Fig. 4

Sanger sequencing reveals highly efficient RELA KO in primary human polydactyly chondrocytes, and expression of NF-κB-dependent inflammatory pathways is lower in KO cells. A Editing efficiency of five different sgRNAs targeting the RELA gene at different locations. B Off-target effects of the best-performing sgRNAs #1, #2 and #5 at the top five in silico-predicted most probable off-target sites. Data are represented as mean ± standard deviation of 3 biological replicates from three donors (n = 3). Statistical significance was determined using a nonparametric one-way ANOVA (* p < 0.05, ** p < 0.01, and **** p < 0.0001). C RT-qPCR of selected inflammatory pathways acting downstream of NF-κB in WT and KO cells following stimulation with IL-1β for 16 h. D Western blot and quantification of RELA in WT and KO cells treated with IL-1β for 30 min compared to housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein levels. RT-qPCR data are represented as mean ± standard deviation of 3 technical replicates from three biological donors (n = 9). Western Blot data are represented as mean ± standard deviation of 3 biological replicates from three donors (n = 3). Statistical significance was determined using a nonparametric Mann–Whitney t-test