Figure 5

MGRN1 does not affect lysosomal competence. (a) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs were fixed and immunostained with anti-CD63 antibody. Enlarged views of the areas within the white boxes (insets) are also shown. Scale bar, 5 μm. (b) Graph plotting data from (a) shows significant increase (~1.5 folds) in average diameter of CD63 positive vesicles for MGRN1-depleted cells, where the vesicles diameter are ~0.78 μm and diameter of vesicles in control cells are ~0.51 μm. Note the presence of intra-luminal vesicles in both the samples, except that the ones with MGRN1 depletion are bigger in size. Approximately 240 vesicles were counted from three independent experiments. ***P≤0.001, using Student’s t-test. Error bars, ±S.E.M. (c) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs subjected to Alexa-Fluor 488 EGF uptake. Cells were washed, fixed at 40 min time point, immunostained for CD63 and imaged. Enlarged views of the areas within the white boxes (insets) are also shown. Insets reveal enlarged views of multiple Alexa-Fluor 488-labeled EGF positive puncta (green) on CD63 positive vesicles with intra-luminal membranes (red). Scale bar, 5 μm. (d) Cell lysates treated with indicated siRNAs were analyzed for the levels of CTSD. The levels of β-tubulin serve as loading control. The low and dark exposures of the CTSD blot indicate the different processed forms of the enzyme. Efficiency of knockdown was confirmed by immunoblotting with anti-MGRN1. The blots are representative of at least three experiments. (e) Histograms plotting data from (d) show significant (~1.5 folds) but similar increase in the levels of mature and immature CTSD, as normalized against the protein levels of β-tubulin (top left) and (bottom left) with the depletion of MGRN1. However, note that the fold change in the mature and immature forms of CTSD are comparable between control and MGRN1 siRNA-treated samples (right). Graph representing three independent experiments. **P≤0.05, n.s., not significant (P=0.75) using Student’s t-test. Error bars, ±S.E.M. (f) Cell lysates were analyzed for CTSD activity. Histogram plotted for the activity of this enzyme in MGRN1 and GFP siRNA-treated samples. To block CTSD activity, cells were either treated with vehicle control or pepstatin A, as indicated. Graph represents average of three independent experiments, performed in triplicate for each cell concentration. Error bars, ±S.E.M.; RFU, relative fluorescence units. (g) HeLa cells treated with irrelevant (GFP) or MGRN1 siRNAs were fixed and immunostained with anti-CTSD antibody. Two fields for each condition are shown. (h) Graph plotting the average diameter of vesicles showed significant increase (~1.4 folds) when MGRN1 is depleted, imaged in (g). Approximately 205 vesicles were counted from three independent experiments. ***P≤0.001, using Student’s t-test. Error bars, ±S.E.M. (i) Lysosomal pH values were measured ratiometrically using LysoSensor yellow/blue DND-160–Dextran. In control cells, the average lysosomal pH was detected as 4.62±0.02, while in cells treated with MGRN1 siRNAs the pH was 4.66±0.01; n.s., not significant (P=0.9), using Student’s t-test. Graph represents average of three independent experiments. Error bars, ±S.E.M.