Fig 2: The tumor suppressor protein p53 regulates G1 duration in response to inherited DNA lesions from the previous S-phase. (A) U-2 OS cells were transfected with siRNAs and exposed to ATRi as indicated. QIBC-derived one-dimensional cell cycle profiles based on total DAPI intensities are shown. (B) QIBC-derived two-dimensional cell cycle profiles based on Cyclin A / DAPI co-staining of U-2 OS cells are shown. Cell cycle staging was performed according to the indicated color code and percentages of cells in individual cell cycle phases are shown on the right. (C) 53BP1 foci were quantified in U-2 OS cells and are depicted as a function of the cell cycle. Note that loss of p53 leads to S-phase entry with elevated amounts of 53BP1 foci (red arrows). (D) Asynchronously growing FUCCI U-2 OS cells were transfected with siRNAs for 24 h and then exposed to ATRi as indicated. Cells were followed by time-lapse microscopy for 72 h at 30 minutes intervals and G1 duration was determined in individual cells.

Fig 3: P1C1TM mediates ADCC of target cells presenting high levels of p53125–134/A24 pMHCs. The ability of P1C1TM to mediate ADCC was assessed using a mutant p53-expressing HLA-A24+ colon carcinoma HT29 cells and b untransduced or HLA-A24-transduced MDA-MB-231. Target cells were incubated with a range of concentrations of P1C1TM and human PBMCs at an effector:target ratio of 15:1 and in the presence or absence of 10 µM exogenous p53125–134 peptides for 16 h. Cytotoxicity was determined by an LDH release assay. Comparison of P1C1TM and the mutant P1C1TM-LALA in their ability to c bind the target p53125–134/A24 pMHC and d mediate ADCC of HT29 cells was done. While the binding avidity between P1C1TM with wild-type Fc and the P1C1TM-LALA mutant with reduced binding to Fc receptor was identical, ADCC was significantly reduced with the P1C1TM-LALA. Data are mean of triplicates ± SEM. ADCC antibody-dependent cellular cytotoxicity.

Fig 4: Model for S-phase commitment in the context of inherited DNA lesions. (A) During unperturbed replication the small number of replication remnants and low amounts of inherited DNA lesions promotes a smooth transition through G1 and rapid S-phase entry. (B) Upon replication stress, an increase in unresolved replication remnants (e.g. stretches of unreplicated DNA) leads to an elevated number of 53BP1-marked inherited DNA lesions. This results in p53-dependent G1 lengthening or, if the damage load is too high, exit from the cell cycle. (C) In a p53-suppressed situation, cells experiencing replication stress enter the next S-phase prematurely with elevated levels of unresolved inherited genomic lesions. Upon oncogene activation during cancer development this may initiate a vicious cycle of damage propagation and accumulation of mutations.

Fig 5: Neural differentiation of JMS patient hiPSCs is impaired and accompanied by activation of p53 signaling(A) Representative bright-field images of neural differentiation of healthy control hiPSCs (WT-DYS0100 and WT-CRL(S)23) and two hiPSC clones from a JMS patient expressing HUWE1 p.G4310R (JMS-cl.1 and JMS-cl.2) at day 11; neural rosette structures are visible.(B) Relative number of rosettes formed in WT and JMS clones (n = 3, biological replicates).(C–F) qRT-PCR analysis of gene expression of OCT4 (C), NES/NESTIN (D), TUBB3/TUJ1 (E), and DCX (F) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates).(G) Immunofluorescence analysis of TUJ1 in WT and JMS hiPSCs at day 13 of neural differentiation.(H) Relative intensity of the TUJ1 signal in WT and JMS clones from experiments like the one in (G) (n = 2, biological replicates).(I–L) qRT-PCR analysis of mRNA levels of the p53 target genes CDKN1A/p21 (I), GADD45a (J), BBC3/PUMA (K), and BAX (L) in WT and JMS hiPSCs and neural cells (collected at day 13) (n = 3, biological replicates).All error bars indicate mean ± SEM; one-way ANOVA followed by Tukey post-test (B); two-way ANOVA followed by Tukey post-test (C–F and I–L); *p = 0.05, **p = 0.01, ***p = 0.001, ****p = 0.0001, n.s. = 0.05. Scale bar: 400 µm. See also Figures S2 and S3.