FoxN1 Regulation of Thymocyte p53 Signaling

FoxN1^flox mice were injected intraperitoneally with TM (2 mg/mouse/day) for 4 successive days.^{28 (link)} On the fourth day after the last TM injection, the mice were killed for flow cytometry assay of p-p53 in TAp63⁺ population. The thymi were torn apart in PBS to release thymocytes, and dissociated by incubation through three enzyme cycles (Collagenase-V/DNase-I) to enrich TECs.^{25 (link), 47 (link)} The single cell suspension of thymic cells was stained with combinations of fluorochrome-conjugated antibodies against cell surface markers: anti-mouse-PE/Cy5-CD45 and PE-MHC-II (M5/114) (BioLegend). Cells were then fixed with 2% PFA/PBS, permeabilized with 0.1% TritonX-100, and intracellularly stained for TAp63 with D-20 antibody (goat), followed by incubation with APC-anti-goat IgG, and then further intracellularly stained with p-p53 antibody (Ser-15, rabbit, Cell Signaling Technology Inc., Cat no. 12571). FoxN1 cDNA vector-injected aged thymi were also subjected to flow cytometry assay to analyze proliferation using intracellular staining of ΔNp63 (BioLegend, no. 619001) and Ki67 (BioLegend, clone 16A8). Data were acquired using a BD LSRII Flow Cytometer (BD Bioscience, San Jose, CA, USA) and analyzed using FlowJo software (FlowJo Home: Tree Star, Inc., Ashland, OR, USA).