These studies confirm previous reports of the association of EGFR and Stat3 in similar intracellular spaces [6,10]. Importantly, the data show for the first time the presence of a heteromeric EGFR, Src and Stat3 complex in the nucleus, as observed by the co-IP and immunoblotting analyses, and confirmed by DANS/DLS analysis. These data contrast the results observed for the normal human pancreatic duct epithelial cells (HPDEC) (Fig. 5A). Immunofluo- rescence staining with laser-scanning confocal microscopy analysis of HPDEC shows a homogeneous distribution of EGFR (red), Src (blue) and Stat3 (green), all of which are strongly outside of the nucleus, with little evidence of co-localization (Fig. 5A). Visualization of the intracellular distribution patterns of EGFR, Src and Stat3 upon inhibition of EGFR or Src tyrosine kinase by immunofluorescence with laser-scanning confocal microscopy indicated that as with the co-IP studies (Fig. 3B), the inhibition of Src or EGFR tyrosine kinase activity alone did not completely eliminate the complex formation. However, Src or EGFR inhibition disrupted the localization patterns. Data shows EGFR, Src and Stat3 distribution in the cytoplasm and the nucleus following 1- or 24-h treatment with EGFR or Src inhibitor (Fig. 5B(ii) and (iii), 1 h, and 24 h, single). Nuclear EGFR levels are diminished, but not abolished (Fig. 5B(ii), single). Thus, EGFR, Src and Stat3 remain localized in the nucleus following tyrosine kinase inhibition. Results further showed persistent EGFR and Src (magenta), and Src and Stat3 (cyan) associations in both the nucleus and cytoplasm following treatment for 1 h with EGFR or Src inhibitor (Fig. 5B(ii) and (iii), 1 h), similar to the data in Figure 3B (1 h). Furthermore, a nuclear EGFR and Stat3 association (yellow) is detected upon 24-h treatment with the EGFR inhibitor (Fig. 5B(ii), 100 nM ZD, 24 h), while nuclear associations of EGFR and Src (magenta), and of Src and Stat3 (cyan) are detected following treatment with Src inhibitor for 24 h (Fig. 5B(iii), 100 nM Das, 24 h). The moderate differences in the observed patterns of complex formation between the co-IP (Fig. 3B) and confocal data may be due to the sensitivity differences between the two approaches. Overall, the findings are consistent with the co-IP data (Fig. 3B) in showing that the inhibition of the kinase activity of EGFR or Src alone is insufficient to completely disrupt all the proteins from the complex. EGFR, Src and Stat3 heteromeric complex regulates the c-Myc gene expression In our previous work, we showed that pancreatic cancer cells were insensitive to the inhibition of EGFR, Src or Stat3 activity alone, in parallel with the observation that the expression of c-Myc was also refractory to the inhibition of EGFR, Src or Stat3 alone, while the concurrent inhibition of aberrant Stat3 activity together with EGFR or Src inhibition strongly suppressed c-Myc expression and induced stronger antitumor cell effects [16]. Those findings suggest a complex regulation of c-Myc induction, which might support the cancer phenotype. Previous report identified only that the nuclear EGFR and Stat3 complex in breast cancer cells induced specific genes, including inducible nitric oxide synthase (iNOS) [10]. Studies were performed to assess the functional significance of the heteromeric complex, particularly in the context of the induction of the c-Myc gene in pancreatic cancer cells. Chromatin immunoprecipitation (ChIP) analysis was pursued to assess the association of the c-Myc promoter with the heteromeric complex. PCR amplification of DNA fragment using a primer against the c-Myc gene promoter and agarose gel electrophoresis showed that each of the anti- EGFR, Src, or Stat3 antibody-chromatin DNA immunoprecipitate contained the c-Myc gene (Fig. 6A(i), EGFR, Src and Stat3). To further confirm this finding, we pursued a modified sequential immunopre- cipitation analysis similar to the one performed in Figure 2B in the A Functional Nuclear EGFR, Src and Stat3 Complex Figure 6. Chromatin immunoprecipitation assay and Western blotting analysis of c-Myc, iNOS, Cyclin D1, and VEGF expres- sion in Panc-1 and Colo-357 cells. (A), Agarose gel electrophoresis of the Polymerase Chain Reaction (PCR)-amplified c-Myc gene fragment from the chromatin DNA precipitated with antibody against EGFR, Src, or Stat3, or with the non-specific IgG; and (B and C), Immunoblotting analysis of whole-cell lysates probing for EGFR or Src (B(i) and C(i)) or c- Myc, iNOS, Cyclin D1 or VEGF (B(ii) and C(ii)), and the effects of siRNA knockdown of EGFR (EGFR siRNA), Src (Src siRNA) or control (con) siRNA, or S3I-201 or Das). Bands corresponding to proteins or c-Myc gene in gel are shown; M, molecular weight marker, EGFR/Src, sequential immuno- precipitation with anti-EGFR and then anti-Src antibody. Data are representative of 3 independent studies, and values are mean and s.d of 3 independent studies; *p-,0.01. doi:10.1371/journal.pone.0019605.g006 context of a ChIP assay, as previously reported [10]. In the sequential ChIP assay in which EGFR chromatin immunecomplex was subjected to a second immunoprecipitation using anti-Src antibody and analyzed by PCR amplification and agarose gel electophoresis, we similarly detected the presence of the c-Myc gene PLoS ONE | www.plosone.org 10 May 2011 | Volume 6 | Issue 5 | e19605