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The production of Adeno-associated virus (AAV) vectors in the lab setting has typically involved expression in adherent cells followed by purification through ultracentrifugation in density gradients. This production method is, however, not easily scalable, presents high levels of cellular impurities that co-purify with the virus, and results in a mixture of empty and full capsids. Here we describe a detailed AAV production protocol that overcomes these limitations through AAV expression in suspension cells followed by AAV affinity purification and AAV polishing to separate empty and full capsids, resulting in high yields of ultra-pure AAV that is highly enriched in full capsids., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Cellular senescence is a form of irreversible growth arrest that cancer cells evade. The cell division cycle protein 20 homolog (Cdc20) is a positive regulator of cell division, but how its dysregulation may relate to senescence is unclear. Here, we find that Cdc20 mRNA and protein expression are downregulated in stress-induced premature senescent lung fibroblasts in a p53-dependent manner. Either Cdc20 downregulation or inhibition of anaphase-promoting complex/cyclosome (APC/C) is sufficient to induce premature senescence in lung fibroblasts, while APC/C activation inhibits stress-induced premature senescence. Mechanistically, we show both Cdc20 downregulation and APC/C inhibition induce premature senescence through glycogen synthase kinase (GSK)-3β-mediated phosphorylation and downregulation of securin expression. Interestingly, we determined Cdc20 expression is upregulated in human lung adenocarcinoma. We find that downregulation of Cdc20 in non-small cell lung cancer (NSCLC) cells is sufficient to inhibit cell proliferation and growth in soft agar and to promote apoptosis, but not senescence, in a manner dependent on downregulation of securin following GSK-3β-mediated securin phosphorylation. Similarly, we demonstrate securin expression is downregulated and cell viability is inhibited in NSCLC cells following inhibition of APC/C. Furthermore, we show chemotherapeutic drugs downregulate both Cdc20 and securin protein expression in NSCLC cells. Either Cdc20 downregulation by siRNA or APC/C inhibition sensitize, while securin overexpression inhibits, chemotherapeutic drug-induced NSCLC cell death. Together, our findings provide evidence that Cdc20/APC/C/securin-dependent signaling is a key regulator of cell survival, and its disruption promotes premature senescence in normal lung cells and induces apoptosis in lung cancer cells that have bypassed the senescence barrier., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Oncogenic K-Ras (K-Ras G12V ) promotes senescence in normal cells but fuels transformation of cancer cells after the senescence barrier is bypassed. The mechanisms regulating this pleiotropic function of K-Ras remain to be fully established and bear high pathological significance. We find that K-Ras G12V activates the angiotensinogen (AGT) gene promoter and promotes AGT protein expression in a Kruppel-like factor 6-dependent manner in normal cells. We show that AGT is then converted to angiotensin II (Ang II) in a cell-autonomous manner by cellular proteases. We show that blockade of the Ang II receptor type 1 (AT 1 -R) in normal cells inhibits oncogene-induced senescence. We provide evidence that the oncogenic K-Ras-induced synthesis of Ang II and AT 1 -R activation promote senescence through caveolin-1-dependent and nicotinamide adenine dinucleotide phosphate oxidase 2-mediated oxidative stress. Interestingly, we find that expression of AGT remains elevated in lung cancer cells but in a Kruppel-like factor 6-independent and high-mobility group AT-hook 1-dependent manner. We show that Ang II-mediated activation of the AT 1 -R promotes cell proliferation and anchorage-independent growth of lung cancer cells through a STAT3-dependent pathway. Finally, we find that expression of AGT is elevated in lung tumors of K-Ras LA2-G12D mice, a mouse model of lung cancer, and human lung cancer. Treatment with the AT 1 -R antagonist losartan inhibits lung tumor formation in K-Ras LA2-G12D mice. Together, our data provide evidence of the existence of a novel cell-autonomous and pleiotropic Ang II-dependent signaling pathway through which oncogenic K-Ras promotes oncogene-induced senescence in normal cells while fueling transformation in cancer cells., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)