Figure 5
CHC22 affects NPY trafficking to the lysosome. (A) Addition of 200 nM BafilomycinA1 (18 hours) or 10 µM E64 and 50 µM Leupeptin for 24 hours resulted in comparable accumulations of NPY in control and knockdown cells. (B) Results of densitometry from at least three repeats. Statistics represent one way ANOVA with Dunnett’s multiple comparison post hoc test (p < 0.05, p < 0.001 are *, *** Respectively). (C) Immunofluorescence experiments with co-staining for Lamp1 and NPY in the presence or absence of BafA1 and CLTCL1 knockdown. In untreated cells Lamp1 and NPY show some overlap but rarely clear co-localisation, however after treatment with BafilomycinA1 and a block of endo/lysosome fusion, clear co-localisation is observed. (D) Graph representing Pearsons coefficient of co-localisation in each case. At least 24 cells tested for each condition. Together, these results indicate, in the absence of CHC22 NPY is accumulating due to trafficking away from the lysosome and when CHC22 is present, NPY is trafficked (and degraded) within the lysosome. Statistics represent Mann-Whitney U Tests (p < 0.05, p < 0.01, p < 0.001 are *, **, *** Respectively). (E) Western blot demonstrating accumulation of NPY in cells with CHC22 knocked down and increased secretion into the media. (F) Western blot demonstrating accumulation of BDNF in cells treated with siRNA and notable increases in secretion of both pro and mature BDNF into the media (quantification in supplement Fig. S4). Uncropped blots are in displayed in Supplementary data files.
