Figure 3
From: Mahogunin regulates fusion between amphisomes/MVBs and lysosomes via ubiquitination of TSG101
Compromise in MGRN1 activity perturbs autophagic fusion events. (a) HeLa cells treated with irrelevant siRNAs (mock siRNAs) were transfected with mCherry-EGFP-LC3B construct; live cells were imaged over indicated time periods to track the fate of vesicles in real time. One representative field, out of 25 cells is shown. Scale bar, 5 μm. (b) Cells treated with MGRN1 siRNAs and transfected with mCherry-EGFP-LC3B construct were imaged similar to (a). One representative field, out of 25 cells is shown. Scale bar, 5 μm. (c) Mock or MGRN1 siRNA-transfected HeLa cells were lysed and immunoblotted to analyze the levels of endogenous LC3 II and p62 in the presence or absence of 300 nM bafilomycin A1. The levels of β-tubulin serve as loading control. Efficiency of knockdown was confirmed by immunoblotting with anti-MGRN1. The blots are representative of at least three experiments. (d) The immunoblots from (c) were analyzed for the levels of LC3 II. Graph shows fold change in LC3 II when normalized against corresponding β-tubulin in the cell lysates, analyzed from three independent experiments. **P≤0.05, n.s., not significant (P=0.18) using Student’s t-test. Error bars, ±S.E.M. (e) Melan a6 and melan md1-nc cells either treated with 50 nM bafilomycin A1 (for 4 h) or left untreated, were lysed and immunoblotted to check for the indicated proteins. This is representative of two independent experiments. (f) Quantification of data from (e) denotes fold change in endogenous LC3 II level when normalized against β-tubulin. (g) HeLa cells treated with mock or MGRN1 siRNAs were transiently transfected with UbG76V-GFP construct (denoted as Ub-GFP). In the last 6 h before harvesting, they were grown in the presence or absence of 10 μM MG132. Cell lysates from these samples were immunoblotted with the indicated antibodies. Faint and dark exposures show accumulation of Ub-GFP with MGRN1 depletion. Ponceaus-S stained membrane shows equal loading of total protein across samples. RNAi efficiency was shown using MGRN1 antibody. (h) HeLa cells transiently co-transfected with GFP-LC3 and empty vector, MGRN1 or MGRN1ΔR were either left untreated or treated with rapamycin for 24 h. Cells transfected with empty vector and GFP-LC3 were used as controls. Following this, cells were either immediately lysed or allowed to recover from the drug treatment for 12 h and then harvested. All samples were immunoblotted using anti-GFP and anti-MGRN1 antibodies. GAPDH was used as loading control. The blots are representative of at least three experiments. (i) Histogram shows fold change in the ratio of GFP-LC3 II/I when normalized against GAPDH from (h). Graph was generated from three independent experiments. **P≤0.05, n.s., not significant (P=0.67) using Student’s t-test. Error bars, ±S.E.M. (j) Cells treated similar to (h) were imaged for the presence of green fluorescent structures. Scale bar, 5 μm. (k) Cells imaged in (j) were scored for total number of GFP-LC3 positive vesicles and average number of such vesicles was calculated. n, total number of cells for the various treatments are indicated in the figure. Error bars, ±S.E.M.
