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. 2014 Jan 30;5(1):e1043. doi: 10.1038/cddis.2014.5

Figure 4.

Figure 4

uPA knockdown affects ERK1/2 survival signaling. (a) Phosphorylation levels of ERK1/2, Akt and regulators of NF-κB signaling (IκBα) in transformed cells surviving TRAIL treatment. BJELR cells were either left untreated (‘control') or treated with TRAIL (1 μg/ml) during 7 or 16 h. Apoptotic cells (‘apop') were collected by successive washes and non-apoptotic attached cells (‘surv') were harvested for western blot analysis. Total and phosphorylated levels of ERK1/2 (Thr202/Tyr204), Akt (Ser 473) and IκBα (Ser32/36) were analyzed. Caspase-8 cleavage (‘Casp-8') was evaluated in each cell population. α-Tubulin, loading control. (b) Phosphorylation status of ERK1/2 and Akt upon uPA knockdown. BJELR cells were left non-transfected (‘mock') or transfected either with pooled siRNAs targeting PLAU mRNA (‘siPLAU') or non-targeting scramble siRNAs (‘scr'). Forty-eight hours after transfection, total and phosphorylated protein levels of ERK1/2 (Thr202/Tyr204) and Akt (Ser 473) were analyzed by western blot analysis. α–Tubulin, loading control. (c) Distribution of total (ERK1/2 Alexa488) and phosphorylated ERK1/2 (pERK1/2-Alexa 488; Thr202/Tyr204) in populations of BJELR cells untreated (‘mock'), scramble transfected (‘scr') and uPA depleted (‘siPLAU') was analyzed by flow cytometry. Images represent the distribution of fluorescence within each sample for a representative experiment out of three independent replicates. Isotypic IgG1 (‘IgG1') was used as control for background florescence. (d) Requirement of ERK1/2 signaling for cell survival upon TRAIL challenge. BJELR cells were either treated for 1 h with 20 μM U0126 MEK1/2 inhibitor or vehicle (DMSO). Apoptosis in untreated samples (‘U0126', ‘DMSO') and upon 3 h of treatment with 1 μg/ml of TRAIL (‘U0126+TRAIL', ‘DMSO+TRAIL') was analyzed by flow cytometry as the percentage of cells with positive labeling for cleaved PARP. Images correspond to one representative experiment out of four independent replicates. Percentage of cleaved PARP-positive cells is indicated in the upper right quadrant