Journal: bioRxiv

Article Title: Nucleotide insufficiency induced by p53 deficiency leads to replication stress driving genomic instability

doi: 10.1101/2025.04.17.649416

Figure Lengend Snippet: a. Left - Representative images of EU (red) and DAPI (Blue) staining, in scr and shp53 cells. Right - quantification of nuclear EU intensity per cell in Scr (101±40; n=383) and shp53 cells (173±76; n=398). EU distribution is divided to tertiaries. The data are representative of three independent experiments with similar results. Statistical analysis was performed using the student’s t-test. b. Left - Representative images of γH2AX (green), EU (red), nuclei (blue) in the same nucleus. Right – Quantification of γH2AX foci in scr (mean foci/cells 1.9; n=383), shp53 (4.1; n=398) and in each tertial of shp53 distribution as shown in A. The mean foci per nucleus in the low (0.5; n=132), mid (3.0; n=132), high (8.7; n=133) tertial. c. Transcription levels quantified using nuclear EU intensity and normalized to scr mean for scr cells (1±0.3; n=708), shp53 cells (1.37±0.3; n=731) and shp53 cells treated with 10-15 μM DRB for 48 hours (1.1±0.4; n=392) or treated with 10ng/ml ActD for 3 hours (1±0.1; n=304). The data are representative of two independent experiments with similar results. Statistical analysis was performed using one-Way Anova test. d. Percentage of nuclei with the indicated number of γH2AX foci for Scr (mean foci/cells 1.9; n=791), shp53 (4.4; n=831), shp53 + 10-15 μM DRB for 48 hours (1.6; n=696) and shp53 + 10ng/ml ActD for 3 hours (1.9; n=769). The data are a summary of two independent experiments. Statistical analysis was performed using Mann Whitney rank-sum test done on the distribution. e. Percentage of cells with the indicated number of micronuclei with the same conditions as d, number of analyzed cells per condition; Scr (n=1126), shp53 (n=1076), shp53 + 10-15 μM DRB for 48 hours (n=945) and shp53 + 10ng/ml ActD for 3 hours (n=934). The Chi-Square test of Independence was performed to compare the distribution of cells with micronuclei between each condition. f. Heatmap representation of log fold changes (log FC) in metabolite levels analyzed using LC-MC in scr, shp53 and shp53 cells treated with 50 μM AUGC for 48 hours, 15 μM DRB for 48 hours or 10ng/ml ActD for 3 hours. The comparisons are shown in the columns (appear at the top of the panel) and include six biological repeats for each condition. In all comparisons the same shp53 data was used. Green - increased metabolite levels compared to the indicated condition. Red - decreased metabolite levels compared to the indicated condition. P-value for each comparison calculated using one-sided t-test and indicated in each box (*p < 0.05, **p < 0.01, the value <0.1 is indicated). g. Hypertranscription fold-change (re-analysis of the TCGA cohort from the study by Zatzman et al.) shown for two groups: WT TP53/MDM2 (mean ± SEM: 2 ± 0.04, n=2273) and TP53-/MDM2+ (mean ± SEM: 2.33 ± 0.03, n=4032). Each circle represents a tumor sample, with the total number of samples per group indicated on the plot. Data are presented as mean ± SEM. Statistical significance was assessed using a non-parametric t-test (Mann-Whitney test) to compare two independent groups, as the data were not normally distributed. P-values less than 0.05 were considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001, **p < 0.0001).

Article Snippet: Finally, slides were mounted using VECTASHIELD Vibrance Antifade Mounting Medium with DAPI (Vector Laboratories, #H-1800).

Techniques: Staining, MANN-WHITNEY, Comparison