Figure_3.tif (3.48 MB)

Cell death induced by autophagy and mTOR inhibition is RIPK-dependent necroptosis.

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posted on 2012-07-26, 00:48 authored by Kevin Bray, Robin Mathew, Alexandria Lau, Jurre J. Kamphorst, Jing Fan, Jim Chen, Hsin-Yi Chen, Anahita Ghavami, Mark Stein, Robert S. DiPaola, Donna Zhang, Joshua D. Rabinowitz, Eileen White

(A) Clonogenic survival assay showing Nec1 pretreatment rescues clonogenic survival. RCC cells were pretreated for one hour with Necrostatin1 (Nec1) followed by the addition of CQ, CCI-779 or combination of CQ and CCI-779 to the media. After 18 hours media was changed to normal culture media and cells were allowed to recover for 5 days. In 4 of 7 RCC cell lines (RCC4, A498, ACHN, 786O), Nec1 pretreatment rescued clonogenic survival from CQ and CCI. (B) Viability graph showing that siRNA knockdown of RIPK3 in RCC4 prevented cell death induced by an 18 hour drug treatment of combination of CQ and CCI-779 as compared to siRNA control siLamin (*p = 0.007). Error bars represent ± S.D. (C) Western blots of RNA knockdown of RIPK3. RCC4 cells were transfected with either Lamin- or RIPK3-siRNA and analyzed for levels of RIPK3 over 2 days. (D) Clonogenic survival assay showing Nec1 pretreatment or knockdown of essential necroptosis protein RIPK3 enhanced survival in the combination of CQ and CCI-779 as compared to cells without Nec1 or with siRNA control siLamin.