Fig. 4: Nucleophagy promotes germline immortality and longevity of the soma by restricting nucleolar expansion.
From: Nucleophagy delays aging and preserves germline immortality
a, Confocal microscopy imaging of day 2 and day 5 adult worms expressing pfib-1FIB-1::GFP, subjected to RNAi-mediated silencing of the indicated genes. Scale bar, 20 μm. b, Western blot analysis and quantification of relative FIB-1::GFP protein level expression of day 3 adult worms subjected to RNAi-mediated silencing of the anc-1, lgg-1, anc-1;lgg-1, rab-7, anc-1;rab-7 and fib-1 genes. Mean ± s.d. of three biological replicates. *P < 0.05, **P < 0.01, ***P < 0.001 using a one-way ANOVA. c, Western blot analysis and quantification of relative FIB-1::GFP protein level expression of day 3 adult worms subjected to RNAi-mediated silencing of the anc-1, daf-2, anc-1;daf-2, bec-1 and anc-1;bec-1 genes. Mean ± s.d. of three biological replicates. NS ≥ 0.05, *P <0.05, **P<0.01 using a one-way ANOVA. d, Life span analysis of RNAi-mediated silencing of anc-1 or bec-1 in a WT and DAF-2-deficient background; n ≥ 131 worms. e, DIC microscopy imaging of day 2 worm gonads subjected to RNAi-mediated silencing of the indicated genes at 25 °C. Arrows indicate the most proximal nucleoli. Scale bar represents 20μm. f, DIC microscopy imaging of day 2 worm gonads subjected to RNAi-mediated silencing of with anc-1, lgg-1 and bec-1 at 25 °C. The zoomed areas indicate each most proximal oocyte. The arrows indicate the nucleoli of the most proximal oocyte. Scale bar, 20 μm. g, Epifluorescence microscopy imaging of WT and anc-1(RNAi)-silenced worms expressing ppie-1mCherry::HIS-58;ppie-2:GFP::PH(PLC1delta1), indicating nuclear (red) and cell membrane (green) boundaries respectively at 25 °C. The numbers indicate oocytes starting from the most proximal (−1) to the most distal oocyte. The gray dashed lines highlight the germ cell area. The red lines highlight tumor-like structure (middle image) or tumor formed (lower image). Scale bar, 20 μm. h, Confocal microscopy imaging of day 2 adult worms expressing plgg-1GFP::LGG-1 and mKate2::ANC-1B, subjected to RNAi-mediated silencing of the ced-9 gene, treated with chloroquine or ultraviolet/chloroquine at 25 °C. The arrows indicate GFP/mKate double-positive cells. Scale bar, 10 μm. i, Quantification of double GFP- and mKate2-labeled germ cells. Mean ± s.d. NS ≥ 0.05, **P < 0.01, ****P < 0.0001 using a one-way ANOVA; n = 15 worm midbody areas. j, Percentage of fertility of indicated worm strains across generations. k, Dissected ovaries from 1.5-year-old mice. The arrows indicate ovarian tumors in nesprin-2−/− female mice.
