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. 2010 Oct 1;18(3):479–492. doi: 10.1038/cdd.2010.118

Figure 1.

Figure 1

Figure 1

Overexpression of 14-3-3ζ proteins block C2-ceramide-induced autophagy. (a) Hela cells were incubated with 10 μM of C2-ceramide for indicated times. Cell extracts were harvested for immunoblot analysis of LC3-I processing into LC3-II, p62/SQSTM1, P-p70S6K, total p70S6K, P-S6, total 6S and 14-3-3 proteins. Tubulin was used as a protein loading control. Endogenous LC3-II/tubulin levels were quantified and the ratio presented as arbitrary units (N=3, *P=0.0006, Student t-test). (b) HEK293T cells transfected with GFP-LC3 were treated as in (a) and GFP-LC3 was analyzed by microscopy. Quantification of GFP-LC3vac cells number and number of GFP-LC3 punctae per cell were performed by counting in a blinded experiment. Columns, average of three different experiments, set up with duplicated samples and counting 6 × 100 cells per sample (quantification of GFP-LC3vac cells with more that five punctae per cell, is presented as a percentage of total cell numbers, N=3, *P=0.0001, **P=0.00087 Student t-test). (c) HEK293T cells transfected with GFP (control) or GFP-14-3-3ζ (GFP-14-3-3ζ) were left untreated or stimulated with C2-ceramide 10 μM for 24 h. Cell extracts were harvested for immunoblot analysis of LC3-I processing into LC3-II, P-p70S6K, total p70S6K, P-S6, total 6S, GFP-14-3-3ζ and total 14-3-3 proteins. Tubulin was used as a protein loading control. Endogenous LC3-II/tubulin levels were quantified and the ratio presented as arbitrary units. Columns, average of three different experiments (*P=0.00037, Student t-test). (d) HEK293T/GFP-LC3 cells were transfected with HA-control or HA-14-3-3ζ were left untreated or stimulated with C2-ceramide 10 μM for 24 h. GFP-LC3 was analyzed. Quantification of number of GFP-LC3 punctae per cell was performed by counting in a blinded experiment. Columns, average of three different experiments, set up with duplicated samples and counting 6 × 100 cells per sample, shown as percentage. Bars=10 μm (*P=0.00059, Student t-test). Western blot analysis shows levels of HA-14-3-3ζ. Tubulin was used as a loading control. A full-color version of this figure is available at Cell Death and Differentiation online