Modeling p53 missense mutations in GBM. A,Trp53^f/− astrocytes were isolated from the cortices of male and female postnatal day 1 mouse pups. The sex of the astrocytes was determined by genotyping PCR (left), and male or female astrocytes from at least 3 pups were combined. B, Astrocytes were stained for the astrocyte lineage marker GFAP to confirm astrocyte purity. Scale bar, 100 μm. C, To create mutant p53–expressing astrocytes, Trp53^f/− astrocytes were transduced with retrovirus to overexpress mutant Trp53-IRES-eGFP, and eGFP-positive cells were sorted by flow cytometry. Sorted eGFP-positive astrocytes were transduced with lentivirus to express Cre-IRES-Puro and selected with 2.5 μg/mL puromycin for 1 week. D, Western blot assay was performed using 50 μg of whole-cell lysates from male and female Trp53^f/− (WT), Trp53 KO, and Trp53^mut/− astrocytes. Trp53^f/− (WT) astrocytes were treated with 20 μg/mL etoposide and 10 μg/mL Nutlin-3a or DMSO control for 8 hours. Mutant p53 astrocytes stably express mutant p53 protein at high levels compared without the treatment needed to stabilize WT p53 protein. E, Loss of the lox-p53-lox (floxed) allele following introduction of Cre recombinase was confirmed by PCR. F, Treatment with Nutlin-3a and etoposide to stabilize p53 results in detectable p53 protein in p53 WT but not p53 KO astrocytes. Trp53^f/− astrocytes were treated with 20 μg/mL etoposide and 10 μg/mL Nutlin-3a or DMSO for 8 hours and were fixed and stained by IF for p53 protein. Scale bar, 200 μm G, IF for GFP (green) and p53 (red) p53 KO and mutant astrocytes. Scale bar, 200 μm.