Fig. 6: Acetate rescues the mitochondrial-Prox1 transcriptional circuit caused by loss of autophagy in LEC. | Nature Communications

Fig. 6: Acetate rescues the mitochondrial-Prox1 transcriptional circuit caused by loss of autophagy in LEC.

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

Fig. 6

a–c RT-qPCR analysis of si CTRL and si ATG5 LEC treated with sodium acetate (AC, 20 mM, 48 h) or vehicle. mRNA expression of Prox1, VEGFR3, and LYVE1 (relative to HPRT). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **p < 0.01 and ***p < 0.001 vs si CTRL, ##p < 0.01 and ###p < 0.001 vs si ATG5. d Representative blots for the indicated proteins in si CTRL or si ATG5 LEC treated with AC or vehicle. 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 < 0.001. e Representative blots for indicated proteins in si CTRL and si ATG5 LEC treated with palmitate (PAL, 500 nM, 48 h) or BSA. 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. f Representative immunofluorescent images of si CTRL and si ATG5 treated with AC or vehicle and stained for mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. g Quantification of mitochondrial fragmentation index (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, *p < 0.05 vs si CTRL, #p < 0.05 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 12 cells analyzed per condition per independent experiment. h Quantification of lipid droplet number per cell. Mean ± SD, N = 4 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, *p < 0.05 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 29 cells analyzed per condition per independent experiment. i Representative super resolution AiryScan images of si CTRL and si ATG5, si CTRL + si DRP1, and si ATG5 + si DRP1 LEC stained for the mitochondrial protein TOMM20 and BODIPY 493/503. Nuclei are stained with DAPI. Scale bars represent 10 µm. j Quantification of mitochondrial index of fragmentation (number of mitochondria/ total mitochondrial area). Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, ****p < 0.0001 vs si CTRL, ####p < 0.0001 vs si ATG5. Mean represents mean per independent experiment, with a minimum of 35 cells analyzed per condition per independent experiment. k Quantification of lipid droplet number per cell. Mean ± SD, N = 3 biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **p < 0.01 vs si CTRL. Mean represents mean per independent experiment, with a minimum of 47 cells analyzed per condition per independent experiment. l–o RT-qPCR analysis in si CTRL, si ATG5, si CTRL + si DRP1 and si ATG5 + si DRP1 LEC. mRNA expression of PROX1, VEGFR3, LYVE1, and CPT1A (relative to HPRT). Mean ± SD, N = 3 (l, n, o) and N = 4 (m) biological replicates analyzed by one-way ANOVA, with Tukey’s test for multiple comparisons, **p < 0.01 vs si CTRL and p = ns vs si ATG5.

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