Fig. 2: Analysis of DNA recombination as a function of the TP53 variant.
a Work-flow of reporter-based recombination measurements. K562(HR3) cells with genomically integrated recombination reporter were electroporated with 10 µg expression plasmid for each TP53 variant. Cells were analyzed 72 h after electroporation using fluorescence-activated cell sorting (FACS) analysis. b FACS gating for recombination measurements. One million (mio.) living cells were first selected in the side scatter versus forward scatter (SSC/FSC) plot, followed by enumeration of EGFP+ cells among these living cells in a Coulter CytoFLEX B3-R1-V0 with APD detectors and GFP-oD1 bandpass filter using the autofluorescence PE/GFP-oD1 plot. Exemplary plots are shown for data obtained with mock- and EV-electroporated cells as well as after electroporation with TP53 WT expression plasmid. c Waterfall plot summarizing recombination results. Columns represent mean values and SEM of recombination frequencies, normalized as fold changes to the means of TP53 WT expressing samples from the same experimental day (mean WT: 3 × 10-5; N = 3-24, n = 8-141). Note that individual values of these measurements are not displayed here for clarity but can be found in the corresponding data presentation in Supplementary Fig. 2c. Relative recombination frequencies after expression of TP53 VUS (blue), functional B/LB control variants (green) and non-functional P/LP control variants (red) are presented. Green and red shaded areas mark functional and non-functional data ranges, leaving the range of intermediate functionality in between (white). Statistically significant differences were calculated by use of Kruskal-Wallis H test followed by Mann-Whitney U test, two-sided. Statistically significant differences (p < 0.0001) of the variant-specific mean fold changes as compared to WT and EV are marked by a and b, respectively. Precise p-values are listed in Supplementary Table 1.
