Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A ) Schematic representation of GFP::LGG-1 fluorescence states in the autophagy pathway. IM, isolation membrane; AP, autophagosome; AL, autolysosome. ( B–E’ ) Adult transgenic WT animals expressing gfp::lgg-1, imaged at Day 1 ( B–E ) and Day 10 ( B’–E’ ) of adulthood. APs (arrows) can be seen in the intestine ( B,B’ ), body-wall muscle ( C,C’ ), pharynx ( D,D’ ), and nerve-ring neurons ( E,E’ ). Dotted lines outline individual intestinal cells ( B,B’ ) and pharyngeal bulbs ( D–E’ ). AB, anterior pharyngeal bulb; TB, terminal pharyngeal bulb. Scale bars = 20 µm. ( F–I ) Quantification of autophagosomes (AP; GFP punctae) in the intestine ( F ), body-wall muscle ( G ), pharynx ( H ), and nerve-ring neurons ( I ) at Days 1, 3, 5, 7, or 10 of adulthood in WT animals. Day 7 was omitted for neurons due to a counting issue at this time point. Data are the mean ± SEM of ≥20 animals combined from three independent experiments per time point. ###p<0.00 and ##p<0.001 for WT control at Day 1, 3, 5, 7, or 10 vs. WT control at Day 1 by Poisson regression. DOI: http://dx.doi.org/10.7554/eLife.18459.002

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Fluorescence, Isolation, Transgenic Assay, Expressing

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–C ) Adult transgenic wild-type (WT) animals expressing gfp::lgg-1(G116A) imaged at Day 1 ( A–C ) and 10 ( A’–C’ ) of adulthood in the intestine ( A ), body-wall muscle ( B ) and pharynx ( C ). Scale bars = 150 µm ( A ), 20 µm ( B ) and 50 μM ( C ). ( D–E ) Quantification of GFP-positive punctae in the intestine, body-wall muscle, and pharynx of WT or daf-2(e1370) animals expressing either gfp::lgg-1 (LGG-1) or gfp::lgg-1(G116A) (LGG-1(G116A)) and raised at 20°C ( D ), or WT or glp-1(e2141) animals expressing either gfp::lgg-1 or gfp::lgg-1(G116A) ( E ) and raised at 25°C until Day 1 of adulthood and then grown at 20°C for remainder of life. Data are the mean ± SEM of combined from two independent experiments per time point with ≥20 animals total. Two additional repeats of WT animals showed similar results (data not shown). ∧ , WT + control vs. glp-1 / daf-2 control at Days 1, 3, 5, 7, and 10; *, glp-1 / daf-2 control vs. WT/ glp-1 / daf-2 + BafA at Days 1, 3, 5, 7, and 10, # , WT/ glp-1 / daf-2 control at Days 3, 5, 7, and 10 vs. WT/ glp-1 / daf-2 control at Day 1. ***/ ∧∧∧ / ### p<0.0001, **/ ∧∧ / ## p<0.001, */ ∧ / # p<0.01 by two-way ANOVA. We note that the pharyngeal counts in WT animals were lower in these experiments compared to others ( – and ); this may be due to the use of different microscopes, strains, and/or experimenters. We note that we also assessed the number of GFP-positive punctae between and near the pharyngeal bulbs to possibly evaluate neurons; however, while we observed very few punctae in this area in animals expressing GFP::LGG-1(G116A) (data not shown), none of the LGG-1 reporters expressed from the endogenous promoter allowed for accurate cell identification, and neurons therefore remain to be fully evaluated. DOI: http://dx.doi.org/10.7554/eLife.18459.003

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Transgenic Assay, Expressing

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–D ) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Days 1, 3, 5, 7, and 10 wild-type (WT) transgenic animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, black lines) or Bafilomycin A (BafA, gray lines). Tissues examined were the intestine ( A ), body-wall muscle ( B ), pharynx ( C ), and nerve-ring neurons ( D ). WT animals were raised at 25°C and incubated at 20°C from Day 1 of adulthood. Data are the mean ± SEM of ≥30 animals combined from three independent experiments per time point. ***p<0.0001, **p<0.001, and *p<0.01 for WT control vs WT + BafA on each day; ###p<0.0001 for WT control at Days 1, 3, 5, 7, or 10 vs. WT control at Day 1 by Poisson regression. DOI: http://dx.doi.org/10.7554/eLife.18459.009

