Autophagosome Proteins LC3A, LC3B and LC3C Have Distinct Subcellular Distribution Kinetics and Expression in Cancer Cell Lines - Fig 1

<p><b>(A)</b> Specificity of various anti-LC3 antibodies tested against recombinant LC3A and LC3B proteins. <b>(B)</b> The LC3A and LC3B processing after 24h of Bafilomycin (100nM) treatment in glioma and breast cancer cell lines, using the LC3A specific ab62720 and of the LC3B specific 5F10 antibodies <b>(C)</b> Double immunofluorecence in the A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the 5F10 antibody that reacts exclusively with LC3B. Noted a clearly distinct expression of LC3A in green vacuoles with perinuclear/nuclear localization and of LC3B red vacuoles that have a diffuse, throughout the cytoplasm, localization. There are no autophagosomes composed of both LC3A and LC3B proteins (c1,c2), whilst LC3A and LAMP2a show extensive colocalization (c3,c4). The distinct identity of LC3A and LC3B autophagosomes was also confirmed under acidic conditions (c5) and after exposure to bafilomycin (c6). <b>(D)</b> Double immunofluorecence in A549 cell line using the ab62720 recognizing exclusively the LC3A protein and the NB600-1384 that reacts with both LC3A and LC3B proteins. Noted that the majority of the perinuclear/nuclear LC3A (green) vacuoles show double immunofluorescence (yellow), a result of double LC3A/LC3B reactivity that produces the NB600-1384 antibody. <b>(E)</b> LC3A and LC3B siRNAs specifically block the expression of the LC3A and LC3B proteins, respectively, in A549 cell line (E1,2,3). In E4 the reactivity of LC3A (ab62720 Ab) and of the LC3B (5F10 antibody) is shown, following silencing of the LC3A or of the LC3B genes, in the A549 cell line.