Volume 40, Issue 4, Pages 533-547 (November 2010) Primary Cilium-Dependent and -Independent Hedgehog Signaling Inhibits p16INK4A  Cleo L. Bishop, Ann-Marie H. Bergin, Delphine Fessart, Viola Borgdorff, Elizabeth Hatzimasoura, James C. Garbe, Martha R. Stampfer, Jim Koh, David H. Beach  Molecular Cell  Volume 40, Issue 4, Pages 533-547 (November 2010) DOI: 10.1016/j.molcel.2010.10.027 Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 1 High-Content Genome-wide siRNA Screen for Modulators of p16 (A) Color coding used to illustrate the number of SD of the experimental siRNA value from the siGLO median. This color-coding system is employed for each phenotypic criterion and is used to define the S (green) and R hits (orange). (B–D) Heat maps of SD scores as described in (A) for each phenotypic criterion following transfection of HMECs with A-pool, Q-pool, Q1, Q2, or Q3 siRNAs for (B) the S and (C) the R hits and (D) the supergrower (SG) hits. A brief function is assigned to each S and R hit (see Experimental Procedures). (E) HMECs stained with DAPI (blue) and αp16 (green) following transfection with control siRNAs (CBX7, siGLO, p16) or A-pool siRNAs targeting representative S (WNT3A, CDH22) and R hits (MOBKL1A, GPR147). Size bar, 200 μm. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 2 Interaction Map and A/Q Analysis (A) Interaction map for Anchor Set (green, orange, yellow) and Set B interactors (white, purple). p16 regulators from Set B are highlighted (purple). Lines represent interactions determined from the BioGrid and HPRD databases. (B and C) Heat maps of SD scores for each phenotypic criterion following transfection of HMECs with A-pool, Q-pool, Q1, Q2, or Q3 siRNAs targeting Set B interactors. Candidates are divided based on the phenotypic criteria into (B) S or (C) R hits and a brief function is assigned to each candidate. (D) HMECs stained with DAPI (blue) and αp16 (green) following transfection with A-pool siRNAs targeting APP, β-catenin, or CBX4. Size bar, 200 μm. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 3 A/Q siRNA Analysis of Selected Hedgehog Pathway Members (A and B) HMECs stained with DAPI (blue) and αp16 (green) following transfection with (A) A-pool siRNAs targeting siGLO, IHH, SHH, FU, or SUFU and (B) A-pool, Q1, Q2, or Q3 siRNAs targeting GSK-3β or treatment with GSK-3β inhibitor XV. Top inset: Heat map of SD scores for each phenotypic criteria following transfection with A-pool, Q-pool, Q1, Q2, or Q3 siRNAs (see Key). Bottom inset: mean mRNA level for (A) SUFU and p16 following transfection with A-pool siRNAs targeting SUFU and (B) GSK-3β and p16 following transfection with A-pool, Q1, Q2 or Q3 siRNAs targeting GSK-3β. Values were normalized to the internal GAPDH control and expressed relative to siGLO control levels (100%). (C) Selected Hh pathway members illustrated with heat maps of SD scores for each phenotypic criteria following transfection with A-pool, Q-pool, Q1, Q2, or Q3 siRNAs. Members of the pathway that localize to the PC are highlighted (pink). Size bar, 200 μm. Error bars = +SD. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 4 Hedgehog and p16 (A) HMEC.pBabe, HMEC.IHH, and HMEC.SHH cultures stained with DAPI (blue) and αp16 (green) after 27 days of culture. (B) Cumulative population doublings (PDs) of HMEC cultures stably transduced with HMEC.pBabe (pBabe), HMEC.IHH (IHH), or HMEC.SHH (SHH) constructs, as a function of time. Arrow indicates time point of images shown in (A). (C) HMECs stained with DAPI (blue) and αp16 (green) either untreated for following treatment with 100 ng/mL SHH-N ligand, 25 ng/mL Wnt3a ligand, or SHH-N and Wnt3a ligand together. (D) Frequency distributions of p16 intensity in HMECs at P6 (gray) or following 24 hr treatment with 100 ng/mL SHH-N ligand (blue), 25 ng/mL Wnt3a ligand (purple), or SHH-N and Wnt3a ligand together (green). Size bar, 200 μm. