FIGURE 4. B2G2-induced death in LNCaP cells is through inhibiting MAP kinase phosphatase expression and activity.

A, LNCaP cells were treated with B2G2 (50 μM), whole cell lysates were prepared at 1, 3, 6 and 24 h time points and analyzed for MKP1, MKP2 and MKP3 expression by immunoblotting. Membranes were stripped and re-probed for β-actin. B, LNCaP cells were treated with B2G2 (50 μM) with or without NAC (10 mM), protein lysates were prepared in phosphatase assay buffer at 6 h post-treatment and MAP kinase phosphatase activity was assessed using p-ERK2 as substrate as detailed in Materials and Methods’ section. LNCaP cells treated with H₂O₂ (1.0 mM) served as negative control in this experiment, whereas, lysate prepared from MKP3-overexpressing LNCaP cells were used as positive control for this assay. Last lane was recombinant p-ERK2 protein. A short and long exposure of the blot is presented here. C, MKP3 expression in vector control and MKP3 overexpressing LNCaP cells was compared by immunoblotting. Membrane was stripped and re-probed for β-actin. D, LNCaP-VC and LNCaP-MKP3 cells were treated with B2G2 (50 μM), whole cell lysates were prepared at 24 h time point and analyzed for MKP3 (long exposure-left panel; short exposure-upper right panel) and p-ERK1/2 (left panel) by immunoblotting. Membranes were stripped and re-probed for total ERK1/2 and β-actin (left panel). E, LNCaP-VC and LNCaP-MKP3 cells were treated with B2G2 (50 μM), total cell number and percentage cell death were determined by trypan blue exclusion assay at 24 h post-treatment. F, LNCaP-VC and LNCaP-MKP3 cells were treated with B2G2 (50 μM), whole cell lysates were prepared at 24 h time point and analyzed for cl PARP. Membrane was stripped and re-probed for β-actin. Data are expressed as of mean ± SEM (n=3) for each treatment. * P < 0.05, significant with respect to control group; # P < 0.05, significant with respect to B2G2-treated LNCaP-VC versus B2G2-treated LNCaP-MKP3 group.