Fig. S1

Fig. S1

Autophagic abnormalities and survival in spns1-mutant fish and worms. (A) Representative transmission electron microscopy images of normal wt or spns1-mutant fish larvae at 84 hpf. Compared with wild-type (wt) control (left), the spns1 mutant (spns1^-/-) (right) accumulates abnormal cytoplasmic inclusions at the hypodermal regions adjacent to yolk sac (ys) (upper two panels) or melanophores (me) (middle two panels), and in the retinal pigment epithelium containing melanophores (me) (lower two panels). Arrows indicate cytoplasmic membranous inclusions. In the right-upper panel, the inset shows a magnified image of the cytoplasmic inclusion surrounded with a dotted square. Scale bar, 2 μm. (B) Modulation of autophagy activity by a mutation in the spns1 homolog (spin-1^-/-; C13C4.5) in C. elegans. Representative images of autophagosomes (EGFP::LGG-1 puncta) in seam cells are shown for wild-type {wt, adIs2122 [lgg-1p::GFP::lgg-1, rol-6(su1006)]} animals and for nematodes carrying a homozygous spin-1 deletion allele {spin-1(ok2087); adIs2122 [lgg-1p::GFP::lgg-1+rol-6(su1006)}. Arrows indicate autophagosomes only in the spin-1^-/- animals. Scale bar, 5 μm. (C) Quantification of EGFP::LGG-1 puncta is shown for the indicated genetic backgrounds and conditions. The count of puncta per seam cell was 0.8936±0.0926 for wt and 1.9899±0.1396 for spin-1(ok2087) L4 larva, respectively [values are the mean ± standard error of mean (S.E.M.) for 94 (wt) and 99 [spin-1(ok2087)] seam cells; more than 20 animals were examined for each strain] (t-test: *p<0.0001). (D) The starvation sensitivity in spin-1(ok2087) mutant worms. Percent of worms surviving to adulthood on NGM plates with OP50 bacteria after incubation in M9 buffer in the absence of food at the L1 larval stage for the indicated times. Error bars are for standard errors of means estimated assuming a Poisson distribution, and similar results were obtained in three independent experiments. (E) Lifespan in spin-1(ok2087) mutant worms is demonstrated by Kaplan-Meier survival analysis. spin-1(ok2087) mutant worms are short lived compared with the wild-type N2 strain on HT115 bacteria. The median lifespan was 12 days for N2 and 10 days for spin-1(ok2087) (log rank test: χ² = 8.834 on one degree of freedom; p = 0.003). Similar results were obtained in 2 experiments with 3 independent replicates each. (F) Shorter lifespan in heterozygous spns1^-/+ adult fish is demonstrated by Kaplan-Meier survival analysis (log rank test: χ² = 54.05 on one degree of freedom; p<0.0001) and validated by Gompertz-Makeham model.