Biomarkers of Response to PI3K Inhibitors Figure 4. Loss of OGT expression increases sensitivity of the MDA-MB-231 and OVCAR-4 tumor cell lines to GDC-0941 and alters the phosphorylation state of Chk2. A, MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. B, Immunoblot analysis of whole cell lysates from MDA-MB-231 cells using anti-OGT or anti-O-GlcNAc antibodies. Cells were treated with 1 mM GDC-0941 or DMSO control for 24 hours, as indicated. C, Immunoblot analysis of whole cell lysates from MDA-MB-231 cells with indicated antibodies. Cell treatments are as indicated. D, Quantitative phosphorylation analysis of Chk2 (T68) in MDA-MB-231 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 1 mM GDC-0941 for 24 hours. Cells were treated with 1 mM GDC-0941 or DMSO control for 24 hours, as indicated. E, OVCAR-4 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 mM GDC-0941. Cell viability was assayed at 0, 24, 48, and 72 hours post-transfection and treatment with GDC-0941. F, Immunoblot analysis of whole cell lysates from OVCAR-4 cells using anti-OGT and anti-OGlcNAc antibodies. Cells were treated with 4 mM GDC-0941 or DMSO control for 24 hours, as indicated. G, Immunoblot analysis of whole cell lysates from OVCAR-4 cells with indicated antibodies. Cell treatments are as indicated. H, Quantitative phosphorylation analysis of Chk2 (T68) in OVCAR-4 cells transfected with OGT-targeting siRNA or non-silencing siRNA, in the presence or absence of 4 mM GDC-0941 for 24 hours. Cells were treated with 4 mM GDC-0941 or DMSO control for 24 hours, as indicated. All above data represent mean 6 SEM (n = 3); *, P,0.05;**, P,0.01;***, P,0.001. doi:10.1371/journal.pone.0046518.g004 PLOS ONE | www.plosone.org 8 September 2012 | Volume 7 | Issue 9 | e46518