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

Fig. 1. Beclin 1-depleted cells are sensitised to necroptosis in an autophagy-independent manner.

Fig. 1

a HT-29 cells depleted of Beclin 1 by the indicated siRNAs were treated with 30 ng/mL TNFα, 1 μM BV6, and 30 μM Z-VAD-FMK (TBZ) in the presence or absence of 45 μM Nec-1 for 4 h. b TC-1 cells depleted of Beclin 1 by indicated siRNAs were treated with 10 ng/mL TNFα, 1 μM BV6, and 20 μM Z-VAD-FMK (TBZ) in the presence or absence of Nec-1 for 3 h. c L929 cells depleted of Beclin 1 by the indicated siRNAs were treated with 5 ng/mL TNFα and 10 μM Z-VAD-FMK (TZ) in the presence or absence of Nec-1 for 3 h. a–c Knockdown efficiencies were determined by western blotting. After inducing necroptosis, cells were stained with annexin V-FITC and 7-AAD, before analysing by flow cytometry. d HT-29, e TC-1, and f L929 cells depleted of autophagy factors by the indicated siRNAs were treated with TBZ or TZ under either normal conditions or autophagic conditions induced by preincubation with HBSS media. After treatment for inducing necroptosis, cells were stained with annexin V-FITC and 7-AAD, before being analysed by flow cytometry. Data are the mean ± standard deviation (S.D.), n = 3, with ns non-significance, *P < 0.05, **P < 0.01, and ***P < 0.001 at each point compared to indicated graph with the two-sided Student’s t test (a–f).