Fig. 1

Total PTEN levels and phosphorylation levels of the PTEN C-terminus in lung cancer cells. The levels of total PTEN and p-PTEN expression were evaluated in five lung cancer cell lines. a Cell extracts were harvested for the analysis of p-PTEN, PTEN, and β-actin by western blotting. Equal amounts (20 μg) of protein were loaded for analysis. The ratio of total PTEN protein to β-actin, b and p-PTEN to total PTEN protein, c is presented as an intensity level relative to that in H441 cells. A representative blot is shown from three independent experiments. Asterisk indicates p < 0.05. (b, c), d Immunostaining for PTEN (green) was performed in H1299 (left) and H358 (right) cells. Nuclear staining was performed by Hoechst33342 (blue). Results are representative of three independent experiments. All images shown are × 200 magnification. e NIS-Elements AR software was used to compare the intensity level of PTEN expression between H1299 (left panels in d) and H358 (right panels in d) cells, compensated by cell numbers, which were counted by Hoechst33342 staining. Data represent the mean ± SE of two independent experiments. Asterisk indicates p < 0.05. To evaluate whether persistent hypoxia might modulate PTEN expression in lung cancer cells in vivo, we examined the tumor sections from Dox-treated nude mice at day 28 after inoculation with H358ON cells carrying GFP. f H–E staining was performed on tumor tissues. g Immunostaining for pimonidazole as a hypoxia marker was performed on a serial section of tumor tissues. An arrow indicates the cells positive for pimonidazole. h Control immunostaining for PTEN on a serial section of tumor samples is shown. i Immunostaining for PTEN was also performed on the serial section of tumor samples. An arrow indicates the cells positive for PTEN. Representative tumor sections are shown from three independent experiments. f–i All images shown are ×200 magnification