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Transgenic Assay, Expressing, Injection, Incubation

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–D ) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Days 1, 3, 5, 7, and 10 glp-1(e2141) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, dark blue lines) or Bafliomycin A (BafA, light blue lines). Tissues examined were the intestine ( A ), body-wall muscle ( B ), pharynx ( C ), and nerve-ring neurons ( D ). The black dashed lines in ( A–D ) show data from wild-type (WT) control animals from for comparison (animals were analyzed in parallel). Data are the mean ± SEM of ≥25 animals combined from three independent experiments. ∧ , WT + control vs. glp-1 control at Days 1, 3, 5, 7, and 10; *, glp-1 control vs. glp-1 + BafA at Days 1, 3, 5, 7, and 10, # , glp-1 control at Days 3, 5, 7, and 10 vs. glp-1 control at Day 1. ***/ ∧∧∧ / ### p<0.0001, **/ ∧∧ / ## p<0.001, */ ∧ / # p<0.01 by Poisson regression analysis. DOI: http://dx.doi.org/10.7554/eLife.18459.015

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Expressing, Injection

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–C ) Quantification of autophagosomes (AP) ( A–B ) and autolysosomes (AL) ( C ) in hypodermal seam cells of adult Day 1 wild-type (WT), daf-2(e1370) , and glp-1(e2141) transgenic animals expressing gfp::lgg-1 ( A ) or mCherry::gfp::lgg-1 ( B–C ) and injected with DMSO (control) or Bafilomycin A (BafA). Data are the mean ± SEM of ≥25 animals combined from at least three independent experiments. ***p<0.0001, *p<0.051 by one-way ANOVA. ( D–E ) Quantification of GFP-positive punctae in hypodermal seam cells of adult Day 1 WT and daf-2(e1370) animals raised at 20°C ( D ), and WT and glp-1(e2141) animals raised at 25°C ( E ) expressing WT gfp::lgg-1 (LGG-1) or mutant gfp::lgg-1 (LGG-1(G116A)). Data are the mean ± SEM from three independent experiments, each with ≥10 animals (one representative experiment shown). ****p<0.0001 by one-way ANOVA. ( F–G ) Quantification of APs and ALs in the hypodermal seam cells of Day 1 WT and daf-2(e1370 mutants expressing mCherry::gfp::lgg-1 raised at 20°C ( F ) or Day 1 WT and glp-1(e2141) mutants expressing mCherry::gfp::lgg-1 raised at 25°C ( G ) and fed from hatching with bacteria expressing empty vector (control) or dsRNA encoding rab-7 . Data are the mean ± SEM of ≥40 animals combined from three experiments. ****p<0.0001 and **p<0.005 by one-way ANOVA. ( H–I ) Quantification of GFP-positive punctae in hypodermal seam cells of adult Day 1 WT, atg-3(bp412) ( H ) and atg-18(gk378) ( I ) animals expressing WT gfp::lgg-1 (LGG-1) or mutant gfp::lgg-1 (LGG-1(G116A)). Animals were raised at 20°C. Data are the mean ± SEM combined from three independent experiments for atg-3 with ≥22 animals combined from two independent experiments for atg-18 with ≥16 animals. ****p<0.0001, ***p<0.001, **p<0.01, and *p<0.05 by one-way ANOVA. DOI: http://dx.doi.org/10.7554/eLife.18459.014

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Transgenic Assay, Expressing, Injection, Mutagenesis, Plasmid Preparation

