LDK down-regulates phosphorylation of targets in the PI3K/AKT/mTOR pathway and causes late mitosis arrest in treated cells. (A-C) Jurkat cells were treated with 10μM LDK for the indicated durations then probed for phospho as well as total proteins by Western blot. Densitometric ratio of phospho to total protein is indicated below the paired panels. (A) LDK treatment reduces phosphorylation of AKT at Thr308 and Ser473. (B) LDK treatment reduces phosphorylation of mTOR. (C) LDK treatment reduces serum-induced phosphorylation of mTOR downstream target, p70S6 kinase, at 6 hours of treatment. Note that LDK did not prevent serum-induced phosphorylation of p70S6K at 3 hours. (cropped, noncontiguous sections of same gels are shown for panels A-C). (D-G) Western blot assessment of LDK-treated Jurkat cells indicates that the G₂/M block occurs after anaphase. (D) Schematic of cyclin A and B1 as well as phospho-histone H3 (pH3) temporal expression patterns observed during the mammalian cell cycle. (E-G) Western blot assessment of LDK (10μM) treated cells indicates that neither cyclin A, cyclin B1, nor pH3 accumulate in treated cells. Treatment durations were 24 hours for E and 16 hours for panels F and G. Dox indicates100nM doxorubicin; Noc, 1μM nocodazolel; and dox and noc, positive control. (H-I) Western blot assessment of Jurkat cells treated with the inhibitors doxorubicin and nocodazole indicates that these inhibitors do not cause dephosphorylation of AKT. (E-I) Vinc indicates vinculin loading control, noncontiguous sections of same gel. Western blots were scanned at room temperature using Epson Expression 1680 scanner and software, 16-bit grayscale acquisition, 300 dpi resolution. Image processing was done using Adobe Photoshop 9.0.2.