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. Author manuscript; available in PMC: 2014 Jun 23.
Published in final edited form as: Cancer Res. 2006 Aug 15;66(16):8007–8016. doi: 10.1158/0008-5472.CAN-05-4451

Figure 2.

Figure 2

Phosphorylated Akt blocks Smad3 translocating to the nucleus in human prostatic epithelial cells. A, cells were incubated with 5 ng/mL TGF-ß1 for 4 hours and processed to give cytoplasmic (C) and nuclear (N) extracts. Specificity was confirmed using histone H1 and ß-actin as nuclear and cytoplasmic markers and loading controls. Analysis of Smad3 localization showed that in nontumorigenic BPH1 cells Smad3 was predominantly localized to the nucleus after TGF-ß1 treatment. In contrast, in BPH1CAFTD1 cells, Smad3 was seen at similar levels in both nuclear and cytoplasmic fractions. Overexpression of myrAkt1 in BPH1 cells (BPH1-Akt) inhibited Smad3 translocation to the nucleus, whereas treatment using the PI3K inhibitors 10 μmol/L Ly294002 (ly) or 100 nmol/L wortmannin (WM) enabled Smad3 to translocate into the nucleus of BPH1CAFTD1. Total and phosphorylated Smad2 localization did not change markedly in response to Akt activation. B, immunofluorescence staining illustrates Smad3 localization in cells with or without TGF-ß1 treatment. Smad3 is visualized in green, whereas nuclei are stained using PI and are shown in red. Without treatment, Smad3 was localized in the cytoplasm in both BPH1 and BPH1CAFTD1 cells. After TGF-ß treatment, almost all of the Smad3 was translocated into the nucleus in BPH1 cells; however, in BPH1CAFTD1 cells, both nuclear and cytoplasmic localization were seen. Pretreatment with wortmannin enhanced nuclear localization of Smad3 in BPH1CAFTD1 cells. C, Smad3 translocation was quantitated using NIH ImageJ software. The average photons locating in the nucleus or in the whole-cell body were measured on a cell-by-cell basis in five random fields. The proportion of the nuclear staining was plotted. BPH1 cells showed much more nuclear Smad3 on TGF-ß1 treatment (25% for pretreatment versus 71% for post-treatment; P < 0.01), but BPH1CAFTD1 did not show significant Smad3 relocalization in response to TGF-ß1 (the nuclear staining proportion was 21% and 24%, respectively; P > 0.05). Wortmannin pretreatment increased the Smad3 nuclear distribution proportion on TGF-ß1 to 61% (P < 0.01).