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Fig. 8. 3-MA treatment does not affect accessibility of LAMP-2-positive vacuoles to fluid-phase endocytosis. M9 cells either untreated (Ctl, A-F), treated with U18666A for 24 hours (U24; G-L) or grown for 3 days in the presence of 10 mM 3-MA with U18666A added for the final 24 hours (U24 + 3-MA; M-R) were incubated for 30 minutes (A-C,G-I,M-O) or 4 hours (D-F,J-L,P-R) in the presence of 5 mg/ml FITC-dextran prior to labelling for LAMP-2. LAMP-2 labelling (A,D,G,J,M,P) is shown in red and FITC-dextran labelling (B,E,H,K,N,Q) in green and merged images are presented in C,F,I,L,O,R. The percentage of LAMP-2/Nile Red-positive swollen vacuoles containing FITC-dextran at 0.5, 1, 2 and 4 hours of FITC-dextran endocytosis was quantified from Mv1Lu and M9 cells treated or not with 3-MA for 3 days and/or U18666A for the final 24 hours, as indicated (S). For all treatment conditions, FITC-dextran does not accumulate in swollen LAMP-2-positive vacuoles at 30 minutes and the majority are accessible after only 4 hours of endocytosis even in the presence of 3-MA. Bar, 5 µm.





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