Fig. 5: Analyses of signalling pathways of AdipoRon-induced cell death in MIAPaCa-2 cells. | Cell Death & Disease

Fig. 5: Analyses of signalling pathways of AdipoRon-induced cell death in MIAPaCa-2 cells.

From: Antidiabetic adiponectin receptor agonist AdipoRon suppresses tumour growth of pancreatic cancer by inducing RIPK1/ERK-dependent necroptosis

Fig. 5

a Western blot analyses of the activation of AMPK, p38 MAPK, Akt and ERK1/2 by AdipoRon. The cells were treated with 100 μM AdipoRon for the indicated time periods. b Effects of various inhibitors on AdipoRon-induced cell death. The cells were pre-treated with vehicle alone (V) or the drug for 1 h and then with 100 μM AdipoRon in the presence or absence of the drug (20 μM SB203580, 5 μM BML-275 or 1 μM Akt inhibitor) for 40 h. c Effect of BML-275 on AdipoRon-induced [Ca2+]mt accumulation. The cells pre-treated with 5 μM BML-275 for 1 h were loaded with Rhod2-AM and MitoTracker Green (MitoT). Vehicle alone or 100 μM AdipoRon was added at time 0. Bar: 50 μm. d Effect of SB203580 on AdipoRon-induced [Ca2+]mt accumulation. The cells pre-treated with 20 μM SB203580 for 1 h were loaded with Rhod2-AM and MitoTracker Green (MitoT). Vehicle alone or 100 μM AdipoRon was added at time 0. Bar: 50 μm. e Effect of BML-275 on AdipoRon-induced phosphorylation of p38 MAPK. f Effect of U0126 on AdipoRon-induced cell death. The cells were pre-treated with vehicle alone (V) or 10 μM U0126 for 1 h and then treated with 100 μM AdipoRon in the presence or absence of 10 μM U0126 for 40 h. g Effect of U0126 on AdipoRon-induced [Ca2+]i accumulation. The cells were loaded with Fluo4-AM. Vehicle alone or 100 μM AdipoRon or 20 μM U0126 was added at time 0. h Effect of U0126 on AdipoRon-induced [Ca2+]mt accumulation. Cells loaded with Rhod2-AM and MitoTracker Green (MitoT) were treated with 100 μM AdipoRon in the presence or absence of 20 μM U0126. Bar: 50 μm. i Effect of various inhibitors on AdipoRon-induced ERK1/2 phosphorylation. The cells were pre-treated with 50 μM Nec-1, 50 μM Nec-1s, 20 μM SB203580 and 5 μM BML-275 for 1 h, and then with 100 μM AdipoRon in the presence or absence of the inhibitors for 30 min. The ratio of p-ERK1/2 to ERK1/2 is shown below. j Effect of Nec-1 on AdipoRon-induced [Ca2+]mt accumulation. The cells loaded with Rhod2-AM were treated with 100 μM AdipoRon in the presence or absence of 100 μM Nec-1 for 6 h. BG: background. k Effect of Nec-1 on AdipoRon-induced mtROS generation. The cells loaded with MitoSOX Red were treated with 100 μM AdipoRon in the presence or absence of 50 μM Nec-1 for 8 h. BG: background. (l) Effect of Nec-1 on AdipoRon-induced [Ca2+]i accumulation. The cells loaded with Fluo4-AM were treated with 100 μM AdipoRon in the presence or absence of 50 μM Nec-1. For b and f, error bars represent standard deviation. *P < 0.001 by Student’s t-test or ANOVA test. ns not significant. Full size images of the western blots presented are shown in Figure S13

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