Fig. 5.

Spermidine oxidation impairs lysosome integrity and function in dSms mutant synapses and SRS fibroblasts. a N¹-acetylspermidine level (mean ± 95% CI) measured from young flies (2–4 DAE) using LC-MS/MS. b Aldehyde content (mean ± S.E.M.; each data point obtained from one extraction of 20 animals, n = 3) measured from third instar larvae using a colorimetric method. c, d Immunostaining of fly laminae labeled with LAMP1 for lysosome membrane and cathepsin L for lysosomal protease. The fluorescence intensity profile is illustrated in a three-dimensional surface plot. LAMP1 (c) and cathepsin L (d) puncta staining intensity is compromised in mutant flies. e, f Western analysis of fly heads (e) and quantification (f mean ± S.E.M.; each data point obtained from one extraction of 10 animals, n = 7 extractions) showing pro- and mature forms of cathepsin L normalized to tubulin control. Full size blots with molecular weight markers are included in Supplementary Fig. 6. g Immunostaining of age-matched control and SRS fibroblasts labeled with LAMP1 and cathepsin D. Note both enlarged (arrow) and fragmented (white arrowhead) LAMP1 pattern presented in SRS fibroblasts. h, i Western blot analysis of human fibroblasts (h) and quantification (i mean ± S.E.M.; each data point obtained from one extraction, n = 5 extractions) showing pro-, intermediate, and mature forms of cathepsin D in SRS fibroblasts with two different mutations compared with control fibroblasts. Full size blots with molecular weight markers are included in Supplementary Fig. 6. j, k Live imaging of LysoSensor yellow/blue probe (j) and LysoTracker red probe (k) in human fibroblasts. Student’s t test. *P < 0.05, **P < 0.01. Scale bar, c, d, 2 µm; g, j, k, 10 µm