Fig. 3: ATGL translocates to contact sites between PER–LD upon fasting.

a Representative time-lapse confocal images for colocalization of ATGL and peroxisome at LD surfaces (arrowhead) during fasting (3 h) in young adult worms expressing DsRED::PTS1 and ATGL-1::GFP. n = 15 for each group. Scale bars, 10 μm. b Percentage of ATGL protein colocalizing with peroxisomes calculated using imageJ software. n = 15 for each group. c Confocal images using adipocytes transfected with mCHERRY::PTS and stained with endogenous ATGL (green), and treated with ISO (1 μM) for 1 h. Arrowhead: colocalzation of ATGL and peroxisome at LD surfaces. DAPI, blue. Scale bars, 10 μm. d Quantification analysis of ATGL colocalized to peroxisomes calculated using imageJ software. n = 15 for each group. e Protease protection assays performed in the absence or presence of Triton X-100. Proteinase K was treated for 30 min in ice. 30 μg of protein from WCE; 20 μg of protein from peroxisome fraction. f, g Representative SIM images and quantification analysis recruited ATGL onto LD surfaces in adipocytes immunostained with endogenous PLIN1 (green) and ATGL (red). Cells were treated with nocodazole (0.05 μg ml⁻¹) under CON or ISO (1 μM) treatment. Scale bars, 5 μm. n = 15 cells for vehicle group; n = 10 cells treated with ISO; n = 11 cells treated with nocodazole; n = 10 cells treated with nocodazole and ISO. Arrowhead: ATGL recruited to LD surfaces in f. h, i SIM images and quantification analysis of recruited ATGL onto LD surfaces in adipocytes immunostained with endogenous PLIN1 (green) and ATGL (red). Cells were c or transfected with siNC or siKIFC3 for 48 h d under CON or ISO (1 μM) treatment. Arrowhead: ATGL recruited to LD surfaces in h. n = 13 cells for siNC group; n = 14 cells treated with ISO; n = 15 cells for siKIFC3 group; n = 14 cells for siKIFC3 treated with ISO. Scale bars, 5 μm. ATGL recruitment was quantified using imageJ. CON control; ISO isoproterenol. Data represent the mean ± SD; ^***P < 0.001 vs. vehicle-CON or siNC-CON or fasting 0 h, and ^###P < 0.001 group in two-way ANOVA followed by Turkey’s post-hoc test.