Figure 3
JNK activity is involved in cell death induced by glucose deprivation. (a) Equal amount of total cell extracts from HIPK2+/+ and siHIPK2 cells cultured in glucose-free medium (glu stv) for the indicated times was analyzed by western immunoblotting with the indicated antibodies. Anti-β-actin was used as protein loading control. (b) RKO-HIPK2+/+ cells were stably transfected with JNK1-APF-HA mutant (dominant negative, DN-JNK1) or with control vector (−). DN-JNK was detected by western immunoblotting with anti-HA antibody; total JNK is shown. (c) HIPK2+/+ control, DN-JNK1-overexpressed cells, and siHIPK2 cells were cultured in glucose-free medium for 24 h before assaying for western immunoblotting with anti-(p)-c-Jun and tot-c-Jun. Anti-tubulin was used as protein loading control (d) HIPK2+/+ control and DN-JNK1-overexpressed cells were cultured as in (c) and cell death was analyzed by trypan blue staining. The result is the mean of three independent experiments performed in triplicate ±S.D. *P=0.001 (e) HIPK2+/+ control and DN-JNK1-overexpressed cells were cultured as in (c) before live cell images were taken. A representative result out of three is shown. (f) ChIP analysis performed with anti-HIPK2 antibody on H1299 cells untreated or treated with glucose-free medium (8 h) PCR analyses were performed on the immunoprecipitated DNA samples using specific primers for HIF-1α promoter. A sample representing linear amplification of the total input chromatin (Input) was included as control. Additional controls included immunoprecipitation performed with nonspecific immunoglobulins (IgG)
