Extended Data Fig. 1: HLH-30 protects against cellular senescence, enabling dormancy and stem cell longevity.
(a) Number of M-phase cells in the distal gonad arms (progenitor zone) of wild type (N2) recovered for 24 hours from 4 or 40 days of ARD. The salmon color indicates worms without M-phase positive cells. Pooled data from 2 independent experiments. (b) Percentage of N2 gonad arms negative for M-phase positive cells (salmon color) upon recovery for 24 hours from 4 or 40 days of ARD. BR = 2. (c) Quantification of germ cell nucleolar areas of N2 worms at 4 or 40 days of ARD. BR = 3. One representative experiment. Each dot represents one nucleolus. (d) Photomicrographs of distal gonad arms of sygl-1::3xFlag worms in N2 background, stained with DAPI (nucleus), anti-3x FLAG (SYGL-1, GSC zone, yellow) and anti-RAD-51 (DNA damage foci, magenta) antibodies. Following 1 hour post 60 Gy irradiation, ad libitum worms were dissected and stained. Scale bar 10 μm. The distal end of the gonad is facing the left side of the image. (e) Representative images of mKate2 labeled mitochondria in GSCs at 48 hours of ARD and 48 hours of refeeding. Images show a single z-layer of the germline, fluorescence and DIC images. Genotypes pie-1p::tomm-20mKate in N2 and hlh-30(tm1978) background. Dashed lines mark the most distal GSC area (30 μm) used for image analysis. Arrowhead show the distal end of the gonad. Scale bar 10 μm (f) Representative photomicrographs of head regions of 48 hour ARD worms stained with SA-β-gal. Genotypes N2, hlh-30(tm1978). 4 worms per genotype. Scale bar 10 μm. (g) SA-β-gal RGB color intensity of head regions of young adult hlh-30(tm1978) worms normalized to N2. Staining control is RNAi again bgal-286. Each dot represents the SA-β-gal intensity of one worm. Pooled data of three biological repeats. One-way ANOVA followed by Turkey’s post-hoc test. (h) Precent mean brood size change of autophagy and lysosomal mutants under ad libitum (grey) or refeeding after 10 days in ARD (blue). Percent change was determined in respect to N2 or him-5 controls. Genotype alleles can be found in the reagent table. Each dots reflect one biological replicate. Kruskal-Wallis followed by Dunn’s post-hoc test. If not stated otherwise: Mean & s.d., Mann-Whitney test (two-sided).