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 5 Regulation of p16 by GLI2 (A) HMECs stained with DAPI (blue) and αp16 (green) following transfection with A-pool siRNAs targeting GLI1, GLI2 or GLI3. Inset: Heat map of SD scores for each phenotypic criteria following transfection with A-pool, Q-pool, Q1, Q2, or Q3 siRNAs. Size bar, 200 μm. (B) Top panel: Western blot analysis using αGLI2-N antibody of protein extracts from HMECs at P6 and P10 (left) and HMEC.pBabe, HMEC.IHH, and HMEC.SHH (right). Full-length and processed forms of GLI2 were detected (arrows). Bottom panel: Loading control. (C) Sequence alignment of the mouse and human p16 genomic sequence. The putative GLI-binding sites are highlighted (red). (D) Full-length GLI2 protein illustrating the N-terminal repressor domain (R), Zinc fingers (Zinc), and C-terminal activator domains (A). The epitopes for the αGLI2-N and αGLI2-C antibodies are shown. GLI2-FL and GLI2-ΔC reporter genes were used in the cotransfection assay shown in (E). (E) Mean relative luciferase activity (RLA) following co-transfection of the GLI2-FL or GLI2-ΔC constructs at the indicated dose with the p16-luciferase construct. (F) αGLI2-N and αGLI2-C ChIP in HMEC cells. The mean relative levels obtained with each antibody for the putative p16 GLI-binding region are indicated (pink). The Ptch GLI-binding region and sequences flanking the putative p16 GLI-binding site (gray) were used as positive and negative controls, respectively. PCR signals were normalized to input and are shown relative to the positive Ptch control. ND = not detectable, error bars = +SD. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 6 Relationship between GLI2 and p16 (A) HMECs stained with DAPI (blue), αp16 (green), and αGLI2-N (red). (B) HMECs stained with DAPI (blue), αp16 (green), and αGLI2-C (red). (C) Contour plots of total p16 nuclear intensity versus total GLI2-N nuclear intensity in individual cells at P6 (left) and P10 (right). (D) Left panel: Mean cell number for HMEC P6 (gray) or HMEC.p16shRNA (purple) following treatment with a range of cyclopamine doses. Error bars = ± SD. Right panel: HMECs stained with DAPI (blue) and αp16 (green) following treatment with 10 μM cyclopamine. (E) Frequency distributions of p16 intensity (left) or GLI2-N nuclear intensity (right) following treatment of HMEC P6 with 0 μM (gray), 7.5 μM (orange), or 10 μM cyclopamine. (F) Frequency distribution of GLI2-N nuclear intensity following treatment of HMEC.p16shRNA with 0 μM (gray), 10 μM (blue, left), 50 μM (blue, middle), 75 μM (orange), or 100 μM (red) cyclopamine. Size bar, 100 μm. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions

Figure 7 Relationship between Primary Cilium and p16 (A) Confocal images of HMECs stained with αIHH (green), αAcTb (red), and DAPI (blue). (B) Mean percentage of PC+ cells +SD in HMECs at P6, P6 cultured to confluence and P10. (C) Left panels: HMECs stained with αSMO (red), αAcTb (green), and DAPI (blue). Right panel: Mean percentage of SMO+ PCs in HMEC cultures at P6. (D) Left panel: HMECs stained with αAcTb (green) αKi67 (red) and DAPI (blue). Right panel: Mean percentage of Ki67+ cells in PC+ population of HMECs at P6. (E) Mean percentage of PC+ cells following transfection of HMECs with control siRNA, p16 siRNA, or in HMEC.p16shRNA stable cell lines. (F) Frequency distribution of p16 intensity in HMECs at P6 for the whole population (gray), PC+ cells (pink) and cells transfected with p16 siRNA (orange). Arrows indicate a PC. (G–I) Mean percentage of PC+ cells in (G) HMECs or HMEC.p16shRNA cultured with 0 μM, 10 μM, or 100 μM cyclopamine (ND = not done); (H) HMEC.p16shRNA versus HMEC.p16shRNA cultured to confluence; and (I) HMEC or HMEC.IHH following transfection with siGLO (gray) or p16 siRNA (orange). Size bar, 10 μm. Error bars = +SD. Molecular Cell 2010 40, 533-547DOI: (10.1016/j.molcel.2010.10.027) Copyright © 2010 Elsevier Inc. Terms and Conditions