Fig 1: Glycogen Is Required for LD Formation(A) Bubble GSEA Unlimited Map (GUM) representation of GSEA transcriptomic analyses. The legend is indicated in the gray box (NES, normalized enrichment score; FDR, false discovery rate).(B, C, D, F, G, and H) qRT-PCR analysis of transcripts of Gys1 (B), Gyg (C), Pygl (D), Scd1 (F), Plin5 (G), and citrate synthase (H) normalized to Ppia in embryonic BAT.(E) ELISA of PYGL in BAT between E14.5 and E18.5.(I) Measure of citrate synthase activity in BAT.(J) PAS staining (left panels) and BODIPY and DAPI staining (right panels) (SB, 10 μm) of primary brown preadipocytes treated at day 0 with siGys1 or a control siRNA (siControl) and analyzed at day 6.(K) Quantification of the number of LDs per nuclei, on images presented in (J) (n = 5 independent experiments, nine images analyzed per condition). Results are expressed as means ± SEM; ∗∗∗p < 0.001, by paired t test.(L) BODIPY and DAPI staining (left panels) and Oil Red O staining (right panels) on Gys1+/+, Gys1+/−, and Gys1−/− mouse embryos at P0 (SB, 10 μm).(M) Quantification of the number of LDs per nuclei from BODIPY-DAPI stainings represented in (L) (n > 5 embryos, three images analyzed per condition). Results are expressed as means ± SEM; ∗p < 0.05 and ∗∗p < 0.01 by one-way ANOVA with Bonferroni post hoc analysis, ∗compared to Gys1+/+ and $ compared to Gys1+/−.(N and O) PAS (N) and Oil Red O (O) staining of hMADS cells treated with siControl or siGys1 and differentiated into brown adipocytes (day 15, SB, 20 μm).(B–I) Results are expressed as means ± SEM; ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001 by one-way ANOVA with Bonferroni post hoc analysis, ∗compared to E14.5, $ to E15.5, # to E16.5, and & to E17.5.