Figure 4(A) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with Oil Red O (ORO) (scale bar= full image-100 μm; hindgut magnification-20 μm). (B) Quantification of hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes stained with ORO (one-way ANOVA with Tukey's multiple comparison test, n=18-26 animals). (C) Quantification of mean intensity of ORO staining in the hindgut of wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes at different time points, starting from L4 stage (one-way ANOVA with Dunnett's post hoc correction, n=18-26 animals). (D) Mean intensity of ORO staining in the whole body of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes plotted against body area. Data information: , bars represent mean ±SEM points represent mean ±SEM. Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (E) Representative images of wt, cest-2.2 O/E and cest-2.2 (lf) animals stained with ORO. Arrows indicate mislocalized lipid droplets. (F) Quantification (mean intensity ± SEM) of ORO staining in hindgut (left panel), foregut (center panel) and whole body (right panel) of 5-day-old nematodes (one-way ANOVA with Dunnett's post hoc correction, n=11-17 animals). Data information: , bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (G) Representative images of 5-day-old wt, gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant animals stained with LipidTOX (scale bar= 100 μm). (H) Statistical analysis of whole-body LipidTOX intensity of 5-day-old nematodes (ordinary one-way ANOVA with Tukey's multiple comparisons test, n=25-37 animals). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (I-J) Representative images of nematodes stained with LipidTOX (I) (scale bar= 100 μm), with quantification (J) (mean intensity ± SEM) of LipidTOX staining. Data information: bars represent mean ±SEM Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (K) Representative thin layer chromatography (TLC) plate of samples from 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes. (L) Densitometry of TLC-separated free fatty acids (FFA) across three biological replicates in 5-day-old mutant animals. Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001. (M) Schematic representation of free fatty acid mass spec analysis. (N) Sum of TLC-separated FFA of 5-day old animals (n=5 biological replicates). (O) Quantification of individual FFA detected after TLC separation in 5-day-old gas-1(fc21) and gas-1(fc21); cest-2.2 O/E mutant nematodes (n=5 biological replicates, n.d.= not detected). Data information: bars represent mean ±SEM ; Across experiments, p value summary is ns= not significant, *p<0.05, **p < 0.01; ***p < 0.001, ****p<0.0001.