Figure 6

Autophagy and endo-lysosomal pathway defects caused by MGRN1 are mediated via TSG101. (a) Experimental logic to establish that MGRN1 interacts with TSG101 to mediate vesicular fusion with lysosomes. (b) HeLa cells treated with the indicated siRNAs were transfected with either empty vector or HA-TSG101. Cell lysates were immunoblotted to analyze the levels of endogenous LC3 II and p62 in the presence or absence of 300 nM bafilomycin A1. GAPDH was used as loading control. Efficiency of knockdown was shown by immunoblotting with anti-MGRN1. indicates HA-TSG101; endogenous TSG101. The blots are representative of at least three independent experiments. (c) Graph shows fold change in LC3 II when normalized against corresponding GAPDH levels; analyzed from three independent experiments. **P≤0.05, n.s., not significant, (P=0.2) using Student’s t-test. Error bars, ±S.E.M. (d) SHSY5Y cells co-transfected with MGRN1 or MGRNΔR construct and either empty vector or HA-TSG101. Cell lysates were immunoblotted to analyze the levels of endogenous LC3 II in the presence or absence of 60 nM bafilomycin A1. GAPDH was used as loading control. Efficiency of all transfections was checked. The blots are representative of at least three independent experiments. (e) Graph shows fold change in LC3 II when normalized against corresponding GAPDH levels; analyzed from three independent experiments. **P≤0.05, n.s., not significant, (P=0.8) using Student’s t-test. Error bars, ±S.E.M. (f) Cells treated with the indicated siRNAs and transfected with HA-TSG101 were subjected to EGF uptake. Lysates were analyzed for the levels of EGFR at specified time intervals. β-tubulin was used as loading control. indicates HA-TSG101; endogenous TSG101. Efficiencies of knockdown and transfection were also checked. (g) In a reverse experiment, HeLa cells were treated with mock or TSG101 siRNAs, followed by transfection of MGRN1 or MGRN1ΔR. Cell lysates were immunoblotted to analyze the levels of endogenous LC3 II and p62 in the presence or absence of 300 nM bafilomycin A1. GAPDH was used as loading control. Efficiency of knockdown was confirmed by immunoblotting with anti-TSG101. Expression of MGRN1 or MGRN1ΔR was checked. Note that the expression of MGRN1ΔR phenocopies TSG101 depletion; also MGRN1 cannot rescue the effects mediated by TSG101. (h) Cells treated with mock or TSG101 siRNAs and transiently transfected with MGRN1 or MGRN1ΔR were subjected to Alexa-Fluor 488 EGF uptake. They were washed, fixed at indicated time points and imaged. Note that disruption of endo-lysosomal pathway by TSG101 depletion could not be salvaged by MGRN1 overexpression. Scale bar, 5 μm. In the presence of TSG101 siRNA, overexpression of MGRN1ΔR generated a more severe qualitative phenotype than MGRN1