Figure 3: Activation of autophagy in 5-FU-treated C. elegans.

(a) Autophagy induction in response to 5-FU measured in a GFP::LGG-1 reporter strain fed control RNAi (L4440) or RNAi targeting the indicated genes (scale bars, 10 μm). (b) Immunofluorescence showing anti-VPS-34 staining in dissected embryos in the absence or presence of 5-FU (scale bar, 5 μm). (c) Western blot analysis of embryonic extracts with and without the addition of 5-FU or the autophagy inhibitor 3-MA, detecting the ~40-kDa GFP::LGG-1 fusion protein and the cleaved ~25-kDa product. (d) F1 survival measured after depletion of BEC-1 (red triangles) and ATG-7 (blue squares) as compared with worms fed control RNAi (black diamonds). The survival curve shows the mean±s.d. for each data point from three independent experiments. (e) Induction of autophagy (% GFP-positive embryos) following depletion of the indicated genes. (f) Immunofluorescence showing anti-VPS-34 staining in N2 and atl-1(tm853) embryos in the absence or presence of 5-FU (scale bar, 5 μm). (g) The fraction (as % of untreated control) of embryos with VPS-34-positive foci following 5-FU treatment. (h) Induction of autophagy (% GFP-positive embryos) in control (GFP::LGG-1) and atm-1(gk186) mutants (atm-1; GFP::LGG-1). (e,g,h) Bar graphs represent mean±s.d. from three independent experiments.