Supplementary Figure 5: MLKL-mediated Calcium influx is involved in plasma membrane rupture during necroptosis, related to Fig. 6.
From: Plasma membrane translocation of trimerized MLKL protein is required for TNF-induced necroptosis
(a) Flow cytometric analysis of intracellular Ca2+ of control shRNA, MLKL shRNA or RIP3 shRNA HT29 cells after treated with TSZ for 4 h with/out NSA or Nec-1 as indicated. Cells were harvested and the Fluo4 fluorescent cells were determined by FACS analysis using Fluo4. (b) HT29 cells were treated with TSZ either in normal DMEM or calcium free DMEM at different time points as indicated. The cell lysates were resolved on non-reducing gel and analyzed by immunoblot with anti-MLKL, anti RIP3, anti-RIP1 or anti-Actin antibodies. * indicates phosphorylated RIP3. (c) Representative image of live HT29 MLKL shRNA cells co-expressing DsRed-MLKL together with RIP3-EYFP treated with TSZ at time 0 and 4 hours in calcium free DMEM. Scale bar, 5 μm. Uncropped images of western blots are shown in Supplementary Fig. 8.