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A ) Schematic representation of mCherry::GFP::LGG-1 fluorescence states in the autophagy pathway. IM, isolation membrane; AP, autophagosome; AL, autolysosome. ( B ) Whole-body expression of mCherry::GFP::LGG-1 in a wild-type (WT) animal at Day 1 of adulthood. Scale bar = 100 µm. Note that the intensity of red fluorescence compared to green is stronger; thus, the red channel was purposely set lower (see Materials and methods). ( C–G’ ) Adult transgenic WT animals expressing mCherry::gfp::lgg-1, imaged at Day 1 ( C–G ) or Day 10 ( D’–G’ ) of adulthood. APs (mCherry/GFP; yellow arrowheads) and ALs (mCherry only; white arrowheads) can be seen in the hypodermal seam cells ( C ), intestine ( D,D’ ), body-wall muscle ( E,E’ ), pharynx ( F,F’ ), and nerve-ring neurons ( G,G’ ). Dotted lines outline individual intestinal cells ( D,D’ ) and pharyngeal bulbs ( F,G’ ). AB, anterior pharyngeal bulb; TB, terminal pharyngeal bulb. Scale bars = 20 µm ( D–G’ ) and ( C ) = 10 μm. ( H ) Intestine of Day 1 WT transgenic animals expressing mCherry::gfp::lgg-1 (green, GFP; red, mCherry) and stained with LysoTracker (light blue). Scale bar = 5 µm. ( I ) Quantification of punctae containing mCherry alone or co-localized with GFP or LysoTracker in the intestine of WT transgenic animals. Data are representative of three independent experiments, each with ≥10 animals. ( J ) Schematic representation of basal AP and AL pool sizes and the effect of inhibiting autophagy by rab-7 RNAi or Bafilomycin (BafA) treatment. ( K ) Quantification of APs and ALs in the intestine of Day 1 WT transgenic animals fed from hatching with bacteria expressing empty vector (control) or dsRNA encoding rab-7 . Data are the mean ± SEM of ≥40 animals combined from three experiments. ***p<0.0001 by Student’s t -test. ( L ) Quantification of APs and ALs in the hypodermal seam cells of Day 1 WT transgenic animals injected with BafA or DMSO (control). Data are the mean ± SEM of ≥30 animals combined from three experiments. ***p<0.0001 and **p<0.001 by Student’s t -test. DOI: http://dx.doi.org/10.7554/eLife.18459.004

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Fluorescence, Isolation, Expressing, Transgenic Assay, Staining, Plasmid Preparation, Injection

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–C ) Immunoblot analysis of lysates from Day 1 transgenic animals expressing mCherry::gfp::lgg-1 or gfp::lgg-1. Blots were probed with anti-LGG-1 ( A ), anti-mCherry ( B ), and anti-GFP ( C ) antibodies. Data are representative of at least two experiments. The lower band of the mCherry::GFP::LGG-1 reporter (*) may be due to cleavage of the N-terminus as it has previously been published that the first 11 amino acids of mCherry are susceptible to cleavage resulting in something that is slightly smaller than the full length protein . ( D–E ) Immunofluorescence to detect endogenous LGG-1 (green) and GFP (D; red) or mCherry (E; red) in dissected intestines of wild-type (WT) animals. Data are representative of at least two independent experiments, each with ≥5 animals. Scale bars = 20 µm. Full-length protein was detected at Day 10 (data not shown). DOI: http://dx.doi.org/10.7554/eLife.18459.005

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Western Blot, Transgenic Assay, Expressing, Immunofluorescence

