Fig. 6: Control and ciliary mutant cells show differences in Wnt signalling.

a Representative immunofluorescent images of nuclear Cyclin D1 (green) staining in control and ciliary mutant KM cells (Phalloidin (cell membrane), DAPI (nuclei)). b Quantification of Cyclin D1 intensity inside the nucleus showed significantly stronger intensities inside the ciliary mutant cells, indicating an upregulation of Wnt signalling (_NWT = 389 cells; _NBbs4^−/− = 330 cells, _NBbs6^−/− = 428 cells; two-sided Mann–Whitney U test pWT vs. Bbs4^−/− = 4.19E-27; pWT vs. Bbs6^−/− = 1.09E-15; pBbs4^−/− vs. Bbs6^−/− = 0.02). c Representative western blot image of Cyclin D1 in control and ciliary mutant KM cells. The experiment was done four times. d Quantification of Cyclin D1 relative band intensity identified a significant difference between control and Bbs6^−/− KM cells (N = 4; unpaired t test pWT vs. Bbs6^−/− = 0.046, Data are presented as mean values + /− SEM), but no difference between both ciliary mutants or WT and Bbs4^−/− cells (N = 4; unpaired t test pBbs4^−/− vs. Bbs6^−/− = 0.767; pWT vs. Bbs6^−/− = 0.056, Data are presented as mean values + /− SEM). e, f Possible schematic representation of EV shedding from control and ciliary mutant cells and its effect on target cells. e Ciliary mutant cells release more MVB-derived EVs with different content from the plasma membrane. Mutant cells exhibit higher levels of WNT signalling (figure created by V.Kretschmer). f Application of the same number of EVs on target cells leads to a Wnt dampening effect. Since less Bbs4^−/− and Bbs6^−/− EVs are taken up than control EVs, it suggests that an individual Bbs4^−/− or Bbs6^−/− EV exerts a stronger Wnt dampening effect (figure created by V.Kretschmer). b Boxplots show median, interquartile range, and maximum and minimum within 1.5 interquartile range. n.s., P > 0.05, *P ≤ 0.05, **P < 0.01, ***P < 0.001.