Figure 1.

Pathways regulated by JG-98. (A) Heat maps demonstrating responses of several signaling pathways to JG-98 in MCF7 and MDA-MB231 cells. Experiment was done using IPAD technology by ActivSignal, Inc. Of note, three gradations of color intensities are presented on the heat map, corresponding to 1.2, 1.8 and 2.4 and higher fold increase or decrease in IPAD values over control. Translation of the IPAD values to actual change in the activity of signaling molecules depends on the target. On average, 1.8 fold change in IPAD values corresponds to 3-fold change in the target activity. (B) Preincubation with 10 μM VER-155008 suppresses signaling responses to JG-98. Experiment was done using IPAD technology by ActivSignal, Inc. (C) Left panel - JG-98 treatment and Bag3 depletion activates ERK1/2. MCF7 cells were treated for 36 h with indicated concentrations of JG-98, and levels of ph-ERK1/2 and total ERK1/2 were determined in cell lysates by immunoblotting with the corresponding antibody. To deplete Bag3 MCF7 cells were infected with Bag3 shRNA retroivirus, as described in our prior publications²³. Right panel - shRNA mediated Bag3 depletion. Bag3 levels were determined by immunoblotting cell lysates with Bag3 antibody. This and all other immunoblots were done three times. A typical immunoblot is shown. (D) Quantification of effects of JG-98 on association between ERK1/2 and Bag3. Association was studied using the ActivSignal IPAD protein-protein interaction method. Histogram represents an average of three independent experiments. SD values are shown, p < 0.01. (E) JG-98 reduces the rate of dephosphorylation of ph-ERK1/2. MCF7 cells were treated with PDB for 7 min to activate ERK1/2 and then further phosphorylation was blocked by incubation of cells with cocktail of inhibitors of respiration (rotenone, 5uM) and glycolysis (2DG 10mM). Cells were collected at indicated time points, and the levels of ph-ERK1/2 and total ERK1/2 were determined as in Fig. 1A. (F) JG-98 downregulates AKT in Bag3-independent manner. MCF7 cells were infected with Bag3 shRNA virus or “empty” shRNA vector (see Materials and Methods) prior to treatment with 2 μM of JG-98 for indicated time periods. Levels of Akt and p-Akt were determined in cell lysates by immunoblotting with corresponding antibodies. (G) JG-98 downregulates c-myc in Bag3-independent manner. MCF7 cells were infected with Bag3 shRNA virus or “empty” shRNA vector as in Fig. 1G and treated with 2 μM of JG-98 for 36h. Levels of c-myc were determined in cell lysates by immunoblotting with corresponding antibody. (H) Preincubation with VER-155008 suppresses effects of JG-98 on ERK, Akt and myc.