Fig. 1: Age-associated lipid droplet accumulation is suppressed by BNA2 overexpression.

a, c Replicative age below panels represents the median age of mother cells determined by budscar counting (see Methods). a Lipid droplets (LDs) in Control (WT, AB18-07) cells visualized by mCherry tagged Erg6p, a known yeast LD protein (magenta) and BODIPY 493/503 (neutral lipid stain, green) (black scale bar: 4 μm). Representative images from four independent experiments. b Total neutral lipid and phospholipid fractions extracted from young (black dots) and aged (blue dots) UCC4925^{12, 13, 19} cells were analyzed by gas chromatography mass spectrometry (n = 6 independent experiments). Paired t-test (two-tailed): **p = 0.0065. c BODIPY stained LDs in Control (WT, AB18-07) and BNA2 overexpression (BNA2-OE) cells (black scale bar: 4 μm). Representative images were taken from cells aged and quantified in one experiment from (d) below. d LDs were stained with BODIPY 493/503 and quantified in Control (WT, AB18-07, black dots) and BNA2-OE (blue squares) cells by flow cytometry. The mean, normalized BODIPY intensities for each time point and strain were determined as in Supplementary Fig. 1 for each individual experiment, and these mean, normalized intensities were averaged for n = 13 independent experiments and plotted ±SEM. Two-way ANOVA multiple comparisons test: ****p ≤ 0.0001. p > 0.05 indicated as ns (not significant). e Replicative lifespan (RLS) determined using microdissection¹⁰. 25 Control cells (WT, AB18-07, black line, median lifespan 25, maximum lifespan 55) and 39 BNA2-OE cells (blue line, median 34, maximum 66) analyzed in n = 1 independent experiment. Log-rank test: p = 0.0116. f RLS determined using the Mother Enrichment Program¹⁹ (see Methods). 2300 Control cells (WT, AB18-07, black line) and 2450 BNA2-OE cells (blue line) analyzed over n = 16 independent experiments. Average median and maximum lifespan (±SEM): Control (25.2 ± 0.3, 41.6 ± 0.6), BNA2-OE (28.9 ± 0.5, 49.9 ± 1.3). Log-rank test: ****p ≤ 0.0001. g, h Cellular NAD⁺ and NADH extracted from Control (WT, AB18-07, black dots) and BNA2-OE (blue squares) cells were quantified by absorbance at 450 nm, averaged, and plotted ±SEM (n = 5 independent experiments). Source data for (b), (d–h) provided as a Source data file.