Figure 2

Altered processing of dual–tagged LC3B when MGRN1 function is either reduced or absent. (a) HeLa cells treated with irrelevant siRNAs (mock siRNAs) or MGRN1 siRNAs, were transfected with mCherry-EGFP-LC3B construct. 72h post transfection, cells were fixed and imaged. Yellow and red vesicles indicate autophagosomes and autolysosomes, respectively. Two fields for each condition are shown. Scale bar, 5 μm. (b) Graph shows the percentage of red vesicles among total vesicles for cells imaged in (a). The number of fluorescent bodies per cell among ~51 cells was calculated from three independent experiments (over 30 fields). It shows a significant decrease (~2 fold) in the percentage of red vesicles (calculated out of the total (yellow±red) vesicles for the entire sample set) using the given equation: percentage of red vesicles=(average number of red vesicles in n cells/average number of (yellow±red) vesicles) × 100. ***P-value≤0.001 using Student’s t-test. Error bars, ±S.E.M. (c) Melan a6 and melan md1-nc cells were transfected with mCherry-EGFP-LC3B and imaged 24 h later. Note that total absence of MGRN1 (as in melan md1-nc cells) closely phenocopies the effects of its partial depletion in HeLa. Scale bar, 5 μm. Two fields for each cell line are shown. (d) Histogram was generated using the same sample set from (c). Histogram shows ~4.1-folds decrease in percentage of red vesicles in melan md1-nc cells, compared with the control melan a6 cells. Ten cells (total number of vesicles, ~250) were analyzed. Error bars, ±S.E.M. (e) HeLa cells transiently co-transfected with various PrP constructs and mCherry-EGFP-LC3B were imaged 24 h later. Scale bar, 5 μm. (f) Histogram plotting the percentage of red vesicles in PrP expressing HeLa cells. Approximately 45 cells were analyzed for each PrP construct. The percentage of red vesicles in the empty vector control or upon overexpression of wild-type PrP was ~57% or 62% of the total number of vesicles, respectively. This number varied between 26 and 32% for all PrP constructs known to increase the amounts of ^CtmPrP generated (that included artificial constructs PrP(AV3), PrP(KH-II) and SA-PrP, as well as the naturally occurring human disease mutation PrP(A117V)). In the presence of Ifn-PrP (which generated enhanced amounts of cyPrP), there were ~33% red vesicles. ***P≤0.001, using Student’s t-test. Error bars, ±S.E.M. (g) Melan a6 and melan md1-nc cells co-transfected with mCherry-EGFP-LC3B and either functional MGRN1 or inactive MGRN1ΔR were imaged. Two fields for each cell line are shown. Scale bar, 5 μm. (h) Graph with results from (g) showing percentage of red vesicles for indicated constructs. Overexpression of MGRN1 in melan md1-nc cells, partially rescued and elevated the percentage of red vesicles to ~39% (that is >3 folds of untransfected cells in Figure 2d). MGRN1ΔR expression could not, however, aggravate the phenotype already exhibited in the untransfected melan md1-nc cells. In melan a6 cells, overexpression of MGRN1 did not drastically affect the percentage of red vesicles. On the contrary, exogenous MGRN1ΔR expression in them affected vesicular fusion with the lysosomes and resulted in a decrease in the percentage of red vesicles. Approximately 10 cells were analyzed. Histogram indicates that overexpression of MGRN1 and MGRN1ΔR exhibit opposite effects. Error bars, ±S.E.M. (i) HeLa cells co-transfected with the indicated PrP constructs, mCherry-EGFP-LC3B and either functional MGRN1 or inactive MGRN1ΔR were imaged. Scale bar, 5 μm. (j) Images taken for each PrP construct were analyzed and plotted for the percentage of red vesicles among total vesicles. The graph indicates that overexpression of MGRN1 partially rescues altered processing of mCherry-EGFP-LC3B, while MGRN1ΔR aggravates this phenomenon. Number of cells analyzed for each PrP construct are indicated. *** P≤0.001, using Student’s t-test. Error bars, ±S.E.M.