RUCova removes unwanted covariance and allows improved classification of apoptotic cells. (A) Scheme of the correlation heatmap, where the lower and upper triangles show the Pearson correlation coefficients between marker values before and after RUCova (using the interaction model $M3(x→i,ji)$ per cell line), respectively. Diagonal unity values are depicted in grey. Pearson correlation coefficients were calculated between the asinh-transformed and z-scored expression values of markers. (B, C) Correlation heatmap with the upper triangle showing the Pearson correlation coefficients between marker values in the UPCISCC131 cell line across cells from 0 and 10 Gy after applying RUCova based on (B) PC1, (C) PC1–PC4. (D) Heatmap of fold changes of asinh-transformed data of measured markers between irradiated and non-irradiated condition for each cell line, and dataset (before or after applying RUCova’s interaction model). (E) Heatmap of percentual difference in the standard deviation $σ$ of each marker’s distribution after applying RUCova relative to the distributions before RUCova (⁠$Δσ=σRUCova−σbeforeσbefore·100$⁠) per cell line and dose levels. Arrows indicate specific cases where $Δσ>0$⁠. (F) Density plots for asinh-transformed expression values of IdU, p-γH2AX, p-p53, and cleaved Casp3 before and after RUCova (y-axis) in cell lines where $Δσ>0$ (arrows in panel E) for 0 Gy (first row) and 10 Gy (second row). (G) Density plot for asinh-transformed expression of cleaved Casp3 in the irradiated (10 Gy) UPCISCC131 cell line, before and after RUCova based on all four PCs. Dashed vertical lines indicate the decision thresholds for apoptotic (higher values of cleaved Casp3) and non-apoptotic populations according to the cleaved Casp3 distribution before and after applying RUCova. (H) Confusion matrix for classification of apoptotic cells in the irradiated UPCISCC131 cell line before and after RUCova (based on all four PCs) according to the decision thresholds in (G). (I) Scatter plots of PC1 vs. asinh-transformed values of cleaved Caspase-3 before (left) and after RUCova (right) for irradiated cells in the UPCISCC131 line. Cells are coloured by apoptotic status. (J, K) Boxplots of asinh-transformed expression of (J) p-γH2AX and (K) p-MEK1/2 before and after RUCova in irradiated UPCISCC131 cells according to their apoptotic status.