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. 2020 May 26;27(11):3065–3081. doi: 10.1038/s41418-020-0561-9

Fig. 6. Beclin 1 but not Beclin 1 ΔCCD prevents necroptosis by suppressing MLKL oligomerisation.

Fig. 6

a–d HT-29 cells stably expressing FLAG-siBECN1#5-resistant Beclin 1 wild type (WT), or Beclin 1 CCD-truncated mutant (ΔCCD) were transfected with siNon and siBECN1#5 for 48 h. a The protein levels were determined by immunoblotting. b, c To analyse necroptotic population, the cells were treated with TBZ in the presence or absence of GSK'963 (TBZG) for 4 h. After treatment, the cells were stained with annexin V-FITC and 7-AAD, followed by flow cytometry analysis. Data are expressed as the mean ± S.D., n = 3, with *P < 0.05 and ns non-significance at each point compared to the indicated graph with the two-sided Student’s t test. d To analyse MLKL oligomerisation, the cells were treated with TBZ in the presence or absence of GSK'963 (TBZG) for 4 h. After treatment, the cell lysates were lysed under non-reducing conditions for MLKL oligomerisation or under reducing conditions, and were then analysed by immunoblotting.