Figure 5. Regulation of cancer cell proliferation by cilia-localized Jouberin.
a. BrdU staining (red) in MEFs transfected with GFP tagged wild-type or ΔRVxP mutant construct (green). Hoechst labels nuclei (blue). Arrows point to GFP positive transfected cells. Quantification of the percent of 200 counted GFP positive cells which stained positive for BrdU is shown at right. **P<0.0005, Chi-square test. b. Staining for cilia in DLD1 cells reveals a lack of cilia. Cilia markers include GT335 (red), Arl13b (green), while Hoechst labels nuclei. c. Western blot analysis of Jbn and Ki67 expression from whole cell lysates of DLD1 cells transfected with Jbn siRNAs. Alpha-tubulin is shown as the loading control. d. BrdU staining (red) of DLD1 cells transfected with Jbn siRNA and treated for 16 hr with BrdU. Hoechst labels nuclei (blue). Quantification of 3000 cells from images acquired and analyzed identically is shown at the right. ***P<10−5, Chi-square test. e. β-catenin staining (green) in DLD1 cells transfected with Jbn siRNA. Hoechst labels nuclei (blue). Arrows point to nuclear β-catenin staining. Quantification of average nuclear staining intensity for β-catenin from over 100 cells in three regions of the well is shown at the bottom. *P<0.05, Student’s t-test. Error bars represent S.E.M. f. Cilia staining using two markers: acetylated tubulin (red) and glutamylated tubulin (green) in human colon CCD-841 cells which exhibited cilia in 75% of cells (over 200 cells examined) whereas DLD1 cells completely lacked cilia. Hoechst (blue) labels nuclei.