Fig. 2: Reduced angiogenic sprouting by autophagy inhibition. | Nature Communications

Fig. 2: Reduced angiogenic sprouting by autophagy inhibition.

From: Angiogenesis-on-a-chip coupled with single-cell RNA sequencing reveals spatially differential activations of autophagy along angiogenic sprouts

Fig. 2: Reduced angiogenic sprouting by autophagy inhibition.The alt text for this image may have been generated using AI.

A Temporal procedure of EC loading (day 0), autophagy inhibitor treatment (day 1), and fixation and imaging (day 4) during 3D angiogenic sprouting assay. B Image processing scheme for morphological feature detection from angiogenic sprouts. Scale bar, 200 μm. C Representative z-projected confocal image of sprouts immunostained with anti-CD31 (green) with or without treatment of autophagy inhibitors. Scale bar, 200 μm. Two times of experiments was repeated independently with similar results. D Quantitative analysis of the average sprout lengths, widths, endpoint counts, and branch point counts in the indicated inhibitor-treated conditions. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, One-way ANOVA with Tukey’s posthoc tests. n = 13 per condition with Control, DMSO Control, and Wort 1 μM; n = 12 per condition with CQ 2 μM, Wort 5 μM, Baf 20 nM, and Baf 50 nM; n = 10, 6, 5 for CQ 10 μM, 3MA 100 μM, 3MA 500 μM, respectively. Biological replicates. Data are mean ± S.D. Source data and exact p-values are provided in a Source Data file. E Quantification of the numbers of sprouts with LC3B puncta in the indicated conditions. *p < 0.05, One-way ANOVA with Tukey’s posthoc tests. n value is same as the data shown in Fig. 2D. Biological replicates. Data are mean ± S.D. Source data and exact p-values are provided in a Source Data file. F Distribution of the average number of pixels with intensities of LC3 puncta over their distances from sprout endpoints in samples treated with (131 sprouts from 5 biological replicate samples) or without (181 sprouts from 7 biological replicate samples) 2 μM CQ. Data are mean ± S.E.M. Source data are provided as a Source Data file. G Representative z-projected confocal images of angiogenic sprouts with or without CQ treatment. Immunostained CD31 (green), LC3B (red), and Nucleus (blue) are shown. A sprout in the yellow-dotted box (left; scale bar, 200 μm) is represented (right; scale bar, 50 μm) in higher magnification. H Representative z-projected confocal image of angiogenic sprouts with treatment of siControl, siATG5, or siATG7. All samples are fixed in day 4 and stained with lectin (green). Scale bar, 200 μm. I Quantitative analysis of the average sprout lengths, widths, endpoint counts, and branch point counts in the ECs transfected with siControl (n = 24), siATG5 (n = 12), or siATG7 (n = 10). Biological replicates. ***p < 0.001, ****p < 0.0001, One-way ANOVA with Tukey’s posthoc tests. Data are mean ± S.D. Source data and exact p-values are provided in a Source Data file.

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