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. Author manuscript; available in PMC: 2017 Jan 1.
Published in final edited form as: J Med Genet. 2015 Oct 21;53(1):62–72. doi: 10.1136/jmedgenet-2015-103250

Figure 3. Mks1 knockdown impairs ciliogenesis in 3D spheroid culture of IMCD3 cells.

Figure 3

(A) Immunostaining of spheroids for cilia (acetylated tubulin, green), tight junctions (ZO1, white), and adherens junctions (β-catenin, red) with DAPI counterstaining (blue) shows loss of cilia after Mks1 siRNA transfection, and rescue by MKS1-WT. (B) Quantification of ciliary frequency in spheroids shows significant differences between control spheroids and spheroids depleted for MKS1 (indicates p<0.0003), and a potential dominant negative effect of transfection with MKS1-p.D19Y (p<0.001). Complete rescue of ciliary frequency was obtained upon transfection with MKS1-WT or MKS1-p.S403L (p<0.01), and a partial rescue upon transfection with MKS1-p.P218S (p<0.06). 50 spheroids were scored per condition. Data was normalized to IMCD3 cells transfected with siControl and empty vector, which was set to 1. Error bars represent SEM (n = 3 experiments), (C) Immunoblot for MKS1 in IMCD3 lysates (siControl) transfected with different MKS1 alleles. Upper band indicates equal endogenous levels of MKS1 in IMCD3 cells. Lower band indicates different MKS1 alleles (not full-length human MKS1 construct). β-actin is used as loading control.