Salubrinal increases basal phospho-eIF2α levels and significantly limits ErbB2-induced acinar growth. (A) Phase-contrast micrographs of control and ErbB2-expressing acini untreated (top panels) or treated with salubrinal (10 μg/ml) from Day 4-day 10 in Matrigel (bottom panels). Note the reduced acinar size of salubrinal treated ErbB2-expressing acini. Scale bars = 60 μm. (B) Western blots showing the increase in phospho-eIF2α levels in empty vector, Wt-ErbB2 or CA-ErbB2 cells untreated or treated with salubrinal under adhered monolayer conditions for the indicated time points. Total eIF2α was used as loading control. (C) Acinar volume was quantified on day 8 in Matrigel after daily treatment with salubrinal (day 4 onwards) for vector, Wt-ErbB2 or CA-ErbB2 acini. Acinar volume was estimated by measure two perpendicular diameters per acinus, assuming an ellipsoid structure. Between 50-100 acini were quantified and results are representative of experiments repeated at least three times. Statistical significance was determined using the unpaired t-test with p < 0.05 defined as statistically significant. (D) Graphs showing the distribution of acinar sizes over several volume ranges to depict the increase in acinar size upon ErbB2 activation, mean ± SD. Note that salubrinal treatment causes a shift in the percentage of acini falling within the smaller size ranges.