The LC3-II comes from a proLC3 ~30KDa protein after cleavage by autophagin Atg4 to produce the active cytosolic form LC3-I. cytoplasm and localized in the nucleolar regions. LC3C was located in the cytoplasm and strongly in the nuclei (excluding nucleoli), where it extensively co-localized with the LC3A and the Beclin-1 autophagy initiating protein. Beclin 1 is known to contain a nuclear trafficking signal. Blocking nuclear export function by Leptomycin B GSK2593074A resulted in nuclear accumulation of all LC3 and Beclin-1 proteins, while Ivermectin that blocks nuclear import showed reduction of accumulation, but not in all cell lines. Since endogenous LC3 proteins are used as major markers of autophagy in clinical studies and cell lines, it is essential to check the specificity of the antibodies used, as the kinetics of these molecules are not identical and may have distinct biological roles. The distinct subcellular expression patterns of LC3s provide a basis for further studies. == Introduction == Autophagy is a major LAMC1 intracellular pathway for the degradation and recycling of long-lived proteins and entire organelles [1,2]. LC3s (MAP1-LC3s) are structural proteins of autophagosomal membranes. The human LC3 gene family has three members, LC3A, LC3B and LC3C, whilst two variants of the LC3A protein have been identified [3,4]. The human LC3 gene family has three members the LC3A, LC3B and LC3C while in a recent paper has been reported five members, LC3A (variant-1, variant-2), LC3B, LC3B2 and LC3C [4]. The form LC3-II is one of the main components of the autophagosome membrane (LC3A-II and LC3B-II, also LC3C-II but not studied here) that resides in both the inner and outer site of the membrane. The LC3-II is derived from a proLC3 ~30KDa protein after cleavage by autophagin Atg4 to produce the active cytosolic form LC3-I. This in its turn is activated by Atg7, GSK2593074A and then transferred to Atg3, a second E2-like enzyme, becoming a membrane-bound form, LC3-II [5]. After autophagosome formation, the LC3-II located in the outer site is released to the cytosol and the LC3-II located in the inner site is degraded GSK2593074A by hydrolases [6]. In this latter form, LC3-II localizes on the spherical autophagosomal and autolysosomal membranes, forming a suitable marker of autophagic activity [6,7]. Whether these three LC3 proteins have a similar biological role in autophagy or other pathways remains obscure. In the literature, most studies focus on an overall LC3 expression, without reporting on the specificity of antibodies used, based on the arbitrary assumption that A and B forms are equivalent. In the current study we examine, after extensively validating antibody specificity, the expression in parallel of the three LC3A, B and C proteins in human cancer cell lines, showing distinct patterns of sub-cellular localization, suggesting a distinct biological role of these sister proteins. == Results == == Identification of LC3A and B specific antibodies == The specificity of the antibodies tested against to the commercial available human recombinant proteins LC3A and LC3B is shown inFig 1A. The anti-LC3A antibodies (ap1805a and ab62720) are specific to the protein LC3A and the anti-LC3B antibodies (5F10 and NB100-2220) are specific to the protein LC3B. The L8918, NB600-1384 and L7543 can bind either to the LC3A or LC3B, but with different sensitivity, while the ab52628 failed to detect either of the two isoforms GSK2593074A under the same conditions. == 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.