A blockade of lysosomal degra dation with NH4Cl resulted in increased levels of both LC3I and LC3II to levels that were similar between mitotic and interphase cells. From this we concluded in a previous report that basal levels of autophagy and mitophagy are robust in both interphase and mitotic cells and most autophagosomes that form during the entire cell cycle are efficiently kinase inhibitor Volasertib degraded through the lysosomal pathway. Treatment with nocodazole and paclitaxel caused dif ferent responses between interphase and mitotic cells. Treatment with either paclitaxel or nocodazole in inter phase cells caused a slight increase in LC3I levels and no change in LC3II levels in the absence of NH4Cl. However, there was no change in both LC3I and LC3II levels in the presence of NH4Cl.

The treatments resulted in a 3 fold increase of LC3I levels, but no change of LC3II levels in mitotic cells. Accumulation of LC3I suggested either an increased synthesis of LC3I through mechanisms related to tran scription, post transcription or translation of LC3 pre cursor and conversion to LC3I or reduced conversion of LC3I to LC3II. The levels of LC3I in interphase cells were slightly increased while the levels of LC3I in mitosis were dra matically enhanced upon paclitaxel or nocodazole treat ment. Although we cannot completely exclude the possibilities that more LC3 mRNA molecules were transcribed or more LC3 I proteins were translated and processed, we believed that such possibilities were unlikely since both mRNA transcription and protein translation are gener ally suppressed in mitosis.

The reduction in total LC3II in either the paclitaxel or the nocodazole arrested mito tic cells relative to control mitotic cells in the presence of NH4Cl further suggested a reduction in net conversion of LC3I to LC3II. Even though punctate foci appeared in more than 16% of paclitaxel treated mitotic cells, no difference in LC3II levels was evident. Thus, the paclitaxel induced GFP LC3 punctate foci are likely made up of aggregates of primarily LC3I. Others using ATG5 deficient cells have also suggested that that punc tate foci containing LC3 do not always represent mature autophagic structures. The ATG5 gene controls the conversion of LC3I to LC3II and its deletion causes accumulation of LC3I. Thus localized accumulation of LC3I on mitochondrial aggregates appears as punctate foci that are less than mature autophagosomes.

We sug gest that the generation of those punctate foci reflect autophagic failure at the initiation stage rather than autophagy independent aggregation. In sum mary, although both paclitaxel and nocodazole impaired the conversion of LC3I to LC3II resulting in accumula tion of LC3I, only in mitotic cells did paclitaxel cause the accumulation of Carfilzomib LC3I in aggregates.