Fig 1.
(A) Specificity of various anti-LC3 antibodies tested against recombinant LC3A and LC3B proteins. (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 (C) 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). (D) 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. (E) 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.
Fig 2.
(A) Confocal double immunofluorescence for LC3A (green) and LC3B (red) in various cell lines. Noted the LC3A accumulation in the perinuclear area, while LC3B is distributed throughout the cytoplasm (A1-7). LC3 aggregates, suggestive of small LC3A+ autophagosomes included in LC3B+ autophagosomes, are occasionally present (boxes in A1,2). Western blots confirm the nuclear presence of LC3A in the nuclei, mainly with the LC3A-II form (A8). (B) Nucleoli are stained with the MIB1/green (B1), the LC3B/red (B2), but not the LC3A/violet antibody (B3); LC3A specific ap1805a and of the LC3B specific 5F10 antibodies were used. This is confirmed in double immunostaining for LC3A/MIB1 (B4), LC3B/MIB1 (B5) and LC3A/LC3B (B6). Noted that LC3B stains the internal nucleolar areas, while MIB1 the peripheral. (C) Actinomycin D damages nucleoli and disrupts the nucleaolar LC3B and MIB1 localization (C1). Acidic conditions (pH = 6.5; C2) or hyperthermia (40°C; C3) do not abrogate the distribution of LC3B in the nucleoli nor of LC3A in the nuclei. Lamin is also used as a control to confirm the presence of nuclei only in the nuclear fraction.
Fig 3.
(A,B,C) 24h incubation with Leptomycin B at 10 ng/ml, results in nuclear accumulation of LC3A, LC3C and Beclin-1 in lung A549,H1299 and glioblastom U87,T98 cell lines. (D) 24h incubation with Ivermectin at 100μg/ml, reduced the nuclear expression of LC3A/Beclin-1 in A549 and H1299 cell lines and increased the perinuclear accumulation of LC3A in the H1299 cell line. (E) Western blots of the nuclear cell fraction confirm accumulation of LC3s and Beclin-1 in lung and glioblastoma cell lines after incubation with Leptomycin B. The reduction of proteins after incubation with Ivermectin was not consistent in all cell lines.
Fig 4.
(A) Cytoplasmic and nuclear staining of LC3C (red) in U87 cell line. (B) Double LC3C (red) and LC3B (green) immunostaining in U87 cells showing lack of co-localization between the two proteins, both in the cytoplasm and the nuclear/nucleolar regions. (C,D) Double LC3C (red) and LC3A (green) immunostaining showing co-localization between the two proteins, mainly in the nuclear area (U87 and T98 cell lines). (E) Western blot for LC3C in the nuclear (N), Pellet (P) and Soluble (S) fraction of U87 and T98 cells. ‘M’ is the marker and Lamin is used as a control to confirm the presence of nuclei only in the nuclear fraction.
Table 1.
Prevalent subcellular patterns of LC3 expression.