Fig. 5: Expression of aberrant ZNF217 induced large phenotypic changes in B cell lymphoma cell lines and interfered with oncogenic signaling pathways.

ZNF217^KO and ZNF217^WT single cell clones were analyzed in cell-based functional assays. a Baseline growth characteristics of RC-K8 (KO: n = 28 and WT: n = 19) and L-428 (KO n = 11 and WT n = 9) were determined by longitudinal WST-1 viability assays after 6 h of serum starvation. Raw data of growth curves shows median and interquartile range of merged replicates of n ≥ 3 independent experiments. Growth curves were fitted with an exponential (Mathusian) growth model and extra sum-of-squares F test was used to compare proliferation/viability. b Bulk cells were transfected with guide RNA and Cas9 and longitudinally analyzed in n = 3 independent experiments for their relative proportion of genotypes by Sanger sequencing. Time points indicate weeks after transfection. Friedman-Test and Dunn’s post test were used, bars show mean (SD). c ZNF217^KO and ZNF217^WT cells were analyzed for their migratory potential in a Boyden Chamber assay after 24 h. Median and 95% CI of means of biological replicates (total n: RC-K8: WT: n = 46, KO: n = 67, L-428: WT: n = 18, KO: n = 22) of n ≥ 3 independent experiments are shown. Statistical significance was determined by Mann–Whitney-Test. d, e Determination of apoptotic/dead cell content in passaging of ZNF217^KO (n = 14) versus ZNF217^WT (n = 8) clones using Annexin-V- and DAPI-based flow cytometry, and cell cycle analysis (WT: n = 14, KO: n = 28). Upper panel shows representative flow cytometry density plots of ZNF217^WT and ZNF217^KO examples of RC-K8. Means of n ≥ 3 independent experiments were merged and error bars show SD. f Proliferation of ZNF217^KO (n = 6) and ZNF217^WT (n = 4) clones upon stimulation in duplicates with an agonist anti-CD40 antibody, IL-4, and IL-21. One representative experiment of three independent ones (total n: KO: n = 14) and WT: n = 16. Bars show median and interquartile range (Welch’s ANOVA test with Dunett’s T3 multiple comparisons test). g–i ZNF217^KO (n = 9) and ZNF217^WT (n = 10) clones were treated in triplicates with anti-CD40 antibody, IL-4, and IL-21. Control cells were moved to control media after 48 h and viability, apoptosis, and cytotoxicity were measured at the indicated time points after removal. Merged data of three independent experiments, bars show median and interquartile range (Kruskal–Wallis test, Dunn’s multiple comparisons test). q indicated by *<0.05; **<0.01, ***<0.001, ****<0.0001.