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–E’ ) GFP (green) and mCherry (red) fluorescence images from Day 1 ( A–E ) and Day 10 ( B’–E’ ) adult wild-type (WT) transgenic animals expressing the mCherry::gfp::lgg-1 reporter. The merged images are shown in . Tissues tested were hypodermal seam cells ( A ), intestine ( B,B’ ), body-wall muscle ( C,C’ ), pharynx ( D,D’ ), and nerve-ring neurons ( E,E’ ). Autophagosomes (AP, mCherry/GFP) are indicated by yellow arrowheads, and autolysosomes (AL, mCherry only) are indicated by white arrowheads. ( F–I’ ) Confocal images of animals with tissue-specific expression of gfp::lgg-1 in the intestine ( F,F’ ), body-wall muscle ( G,G’ ), pharynx ( H,H’ ), and nerve-ring neurons ( I,I’ ) at Day 1 ( F–I ) and Day 10 ( F’–I’ ) of adulthood. Arrows indicate APs (GFP punctae). Dotted lines outline individual intestinal cells ( B,B’,F,F’ ) and pharyngeal bulbs ( D,D’,E,E’,H,H’,I,I’ ). AB, anterior pharyngeal bulb; TB, terminal pharyngeal bulb. Scale bars = 20 µm except in ( A ) = 10 µm. DOI: http://dx.doi.org/10.7554/eLife.18459.006

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Fluorescence, Transgenic Assay, Expressing

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A ) Schematic of the autophagy steps with rate constants α, β, and γ. IM, isolation membrane; AP, autophagosome; AL, autolysosome; L, lysosome. ( B ) Definition of autophagic flux at steady state. The rate of AP formation ( dAP/dt ) and AL formation ( dAL/dt ) at steady state is equal to 0 allowing for derivation of equations (1) – (3) for AP pool size ([AP]) and AL pool size ([AL]). The overall autophagic flux (‘FLUX’) at steady state is equivalent to the rates of the individual autophagy steps (equation (4)). ( C ) Derivation of the relationship between rate constants β and γ and the ratio of AP to AL based on the observation made in this study that [AP] < [AL] in C. elegans . We used the steady-state equations derived in ( B ). Since AP and AL pool sizes were quantified using the same tissues from animals of the same genotype and age, [IM] and α are equivalent for equations (1) and (3). Thus, the ratio [AP]:[AL] is inversely related to the ratio of the rate constants γ:β, suggesting that γ < β (equation (5)). ( D ) Relative β and γ rates following Bafilomycin A (BafA) treatment (β BafA and γ BafA ) compared with control treatment (β control and γ control ) can be calculated using equations (1) – (3) derived from steady-state conditions in ( B ). [IM] and α are assumed to be constant in control and BafA-treated animals since AP and AL pool sizes were quantified using animals of the same genotype and age. β BafA and γ BafA are inversely related to the change in AP or AL number following BafA treatment (equation 6). Thus, a hypothetical threefold increase in APs and twofold increase in ALs suggests a corresponding threefold decrease in β BafA and twofold decrease in γ BafA , demonstrating that BafA inhibits both the AP to AL step and the AL to degradation step, as indicated in the diagram. ( E ) Hypothetical calculation of rate constants. In WT animals, we arbitrarily set the rate of autophagic flux (i.e. [IM]α) to 12). In daf-2 mutants, we envisioned three scenarios: (1) no change in flux, where [IM]α = 12, similar to WT, (2) a hypothetical twofold increase in flux where [IM]α = 24, and (3) a twofold decrease in flux where [IM]α = 6. We then used our observed AP and AL data from WT animals and daf-2 mutants expressing the mCherry::GFP::LGG-1 reporter (red numbers) to show that different rate constants β and γ (blue numbers) can be calculated for each scenario using the flux equation in ( B ). Thus, it is necessary to quantify the individual rates (i.e. [IM]α, [AP]β, or [AL]γ) or the rate of overall autophagic degradation to determine autophagic flux. The same conclusion can be reached by solving the equations in ( B ). DOI: http://dx.doi.org/10.7554/eLife.18459.007

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Isolation, Derivative Assay, Expressing

