Article Category: Cancer 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.

Reference:

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.


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

Optical Probes Icon

IRDye 800CW YC-27 (P/N 926-27000) 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® Impulse 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 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.

Normalize Protein Concentrations with Nucleic Acid Stains in In-Cell Western™ Assays

In-Cell Western Assays - Fluorescent ImmunoassaysWhen performing the In-Cell Western assay, one fluorescence channel is often used for normalization. This allows quantification data for the protein of interest to be corrected for well-to-well variation in cell number, thereby increasing the overall accuracy of the assay. In many cases, a second protein is chosen for normalization.

LI-COR has a protocol for Nucleic Acid Stains Used as Normalizing Agents in ICW Procedures that describes the use of a DNA stain in the 700 nm channel as an alternative for normalization. Choosing to normalize with a DNA stain eliminates the need to identify and optimize an additional primary antibody for the assay, and avoids any issues that may arise if levels of the normalization protein are affected by the cell treatments used.
HeLa Cell Dilution Series to Measure Linearity of Various DNA Stains

The linearity of the DNA stains was evaluated with respect to cell number. HeLa cells were serial diluted in a 96-well plate at a starting concentration of 1.5 x 105 cells per well. Odyssey Blocking Buffer (PBS) was used to block for approximately 2 h before staining. DNA stains were diluted with fresh Odyssey Blocking Buffer at the recommended levels listed above. Blocking buffer was removed from the plate and blocking buffer plus DNA stain was added (50 μl per well). The plate was allowed to incubate at room temperature on a rotary shaker for 1 hour followed by 4X washes in 1X PBS + 0.1% Tween®-20. Care should be taken to protect the plate from light to insure the highest sensitivity of the stain. The linearity for SYTO® 60 and TO-PRO®-3 were very good with R2-values of 0.99 and 0.98, respectively (figure above).

For more information on In-Cell Western Assays, visit our website.

Request a LI-COR Catalog and Applications Handbook . It includes all of the LI-COR BIO products (Odyssey® family of imaging systems and Pearl® Impulse Small Animal Imaging System, reagents and accessories) plus technical notes, application workflows, and troubleshooting for key applications.

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.

How Can You Use Quantitative Cell Signaling Analysis in Your Research? 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 Seeding Microplate example

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, D.et 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 Assayy? 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!