Nucleotide depletion reveals the impaired ribosomebiogenesis checkpoint as a barrier against DNA damage

Many oncogenes enhance nucleotide usage to increase ribosome content, DNA replication, and cell proliferation, but in parallel trigger p53 activation. Both the impaired ribosome biogenesis checkpoint (IRBC) and the DNA damage response (DDR) have been implicated in p53 activation following nucleotide depletion. However, it is difficult to reconcile the two checkpoints operating together, as the IRBC induces p21‐mediated G1 arrest, whereas the DDR requires that cells enter S phase. Gradual inhibition of inosine monophosphate dehydrogenase (IMPDH), an enzyme required for de novo GMP synthesis, reveals a hierarchical organization of these two checkpoints. We find that the IRBC is the primary nucleotide sensor, but increased IMPDH inhibition leads to p21 degradation, compromising IRBC‐mediated G1 arrest and allowing S phase entry and DDR activation. Disruption of the IRBC alone is sufficient to elicit the DDR, which is strongly enhanced by IMPDH inhibition, suggesting that the IRBC acts as a barrier against genomic instability

Analysis of autophagic vesicles in mitotic cells

The detection of autophagic vesicles in interphase cells is well characterized with markers such as LC3, SQSTM1 (also known as p62) and LAMP2, which are commonly used in immunofluorescence and biochemistry assays to evaluate the status of autophagy in adherent cells. During mitosis, cells undergo important morphological changes which alter the position of the central plane, therefore the imaging on dividing cells has to be specifically designed. Here, we describe a method to label and image autophagic vesicles in mitotic cells to systematically analyze their number, morphology and distribution