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Fig. 7. Autophagy in cells containing a FAG. (A) Vero cells were transfected with LC3-GFP plasmid and cultured in medium without FBS, but containing pepstatin A (Pep, 10 µM) plus E-64-d (10 µM). (B) LC3-GFP accumulated around the FAG in transfected cells cultured in complete medium without lysosomal inhibitors. (C) Representative experiment of an immunoblot using anti-LC3 antibody in cells treated with (to induce FAG formation) or without Jpk (+/–Jpk) and grown in medium with or without (4 hours) serum (+/–FBS) in the presence of lysosomal inhibitors. The positions of endogenous LC3-I and LC3-II are indicated. Fold increases in the ratios of LC3-II to tubulin (calculated as described in the Materials and Methods) from three independent experiments were 2.4±0.1 (Jpk–/FBS–), 3.8±0.3 (Jpk+/FBS–), 1.0 (control, Jpk–/FBS+) and 2.4±0.2 (Jpk+/FBS+). All differences were statistically significant at P
0.005 using the Student's t-test. (D) Ultrastructure of a FAG-containing cell (the FAG is limited by broken line), in which autophagic vacuoles appeared tightly associated with the FAG. (E) MDC-containing vesicles accumulated around the FAG. (F,G) During clearance of the FAG after Jpk removal (–Jpk for 16 or 24 hours), the number of MDC-containing vesicles and lysosomes (stained with MDC and/or anti-LAMP2 antibodies) increased and were associated with F-actin punctae and F-actin amorphous aggregates (arrowheads in G). (F-I) Rpc (200 nM) added after Jpk removal accelerated the dissolution of the FAG (compare H with F and I with G). Inset in H shows the tight association and partial colocalization of F-actin punctae (red) with lysosomes and/or MDC-containing vesicles (green and blue, respectively). (J) The number of autophagic vacuoles increased during the dissolution of the FAG (–Jpk for 36 hours). (K-M) Some autophagic vacuoles contained fragments of microfilaments arranged in parallel (arrow in K; high magnification in L; M is an enlargement of the boxed area in L). Scale bars: 10 µm (A,B,E-I), 1 µm (J), 200 nm (D), 100 nm (K), 30 nm (L), 10 nm (M).
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