Extended Data Fig. 4: Lysosomal SPP-8 suppresses NFYB-1 dependent mitochondrial dysfunction (Related to Fig. 4).
From: NFYB-1 regulates mitochondrial function and longevity via lysosomal prosaposin
a, spp-8i modestly increases WT and nfyb-1(cu13) lifespan (N = 2, n = 120 per repeat, per condition). b, spp-8 mRNA levels are unaffected by nfya-1 and nfyc-1 knockdown AL day 1, quantitated by qPCR (n ≥ 1000). c, skn-1 and dve-1 knockdown diminished elevated spp-8 mRNA in nfyb-(cu13) AL day 1, quantitated by qPCR (n ≥ 1000). d, e, Representative immunoblot of 1 kb pspp-8::NLS::GFP, shows skn-1 dependent regulation of spp-8 in nfyb-1(cu13) compared to AL WT day 1 (n = 100 per repeat, per condition) with quantitation and actin loading control. f, g, spp-8i rescues the reduced mitochondrial circularity and cross-sectional area of nfyb-1(cu13) mutants in AL day 5 (n ~ 30), quantitated47. h, spp-8i has no effect on phsp-4::gfp expression in indicated genotypes, quantitation by biosorter, AL day 1 (n ≥ 400). i, spp-8i has no effect on nfyb-1(cu13) regulated ER genes, quantitated by qPCR, AL day 1 (n ≥ 1000). j, k, Confocal microscopy images show endogenously tagged mKate2::NFYB-1 in intestine, co-localizes with LMP-1::GFP, AL day 1. Percentage colocalization as measured using Manders Colocalizaton Coefficient (Costes significance test was done for colocalization, refer method section for detail), AL day 1, scale bar = 10 μm (n ~ 30). l, nfyb-1(cu13) mutation leads to less acidified lysosomes in comparison WT, stained with LysoTracker Red in AL day 1 (n ~ 30). b-l, All data represent N = 3 independent biological replicates, n = total worms per condition from three replicates unless stated otherwise. Error bar shows mean ± s.e.m, statistics determined by b-c, e-h, one-way ANOVA, i and l, two-sided t-test ns: not significant, *P < 0.5, **P < 0.01, ***P < 0.001. a, Two-sided Mantel–Cox log-rank test, refer Supplementary Table 2 for statistics.
