Figure 5.

Subcellular Distribution and Catalytic Activity of PTEN-L and lclPTENL

(A) Immunofluorescence was performed on U87MG cells expressing either PTEN-L or lclPTEN-L that were treated without or with doxycycline for 48 hr. PTEN immunofluorescence is shown pseudocolored in orange. Nuclei are stained with DAPI. Immunofluorescence for the top and bottom panels was performed at different times and is for comparison of subcellular distribution rather than expression levels. (B) Total cell lysates from U87MG cells expressing PTEN, PTEN-L, or lclPTEN-L, without or with 48 hr doxycycline treatment, were probed with antibodies to phosphorylated and total Akt. A representative blot from three independent experiments is shown. Quantitation of data from the three experiments is shown in Figure S1. (C) U87MG cells expressing doxycycline-inducible PTEN, PTEN-L, or lclPTEN-L were treated with or without doxycycline for 48 hr. For the last 2 hr of the 48-hr induction, EdU was added to the media. Cells were then fixed and proliferation was assessed by determining the percentage of EdU⁺ cells in 10 randomly chosen fields of view (between 500 and 1,000 cells total). Error bars show the mean ± SE. *p < 0.05 by the Mann-Whitney rank-sum test.