibitor PD168393 in A431 cells responding to epidermal growth factor (EGF).

Check here for future blog posts on other applications of quantitative cell signaling analysis!

Seeding Cells in Microplates for In-Cell Western™ Assays – Hints & Tips

In-Cell Western Assays - Fluorescent Immunoassays
One of the first steps in an In-Cell Western Assay experiment is to seed cells into the wells of a tissue culture microplate. Cell density is more important for some cell lines than others. In particular, cells that depend more on extracellular activity for proliferation (such as epithelial cells) are affected to a greater extent by initial growth conditions. There are three factors to consider when seeding cells:

  1. Plates: For most adherent cells that stick to wells tightly (e.g. A431, HeLa, HEK293, CHO), we recommend regular tissue culture microplates with low auto-fluorescence, such as Nunc P/N 167008. For adherent cells that could detach from wells during In-Cell Western assay wash steps (e.g. NIH/3T3), we recommend Poly-D-lysine coated 96-well microplates.
  2. Cell seeding density: Typically, 15,000 to 40,000 cells are seeded per well. Two to three days are usually required for cells to reach the appropriate confluency, depending on growth rate. Seeding with low cell numbers is recommended if you plan to culture for several days before use. Plates seeded with higher cell numbers will be ready to use earlier.
  3. Confluence: To obtain maximal fluorescent signals, complete or near complete confluency is recommended for cells that stick to wells tightly. For cells that adhere loosely to wells, such as NIH3T3, 70% confluency should be used. Please note that cell type and experimental conditions may affect the acceptable level of growth confluency.

The example below illustrates the importance of cell seeding density for A431 cells. As shown in the corresponding graph, cell growth is greatly inhibited when there are too few neighboring cells.

cell seed plate

Graph showing why Seeding Plates for ICW Assays is Important

Use Quantitative Cell Signaling to Study Apoptosis

AGAIN with the quantitative cell signaling! YES! because it is so versatile!! I am sure you will find that this will become a valuable technique to use in your research.

This quantitative immunofluorescent assay – the one that we call an In-Cell Western™ (ICW) Assay – can be used to study a variety of mechanisms. Here is an example of an ICW used to study apoptosis.

As you may already know, there are two major apoptosis signaling pathways: the death receptor (extrinsic) pathway and the mitochondrial (intrinsic) pathway. Under most circumstances, activation of either pathway leads to proteolytic cleavage and activation of caspases, a family of cysteine proteases that act as common death effector molecules. The In-Cell Western Assay is a very helpful research tool for scientists who are quantifying cell signaling.

Time Course of Caspase-3 Activation in SP2 Cells Performed using an In-Cell Western Assay

Figure 1. Time course of caspase-3 activation in S2 cells. (A-C) In-Cell Western analysis of S2 cells treated with Actinomycin D (Act D) to induce apoptosis. Each time point was measured in triplicate and stained for anti-active-caspase-3 (A; green) and f-actin (B; red, stained with near-infrared fluorescent phalloidin). Panel C shows merged pseudocolor images. (D) Active-caspase-3 protein levels from (A) were quantified and normalized to f-actin levels in (B) for each time point. The active caspase-3:f-actin ratio at 0min Actinomycin D exposure was designated as 1, and all other ratios are shown relative to this value. Error bars represent the standard error of each independent measurement. Exposure of S2 cells to Actinomycin D increased the relative levels of active caspase-3 over time. Reprinted with permission from Bond, al. Biol Proced Online. 10(1):20-28(2008).

Here is our complete apoptosis assay example protocol of the HeLa cellular response to anisomycin treatment (detailing the seeding, induction, and detection).

Quantitative Cell Signaling Analysis – What’s all the Buzz About?

In-Cell Western Assays for Quantitative Cell Signaling Analysis

What’s all this BUZZZZ you are hearing about being able to quantitate cell signaling in plate-based assays? If you are at AACR in Chicago this week, stop by Booth 3800 (LI-COR® Biosciences) and we can tell you all about the In-Cell Western™ Assay – and how you can use this method to quantitate signaling, look at levels of protein phosphorylation, perform RNAi studies, monitor gene expression levels, conduct cell proliferation assays, and more. Imaging can be performed on the Odyssey® CLx, Odyssey Classic, or the Odyssey Sa Infrared Imager (the Sa also has the option for automation and barcode reading). And, if you can’t make it to AACR, stay tuned here and I will be blogging about this topic over the next week or so.

Okay, let’s start at the beginning. So what – exactly – is an In-Cell Western Assay? Well, some call it a cytoblot. To others, it’s a cell-based ELISA or an In-Cell ELISA (ICE Assay). To LI-COR, it’s a In-Cell Western Assay (we call it an ICW, for short) and is a quantitative immunofluorescence assay performed in microplates (96- or 384-well format). It combines the specificity of Western blotting with the reproducibility and throughput of ELISA.

In a nutshell, the basic steps are:

  • Culture cells in microplates
  • Treat cells
  • Fix and permeabilize
  • Stain with primary antibodies – 1 or 2 protein targets per well
  • Stain with IRDye secondary antibody conjugates
  • Image microplate and quantify fluorescent signals from cell populations in each well
  • Quantify relative protein levels
  • Normalize to correct for well-to-well variation

That doesn’t sound too difficult, right? Of course, just like any scientific technique, there are things to keep in mind to make sure your experiment gives the best, clearest, most accurate and reproducible results it can. In the next posts, I’ll share some of the technical tips to keep in mind – plus examples of how your research colleagues have used In-Cell ELISAs in their published papers.

In the meantime, here is the ICW Brochure, which includes a little more info on the technique and some examples with data. We also have a video introduction to In-Cell Western Assays – for those that like the movies!