Fig. 4: LEC-autophagy is required for the traffic and release of lipids for mitochondria fatty acid oxidation. | Nature Communications

Fig. 4: LEC-autophagy is required for the traffic and release of lipids for mitochondria fatty acid oxidation.

From: Lipid droplet degradation by autophagy connects mitochondria metabolism to Prox1-driven expression of lymphatic genes and lymphangiogenesis

Fig. 4

Representative super resolution AiryScan live images (a) and quantification of colocalization of fluorescently labeled (C16:0) palmitate (BODIPY-PAL) in combination with NILE RED (Manders M1 coefficient) (b) and quantification of lipid droplets (c) in si CTRL and si ATG5 LEC pulsed with BODIPY-PAL (overnight incubation) and imaged at 0 h and after 24 h chase period. Zoomed boxed areas show BODIPY-PAL (green) colocalization with NILE RED-stained lipid droplets (magenta). Mean ± SD, N = 3 biological replicates with a minimum of 19 cells per condition in total analyzed using one-way ANOVA with Tukey’s test for multiple comparisons (b) and Kruskal–Wallis test with Dunn’s multiple comparisons test (c), **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs si CTRL (0 h). Scale bars represent 10 μm. d–j Representative super resolution AiryScan live images (d) of si CTRL and si ATG5 LEC pulsed with BODIPY-PAL (overnight incubation) and imaged after 6 h and 24 h chase period. Mitochondria were labeled with MitoTracker deep red (30 min), before imaging (e, h), mitochondrial circularity (f, i), and index of mitochondrial fragmentation (number of mitochondria/ total mitochondrial area) (g, j). Zoomed boxed areas show colocalization of the mitochondria (magenta) with BODIPY-PAL-signals (green). Mean ± SD, N = 3 biological replicates with a minimum of 23 cells per condition in total analyzed using unpaired two-tailed Student’s t test (e, f, h) or two-tailed Mann-Whitney test (g, i, j), **p < 0.01 and ****p < 0.0001 vs si CTRL. Scale bars represent 10 μm. k Oxygen consumption rate (OCR) of LEC isolated from WT or LEC-Atg5−/− mice. l ATP-linked respiration. m Maximal respiration and n Spare respiratory capacity from (k). Mean ± SD, N = 3 biological respiration using unpaired Student’s t test (two-tailed), ****p < 0.0001. o OCR of si CTRL or si ATG5 LEC measured in ß-oxidation assay medium. Etomoxir (Eto, 40 μM) was added 15 min prior to baseline measurement in ß-oxidation assay medium. p Maximal respiration and q Spare respiratory capacity from OCR in (o). Mean ± SD, N = 4 biological replicates analyzed using one-way ANOVA with Tukey’s test for multiple comparisons, *p < 0.05 and **p < 0.01 vs si CTRL. r Fatty acid oxidation (FAO) assay of si CTRL and si ATG5 LEC supplemented with 2 mCi/mL [9,10-3H]-palmitic acid for 18 h. Mean ± SD, N = 4 biological replicates analyzed using one sample t test (two-tailed), **p = 0.003.

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