Fig. 2: Osimertinib induces vacuolization and paraptosis-like cell death in GBM cells.

A LN-229 and U87MG cells were treated with vehicle or varying concentrations of Osimertinib for 24 h. Immunoblotting (IB) analysis for Caspase-3, PARP, and GAPDH, 5 μM Dox-treatment as a positive control. B LN-229 and U87MG cells were treated with vehicle or 5 μM Osimertinib for varying time. IB analysis for Caspase-3, PARP, and GAPDH, 5 μM Dox-treatment as a positive control. C LN-229 and U87MG cells were treated as in B. Cell morphology was examined by phase-contrast microscopy in C, (scale bar = 20 μm). The numbers of vacuolated and non-vacuolated cells were counted manually, and the ratio of vacuolated cells was calculated and shown as mean ± SD, n = 6; ***p < 0.001. D Analysis of the morphological changes in Osimertinib-treated cells by electron microscopy Electron microscopy was performed in LN-229 cells treated with 5 μM Osimertinib or DMSO for 24 h. Red arrows indicate dilated mitochondria and green arrows indicate dilated ER. Bar, 1 μm. E LN-229 and U87MG cells were exposed to Osimertinib (5 μM) in the absence or presence of CHX (10 μM), Z-VAD-FMK (5 μM), BafA1 (2.5 μM) for 24 h. Cell morphology was examined by phase-contrast microscopy (scale bar = 20 μm). The numbers of vacuolated and non-vacuolated cells were counted manually, and the ratio of vacuolated cells was calculated and shown as mean ± SD, n = 6; **p < 0.01, ***p < 0.001. F LN-229 and U87MG cells were exposed to Osimertinib (5 μM) in the absence or presence of CHX (10 μM) for 24 h. Cell lysates were analyzed by IB using the indicated antibodies. G LN-229 and U87MG cells were exposed to Osimertinib (5 μM) in the absence or presence of Z-VAD-FMK (5 μM), BafA1 (2.5 μM) for 24 h. Cell lysates were analyzed by IB using the indicated antibodies. H LN-229 and U87MG cells were treated with vehicle or 5 μM Osimertiinb for the indicated times and cell lysates were analyzed by IB using the indicated antibodies. I LN-229 and U87MG cells were vehicle-treated or treated with varying concentrations of Osimertinib (2.5, 5, and 10 μM) for 24 h. Cell lysates were analyzed by immunoblotting (IB) using the indicated antibodies. J LN-229 and U87MG cells were treated the same as in G, protein levels of CHOP, Bip, and GAPDH were analyzed by IB. All experiments in this figure were performed three times with comparable results.