ErbB2 overexpression does not modulate phospho-eIF2α signaling under basal growth conditions. (A, B) Western blots of whole-cell lysates from monolayer cultures of MCF10A cells expressing empty vector, Wt-ErbB2 or CA-ErbB2 constructs for phospho-and total PERK (A, top panels) or phospho- and total eIF2α (A, bottom panels) or ATF4. Total eIF2α or GAPDH was used as loading control respectively. Bottom panels depict relative intensities quantified by scanning densitometry and normalized to vector control. The averages of three or more independent experiments are shown; ± SEM. (C) Equatorial confocal sections of 10-day vector control (a) or wild type (Wt) ErbB2/Neu (b) or constitutively activated (CA) ErbB2/Neu (c) structures labeled with an anti-CHOP antibody (green) or DAPI nuclear stain (blue). Arrows point to CHOP-positive cells in the acinar structure. Scale bars = 25 μm. (D) Graph showing the percentage of cells per acinar structure that stained positive for CHOP. Values are representative of four independent experiments performed on different days (8-10) of growth in Matrigel; mean ± SEM. Statistical significance was determined using the unpaired t-test with p < 0.05 defined as statistically significant. N.S - not significant.