Fig. 1: Autophagy is essential for lymphatic endothelial cell homeostasis. | Nature Communications

Fig. 1: Autophagy is essential for lymphatic endothelial cell homeostasis.

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

Fig. 1

a Representative blots for indicated proteins of si CTRL, si ATG5, and chloroquine (CQ 25 µM, 48 h) treated LEC. Densitometric quantification is indicated beneath the blots. Mean ± SD, N = 3 biological replicates analyzed using one-way ANOVA, with Tukey’s test for multiple comparisons *p < 0.05 and ***p < 0.001 vs si CTRL. Representative images (b) and quantification (c) of si CTRL, si ATG5 and CQ-treated LEC after GFP-LC3 transfection. Nuclei are stained with DAPI. Scale bars represent 10 µm. Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, *p = 0.02 and ***p = 0.0002 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 19 cells analyzed per condition per independent experiment. Representative images (d) and quantification (e) of si CTRL and si ATG5 LEC spheroids upon stimulation with VEGF-C (100 ng/mL, 48 h). Scale bar represents 100 µm. Mean ± SD, N = 4 independent donors analyzed using unpaired Student’s t test (two-tailed), **p = 0.004. Mean represents mean per independent experiment, with a minimum of 10 spheroids analyzed per condition per independent experiment. f [3H]Thymidine incorporation assay into DNA of si CTRL and si ATG5 LEC upon stimulation with VEGF-C. Mean ± SD, N = 3 biological replicates analyzed using unpaired Student’s t test (two-tailed), *p = 0.01. g Quantification of scratch wound healing for si CTRL and si ATG5 LEC monolayers in the presence of 500 μg/ml Mitomycin C. Mean ± SD, N = 3 analyzed using unpaired Student’s t test (two-tailed), ** p = 0.001. h Flow cytometry analysis of cell death by propidium iodide (PI) of si CTRL and si ATG5 LEC. Mean ± SD, N = 3 biological replicates analyzed using unpaired Student’s t test (two-tailed), p = 0.97. i Representative images of mixed spheroids containing equal amount of fluorescently green labeled si CTRL and red labeled si ATG5 LEC. Scale bar represents 100 µm. j Representative immunofluorescent images of whole corneal mounts (dashed lines) of wild type (WT) and LEC-Atg5 knock out mice (LEC-Atg5−/−) stained for LYVE1, 8 days post corneal cauterization. Scale bar represents 1 mm. k Representative immunofluorescent images of corneal sections dissected from WT and LEC-Atg5−/− mice stained for LYVE1 and CD31, 8 days post corneal cauterization. Scale bars represent 100 µm. l Quantification for the number of end points, number of branch points, average of cumulative length, and surface density for LYVE1+ lymphatic vessels. Mean ± SD, N = 5 corneas for WT and LEC-Atg5−/− analyzed using unpaired Student’s t test (two-tailed), **p < 0.01. m Quantification for the number of end points, number of branch points, average of cumulative length, and surface density for CD31+ blood vessels. Mean ± SD. N = 5 corneas for WT and LEC-Atg5−/− analyzed using unpaired Student’s t test (two-tailed), p = not significant.

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