Figure 3. Cytoplasmic enrichment of FoxO1 in response to serum and stretch in hGF. (A) hGF were cultured in serum-free medium on glass immunohistochemistry chambers for 24 hrs. Cells were then fixed as described in MATERIALS & METHODS and stained with anti-FoxO1 antibody; mounting medium contained DAPI for identification of nuclei. a: DAPI nuclear staining. b: FoxO1 staining. c: superimposed image illustrating that the most intense FoxO1 staining corresponds to the nuclear area. (B) hGF were cultured in 10% FBS-containing medium on glass immunohistochemistry chambers for 24 hrs. Cells were then fixed and stained as in Fig. 3A. The superimposed image (c) indicates that FoxO1 is evenly distributed in the cytoplasm. (C) hGF were cultured in serum-free medium for 24 hrs and kept under static conditions for an additional 24 hrs. The bottom areas of the silicone stretch chambers were then cut and placed on glass slides prior to being mounted. Immunohistochemistry was then performed in a manner identical to that described in Figs. 3A, 3B. The right panel illustrates that most of the immunofluorescent signal is localized to the nucleus. (D) hGF were cultured in serum-free medium for 24 hrs prior to being stretched for an additional 24 hrs. The chambers were treated as in Fig. 4A. Immunohistochemistry was then performed in a manner identical to that described for Figs. 3A, 3B. The right panel illustrates that the immunofluorescent signal is distributed evenly throughout the cytoplasm. (E) Negative control for Figs. 3 and 4. Cells were cultured in serum-containing medium for 24 hrs, fixed, and processed as indicated in MATERIALS & METHODS in the absence of a primary antibody. The image represents superimposed images of DAPI and FoxO1 taken with the same settings as in Figs. 3 and 4. This Fig. illustrates very faint cytoplasmic straining and demonstrates the specificity of the FoxO1 antibody used in the immunofluorescent studies. Magnification: 80X.