Figure 1. Aurora kinase A inhibition induces cytotoxicity, apoptosis, DNA damage, and immunogenic cell death in human papillomavirus (HPV)-positive murine and human cancer cell lines in vitro. (A–C) Murine and human cancer cells were treated with 300 nmol/L alisertib for 48 hours before being subjected to lysis and immunoblotting with the indicated antibodies (A), annexin-FITC staining to measure apoptosis (B), or a CellTiter-Glo assay to measure cell viability (C). (D–E) Murine (D) and human (E) cells were treated with 300 nmol/L alisertib for 48 hours before immunoblotting with antibodies that indicate the presence of pyroptosis, DNA damage, and immunogenic cell death (ICD). To determine the amount of released HMGB-1 (D, E), cytochrome C (D), and lactate dehydrogenase (LDH) (E), equal volumes of conditioned medium were subjected to immunoblotting analysis; Ponceau S staining was used as a loading control. (F) Calreticulin (CRT) expression on the cell surface was analyzed by flow cytometry on murine and human cell lines treated with 300 nmol/L alisertib for 48 hours. The significance of differences was determined using an unpaired, two-tailed Student’s t-test. *p≤0.05, **p≤0.001, ***p≤0.0001. CL, cleaved; DMSO, dimethyl sulfoxide; FL, full; GDSME, gasdermin E; MFI, mean fluorescence intensity; UM-47, UMSCC47.