Fig. 2: Phosphorylation-deficient PA28γ-T23 knock-in mice are resistant to 4NQO-induced HNSCC in vivo, whereas the phosphomimetic mutation enhances susceptibility.

a Schematic diagram of gRNA target sequences and partial donor oligo sequences in CRISPR/Cas-mediated genome engineering for generating the C57BL/6 mouse model with a point mutation (T23A or T23D) at the PA28γ gene locus. The gRNA specifically targets the exon 2 region of the PA28γ gene, which encodes T23. The donor oligo introduced the T23A (ACA to GCA) or T23D (ACA to GAC) mutation into exon 2 by homology-directed repair. In addition, a synonymous mutation R21 (CGG to CGC) was introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. b Sanger sequencing results derived from genomic DNA of PA28γ_T23A and PA28γ_T23D mice. c Experimental schematic illustrating the strategy for inducing HNSCC in WT, PA28γ_T23A, and PA28γ_T23D mice (n = 16/group) using the carcinogen 4NQO. d Representative images of tongue lesions in 4NQO-induced HNSCC mouse models at week 24. The dotted circles indicate macroscopically prominent lesions. e The number (Left) and volume (Right) of macroscopic prominent lesions in the tongues were quantified in week 24 of the 4NQO-induced HNSCC experiment. Data shown represent the mean ± SD for n = 16 mice; statistical significance was assessed by two-sided unpaired t-test. f Survival analysis using the Kaplan-Meier method on over 10% body weight loss in WT, PA28γ_T23A, and PA28γ_T23D mice during weeks 16–24 of the 4NQO-induced HNSCC experiment. Statistical significance was assessed by the log-rank (Mantel-Cox) test. g Representative H&E staining of severe dysplasia or carcinoma in-situ, microinvasive carcinoma, and invasive carcinoma in tongue lesions (Left). Scale bars, 100 μm. The tongue lesions of three mouse groups were evaluated and compared (Right). Statistical significance was assessed by two-sided Fisher’s exact test. Source data are provided as a Source Data file.