Figure 4 | Cell Death & Disease

Figure 4

From: Eleostearic acid induces RIP1-mediated atypical apoptosis in a kinase-independent manner via ERK phosphorylation, ROS generation and mitochondrial dysfunction

Figure 4

RIP3 is not involved in ESA-mediated apoptosis. (a) RIP1 was ubiquitinated by TNF-α (100 ng/ml) along with Z-VAD-fmk (20 μM) in PC12 cells. TNF-α alone did not undergo ubiquitination. (b) RIP1 uniquitination was not observed after ESA (2 μg/ml) stimulation. (c) RIP1 was not ubiquitinated by ESA in the presence of Nec1 (50 μM) and Z-VAD-fmk (20 μM). (d) RIP3 was expressed in 3T3 fibroblasts, and associated with RIP1 when the cells were treated with both TNF-α (25 ng/ml) along with z-VAD-fmk (50 μM). Association of RIP3 with RIP1 occurred a few hours after stimulation. At the same time, both RIP proteins were degraded. Nec1 (36 μM) inhibited association of RIP1 with RIP3. TNF-α alone did not induce the interaction. (e) Involvement of autophagy was examined. LC3-II derived from autophagosomes was not observed in cells treated with ESA (2 μg/ml) alone or with z-VAD-fmk (20 μM). Cells cultured in Hank’s buffer for 1 h produced the autophagic LC3-II band, which was used as a positive control.

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