Fig. 2: Autophagy deficiency impairs expression of lymphatic markers.

a, b Representative blots for the indicated proteins in si CTRL, si ATG5 or CQ-(25 µM, 48 h) treated LEC. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 vs si CTRL. c Representative blot for the indicated proteins in si CTRL and si ULK1 LEC. Densitometric quantification is indicated beneath the blots. Mean ± SD, N ≥ 3 biological replicates analyzed using unpaired Student’s t test, *p < 0.05, **p < 0.01 and ***p < 0.001. RT-qPCR analysis of si CTRL, si ATG5, or CQ-treated LEC. mRNA expression of VEGFR3, LYVE1, PROX1, and NR2F2 (relative to HPRT). Mean ± SD, N = 3 (d, g), N = 4 (e) or N = 6 (f) biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0001 vs si CTRL. h–k RT-qPCR analysis of si CTRL and si ULK1 LEC. mRNA expression of VEGFR3, LYVE1, PROX1, and NR2F2 (relative to HPRT). Mean ± SD, N = 3 (i, j), N = 4 (h, k) biological replicates analyzed by unpaired Student’s t test (two-tailed), *p < 0.05, **p < 0.01, ****p < 0.0001. l Representative immunofluorescent images of LYVE1+ lymphatic vessels (dashed lines) from corneal sections dissected from wild type (WT) and LEC-Atg5 knock out mice (LEC-Atg5^−/−) stained for-VEGFR3. Nuclei are stained with DAPI. Scale bar represents 100 µm. m Quantification of VEGFR3 fluorescent intensity (arb. unit, arbitrary unit) per vessel area. Mean ± SD, N = 3 corneas with a minimum of 24 images analyzed per condition, analyzed by unpaired Student’s t test (two-tailed), ****p < 0.0001.