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. 2017 Aug 28;36(50):6850–6862. doi: 10.1038/onc.2017.295

Figure 2.

Figure 2

Vps34 is cleaved by active caspase 8. (a) Active caspase 8 eliminated SUMOylated Vps34 and total Vps34. Immunoblot of His-SUMO1-conjugated Vps34 and total Vps34 in HEK293T cells transfected as indicated for 24 h, and treated with LBH589 for 24 h. (b) Except z-IETD-FMK (20 μM), neither MG132 (10 μM) nor chloroquine (20 μM) blocked abrogation of F-Vps34 caused by active caspase 8. Immunoblot analyses were performed on transfected HEK293T cells, as indicated. (c) Active caspase 8 degraded Vps34 in a dose-dependent manner. Vps34 was extracted from HEK293T cells transfected as indicated. (d) Immunostaining of endogenous Vps34 (green) colocalized with active caspase 8 (red) in MCF7 cells treated with LBH589 and/or z-IETD-FMK, for 18 h. (e) Abrogation of endogenous Vps34 cleavage by caspase inhibitors in LBH589-treated MCF-7 cells. The red arrow denotes cleaved Vps34. (f) Upper, immunoblot analyses of variant mutant of Vps34 expression in HEK293T cells with the presence of LBH589 for 24 h. Lower, the location of predicted cleavage sites in Vps34 structure of scheme. (g) Caspase 8 cleavage of Vps34 at the site of Asp285. Immunoblot of the variant mutant effects on Vps34 expression in HEK293T cells cotransfected with caspase 8 treated with 50 nM LBH589 for 24 h. (h) In vitro cleavage of Vps34 by active caspase 8. The red arrow denotes cleaved Vps34. (i) Multiple alignment of Vps34 shows conservation of the caspase 8 cleavage site in the SDHD motif. DAPI, 4', 6-diamidino-2-phenylindole.