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A,B ) Fluorescence microscopy of wild-type (WT) animals expressing mCherry::gfp::lgg-1 treated with LysoTracker and injected with DMSO (control) ( A ) or Bafilomycin A (BafA) ( B ) at Day 1 (D1) of adulthood. White arrowhead, mCherry/LysoTracker punctae; yellow arrowhead, mCherry/GFP punctae. ( C ) Quantification of autophagosomes (AP, mCherry/GFP punctae) and autolysosomes (AL, mCherry-only punctae) in hypodermal seam cells of transgenic WT or cst-1(tm1900) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control) or BafA. Data are the mean ± SEM of ≥35 animals combined from two experiments. ***p<0.0001 and **p<0.001 by one-way ANOVA. ( D ) Quantification of punctae containing mCherry alone or co-localized with GFP or LysoTracker in the intestine of Day 1 WT (data from ) and daf-2(e1730) transgenic animals raised at 20°C or Day 1 WT and glp-1(e2141) transgenic animals raised at 25°C. Data are representative of three independent experiments, each with ≥10 animals. DOI: http://dx.doi.org/10.7554/eLife.18459.008

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Fluorescence, Microscopy, Expressing, Injection, Transgenic Assay

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–D ) Quantification of autophagosomes (AP) in the intestine ( A ), body-wall muscle ( B ), pharynx ( C ), and nerve-ring neurons ( D ) of Day 1 wild-type (WT) animals expressing gfp::lgg-1 and analyzed at two hours or 24 hours after injection of DMSO (control) or Bafilomycin A (BafA). Data are the mean ± SEM of 15 animals combined from two experiments ***p<0.0001, **p<0.001, *p<0.01 by ANOVA. We note that the BafA treatments in the pharynx were negative in this data set; while other 2 hours incubations caused changes in pharyngeal tissues of WT animals at this and other time points, the response to BafA may be more variable in the pharynx. ( E–H ) Eggs of WT transgenic animals expressing mCherry::gfp::lgg-1 were allowed to hatch at 20°C overnight and left at 20°C or incubated at 25°C until Day 1 of adulthood. Animals were then moved to 20°C for the rest of their lifespans. Animals were imaged at Day 1 or Day 3 of adulthood. APs (mCherry/GFP punctae) and ALs (autolysosomes; mCherry-only punctae) were quantified in the intestine ( E ), body-wall muscle ( F ), pharynx ( G ), and hypodermal seam cells ( H ). Data are the mean ± SEM of 15 animals combined from two experiments ***p<0.0001, **p<0.001, *p<0.01 by one-way ANOVA. DOI: http://dx.doi.org/10.7554/eLife.18459.010

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Expressing, Injection, Transgenic Assay, Incubation

Journal: eLife

Article Title: Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging

doi: 10.7554/eLife.18459

Figure Lengend Snippet: ( A–D ) Quantification of autophagosomes (AP) and autolysosomes (AL) in adult Day 1, 3, 5, 7, and 10 daf-2(e1370) animals expressing mCherry::gfp::lgg-1 and injected with DMSO (control, dark green lines) or Bafilomycin A (BafA, light green lines). Tissues examined were the intestine ( A ), body-wall muscle ( B ), pharynx ( C ), and nerve-ring neurons ( D ). The black dashed lines in ( A–C ) show data from wild-type (WT) control animals from for comparison (animals were analyzed in parallel). The black dashed line in ( D ) shows data from WT animals incubated at 20°C for their entire lifespan. Data are the mean ± SEM of ≥25 animals combined from three independent experiments. ∧ , WT control vs. daf-2 control at Days 1, 3, 5, 7, and 10; *, daf-2 control vs. daf-2 + BafA at Days 1, 3, 5, 7, and 10; # , daf-2 control at Days 3, 5, 7, and 10 vs. daf-2 control at Day 1. ***/ ∧∧∧ / ### p<0.0001, **/ ∧∧ / ## p<0.001, */ ∧ / # p<0.01 by Poisson regression. See also for quantification of APs in gfp::lgg-1 transgenic animals. DOI: http://dx.doi.org/10.7554/eLife.18459.013

Article Snippet: Primary antibodies were mouse anti-GFP (diluted 1:100; Santa Cruz Biotechnology, Dallas, TX), mouse anti-mCherry (diluted 1:50; Clontech, Mountain View, CA), and rabbit anti-LGG-1 (diluted 1:100; sample kindly providd by Abgent, San Diego, CA); they were diluted in block and incubated overnight at 4°C.

Techniques: Expressing, Injection, Incubation, Transgenic Assay