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. 2017 Aug 23;8(46):80770–80789. doi: 10.18632/oncotarget.20415

Figure 4. DAPK-mediated Beclin-1 phosphorylation contributes to BetA-induced autophagic cell death.

Figure 4

(A) IM-9 and IM-9/ Bcl-2 cells were treated with BetA for different periods of time. Treated cells were lysed for detecting DAPK phosphorylation (p-DAPK), total DAPK (t-DAPK), Beclin-1 phosphorylation (p-Beclin-1) and total Beclin-1 (t-Beclin-1) by Western blotting, with β-Actin serving as a loading control. Arrowhead referred to the upshifted Beclin-1. (B) IM-9/ Bcl-2 cells were transfected with Ctrl vector or HA-Beclin-1 T119A for 48 h, and then cells were lysed for detecting LC3-I and LC3-II levels by Western blotting, with β-Actin serving as a loading control. (C) IM-9/ Bcl-2 cells were treated with BetA for 48 h, and then treated cells were collected for MDC staining by flow cytometry. Representative results of three experiments with consistent results are shown. (D) IM-9/ Bcl-2 cells were treated as described in B, and cell viability was determined as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean ± S.D. **,P<0.01, the compared groups: 48 h & 0 h under the same vector treatment in IM-9 cells; Beclin-1T119A & Ctrl vector treatment at 24 h or 48 h in IM-9/Bcl-2 cells). (E) Detection of DAPK activity. DAPK was immunoprecipitated with anti-DAPK antibodies from BetA-treated cells and an in vitro kinase assay was performed using MLC (2 mg) as a substrate in the presence of EGTA, 0.1 or 1 nM calmodulin (CaM). Kinase activity, calculated by quantifying the relative MLC phosphorylation levels after normalization to DAPK protein levels (n=3, mean ± S.D. *,P<0.05). (F) IM-9/Bcl-2 cells were transfected with Flag-DAPK S308D or Flag-DAPK ΔCaM for 48 h, and then cells were lysed for detecting p-Beclin-1, LC3-I and LC3-II levels by Western blotting, with β-Actin serving as a loading control. (G) IM-9/Bcl-2 cells were transfected with Flag-DAPK S308D or Flag-DAPK ΔCaM for 48 h, and then treated with BetA for 48 h. Cell viability was determined as described in Materials and methods. Graphs showing results of quantitative analyses (n=3, mean ± S.D. *,P<0.05). (H) Indicated cells were transfected with HA-tagged Beclin-1 (WT) or HA-tagged T119A Beclin-1 mutant with or without Flag-tagged activated DAPK (ΔCaM). Beclin-1 was immunoprecipitated using HA antibodies, and the co-immunoprecipitated proteins, as well as the total cell extracts, were blotted using the indicated antibodies. All data are representative of three independent experiments.