Fig. 6: Notch localized at endosomal, autophagosomal and lysosomal compartments.

A Schematic representation of endosomal maturation and fusion with autophagosomes. Endosomes mature into multivesicular bodies, in which limiting membrane the Notch Intracellular Domain (NICD) can be cleaved and enter the nucleus (nucleus not shown). Alternatively, the NICD can be sorted to intraluminal vesicles of the multivesicular body that fuses with autophagosomes to give rise to amphisomes, which fuse with lysosomes, resulting in autolysosomes where the NICD is degraded. B–F High-resolution (AiryScan) confocal images of Notch associated to endosomal or autophagic vesicles in the lymph gland. Part of Notch protein (cyan, white arrowheads) localized in Rab5-positive early endosomes (red, yellow arrowheads) (B) Rab7-positive late endosomes/multivesicular bodies (red, yellow arrowheads) (C), amphisomes positive for Rab-7 (green, yellow arrowheads) and Atg8a (red, pink arrowheads) (D) or autolysosomes decorated with Lamp (green, yellow arrowheads) and Atg8a (red, pink arrowheads) (E). F After autophagy inhibition through the expression of Atg1^RNAi, Rab7-positive vesicles significantly enlarged (red, yellow arrowheads) and contained increased amounts of Notch protein (cyan, white arrowheads). G Quantification of the size of Rab7-positive vesicles in the experiments of panels (C, F) and after silencing the indicated autophagy genes. Wild type, n = 1431 vesicles; Atg1^RNAi, n = 753; Atg17^RNAi, n = 628; Vps15^RNAi, n = 713; Atg18^RNAi, n = 1085. H Quantification of Notch in Rab7 vesicles in the experiments of panels (C, F) and after silencing of the indicated autophagy pathway genes. Wild type, n = 966 vesicles; Atg1^RNAi, n = 1989; Atg17^RNAi, n = 1564; Vps15^RNAi, n = 1857; Atg18^RNAi, n = 1048. Boxes represent the interquartile range, the central line indicates the median, and whiskers extend to 10^th and 90^th percentiles. Mean values are shown in G as a dash. The statistical analysis performed in both cases was Kruskal-Wallis test followed by Dunn’s test for treatment versus control comparisons (p = 0.0067 for Atg18 ^RNAi in G; p < 0.0001 for every other comparison). Source data for plots are provided as a Source Data file. **p < 0.01; ****p < 0.0001.