Figure 7

TAK1 induces cytotoxic autophagic cell death.

(A) Eye discs of third-instar larvae of the indicated genotypes were stained with LysoTracker Red to detect autophagic activity and with an active caspase-3 antibody to detect apoptosis. Adult eye phenotypes of the indicated genotypes were visualized. Scale bars indicate 100 μm. (B) The number of puncta in each immunofluorescent image was quantified. Bars indicate means ± s.d. of three independent experiments. (C) The apoptosis inhibitor p35 had little influence on the dTAK1-induced eye phenotype. Immunostaining of third-instar larval eye discs with 1 μM LysoTracker Red shows many punctate structures. The eye discs were stained with active caspase 3 to allow detection of apoptosis. The adult eye phenotype of flies co-expressing dTAK1 and the effector caspase inhibitor p35 was visualized. Scale bars, 100 μm. (D) HEK 293T cells were transfected with the indicated constructs for 24 h and cytotoxicity was then measured using a LDH release assay. The error bars represent the means ± s.d. of three independent experiments. (E) HEK 293T cells were transfected with the indicated constructs for 48 h. Luminogenic caspase 3/7 substrates were added and the cells were then incubated for 1 h at room temperature. Caspase activity was analyzed using a luminometer. Bars represent means ± s.d. of three independent experiments. (F) HEK 293T cells were transfected with the indicated constructs and left untreated or treated with 100 nM bafilomycin A1. The cells were analyzed for cytotoxicity using a LDH release assay. The values represent the means ± s.d. of three independent experiments. (G) The level of TAK1-induced cell death is restored by treatment with the autophagy inhibitors bafilomycin A1 and 3-MA. The cells were pre-treated with 20 nM bafilomycin A1 or 2 mM 3-MA for 1 h. After drug treatment, the cells were transfected with either a control plasmid or a TAK1 plasmid. 48 hours after transfection, cells were harvested and cell viability was assessed using a trypan blue exclusion assay. The values represent the means ± s.d. of three independent experiments. * P < 0.05, ** P < 0.01 and *** P < 0.001.