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Fig. 1. LC3 and Rab7 distributions changed during amino acid starvation. Triple immunofluorescence staining of LC3, Rab7 and lysobisphosphatidic acid (LBPA) in HeLa cells. The cells were grown on coverslips and either fixed without treatment (0 h) or incubated in serum and amino acid free medium for 1 to 4 hours as indicated on the left. The cells were fixed with 4% paraformaldehyde and permeabilised with saponin. Optical sections are shown. Overlay of LC3 (green) and Rab7 (red) is shown in the right column. Arrows and inserts in the 2 h and 4 h rows indicate large LC3-positive structures, which started to appear after 2 hours starvation. These structures were also positive for Rab7, as indicated by the arrows in the Rab7 column and yellow colour in the merge column. At later time points, LBPA was also present in these structures, as indicated by small arrowheads, and yellow colour in the inserts, in the LBPA column (LC3 is green, LBPA red). LC3 and Rab7 also colocalised in small vesicles that probably represent newly formed autophagosomes (large arrowheads in 1 h and 2 h cells). Bar, 10 µm.
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