Figure 1

Compromised function of MGRN1 affects markers of late endocytosis/lysosomes and autophagy. (a) HeLa cells treated with irrelevant siRNAs (GFP siRNAs) or MGRN1 siRNAs, co-immunostained for CD63 and MGRN1 were imaged. Note a qualitative increase in size of vesicles with the functional depletion of MGRN1. The channels for acquiring the images are indicated. Enlarged views of the areas within the white boxes are also shown (insets). Scale bar, 5 μm. The immunoblot on the right shows efficiency of siRNA-mediated MGRN1 knockdown. (b) HeLa cells similarly treated as in (a) were lysed and immunoblotted for autophagy and lysosomal proteins. The levels of GAPDH and β-tubulin serve as loading controls. The blots are representative of at least three experiments. Efficiency of knockdown was checked using anti-MGRN1 antibody. Note that the antibody used against LC3, detects only endogenous LC3 II. (c) The immunoblots shown in (b) were analyzed for the levels of LC3 II. Graph shows results from three independent experiments. **P≤0.05, using Student’s t-test. Error bars, ±S.E.M. (d) Melanocytes, melan a6 and melan md1-nc cells were stained with lysotracker and imaged. Lack of MGRN1 causes enlargement of acidic vesicles. Two fields for each cell line are shown. Insets a (melan a6) and b (melan md1-nc) reveal enlarged views of the vesicles. Scale bar, 5 μm. (e) Graph shows the size distribution of lysotracker positive vesicles imaged in (d). Over 1000 vesicles from at least 25 cells are represented for each cell line. The horizontal line in each box shows the median value and the white square inside each box is the mean. The upper and lower boundaries of individual box show the upper and lower quartiles, the whiskers are S.D. Outlier values are shown outside the whiskers. (f) The average diameter of vesicles analyzed in (e) shows a minor but significant increase in size in melan md1-nc cells as compared with the control, melan a6 cells. ***P≤0.001, using Student’s t-test. Error bars, ±S.E.M. (g) MGRN1 or MGRN1ΔR was ectopically expressed in melan a6 and melan md1-nc cells. The samples were stained with lysotracker and imaged. Enlarged views of the areas within the white boxes (insets) are also shown. Scale bar, 5 μm. (h) The effect of MGRN1 or MGRN1ΔR overexpression in melanocytes was plotted (mean±S.E.M.) for ~100 vesicles. Note that the overexpression of MGRN1 partially rescues the enlarged lysosomal/vesicular phenotype, while MGRN1ΔR aggravates it. **P≤0.05, ***P≤0.001 using Student’s t-test. Error bars, ±S.E.M.