Technical noteHousekeeping genes; expression levels may change with density of cultured cells
Introduction
Western immunoblotting is a powerful technique to detect and characterize a multitude of proteins. Since the first trials in 1979 (Towbin et al., 1979), Western blotting methods have been used extensively to examine protein levels in different cells or tissues (Kurien and Scofield, 2003). Proteins are usually resolved by sodium-dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) at first, then transferred electrophoretically to a membrane. The subsequent detection of the membrane-bound proteins using specific antibodies has evolved into a powerful tool for cell biology. The experimental protocol invariably involves the comparison of levels of a given protein or its modifications under study between different cell preparations. For this reason, it is necessary to ensure that all lanes of the gel were loaded with equal amounts of total protein and this can be achieved by determining protein concentrations in the cell lysates. However, as an internal control, and to take protein degradation into account, it is also necessary to probe for a protein which is not expected to change with the different conditions (“housekeeping” gene product). In fact, antibodies to a number of such proteins have been commercially developed. Most are also very abundant proteins in the cell, such as β-actin, heat-shock protein-90 (Hsp90), Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and α-tubulin, to name a few.
Cells in normal tissues or in tumors have extensive opportunities for adhesion to their neighbors in a three-dimensional organization. This is reproduced in vitro by culturing cells in dishes to high densities; such dense cultures, albeit only two dimensional, may in part mimic some of the physiological signals that occur in vivo. In fact, cell to cell adhesion in cultured, normal epithelial cells and fibroblasts triggers an increase in activity of the Rac1 and Cdc42, Rho family GTPases (Etienne-Manneville and Hall, 2002), which plateaus at a confluence of ∼ 90%. Rac1/Cdc42, in turn, were found to cause a dramatic increase in the phosphorylation of the Signal transducer and activator of transcription-3 at tyr705 (Stat3-ptyr705) (Arulanandam et al., 2009). The peak is usually at 1–2 days after confluence, depending upon the cells' growth rate (Vultur et al., 2004, Raptis et al., 2009). In such experiments, it is important to employ a gene product whose levels are not affected by cell density, as a control for protein loading.
In our search for a suitable marker, we examined the effect of cell density upon the levels of a number of commonly used housekeeping gene products. The results showed that the levels of GAPDH and α-tubulin increased gradually with cell density starting at 10% confluence and plateauing at ∼ 90%. Hsp90 and β-actin levels on the other hand did not change with cell density, from 10% confluence up to 5 days after confluence, indicating that these proteins may be appropriate controls. This is the first report on the effect of density of cultured cells upon the levels of certain commonly used, housekeeping gene products.
Section snippets
Cell growth, protein extraction, and Western blotting
Mouse NIH3T3 fibroblasts (subclone DL+10) were previously described (Vultur et al., 2004). They were grown to different densities in Dulbecco's modification of Eagle's medium supplemented with 5% calf serum. Cells were scraped with a rubber policeman into 1.8 mL microcentrifuge tubes, washed with cold phosphate-buffered saline (PBS) and the cell pellet resuspended in the different extraction buffers. Three buffers were used: NP-40 [50 mM Hepes pH 7.4, 150 mM NaCl, 10 mM EDTA, 10 mM Na4P2O7, 1%
Results and discussion
Mouse NIH3T3 fibroblasts were grown in tissue culture dishes at different densities, ranging from 10% confluence to 5 days after 100% confluence (Fig. 1A). To eliminate any effects of extraction efficiency differences, we used three commonly employed lysis buffers. In initial experiments, cells were lysed directly on the plate and protein determination conducted on extracts clarified by centrifugation. However, significant amounts of serum proteins (mainly bovine serum albumin) present in the
Conclusions
In this communication we examined the levels of four gene products which are commonly used as loading controls in Western blotting experiments. The results revealed that levels of α-tubulin and GAPDH increased significantly with cell confluence from 10% to 100%, which shows that these proteins may be unsuitable as loading controls for Western blotting experiments requiring growth of cells to subconfluence. On the other hand, our results demonstrate that Hsp90 and β-actin levels were essentially
Acknowledgements
The financial assistance of the Canadian Institutes of Health Research (CIHR), the Canadian Breast Cancer Foundation (Ontario Chapter), the Natural Sciences and Engineering Research Council of Canada (NSERC), the Ontario Centres of Excellence, the Breast Cancer Action Kingston, the Clare Nelson bequest fund and the Canadian Breast Cancer Research Alliance through grants to LR is gratefully acknowledged. RA was supported by a Canada Graduate Scholarships Doctoral award from CIHR, the Ontario
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The first two authors contributed equally to this work.