Supplemental Information. Supernumerary Centrosomes. Nucleate Extra Cilia and Compromise. Primary Cilium Signaling. Current Biology, Volume 22
|
|
- Brittany Flynn
- 6 years ago
- Views:
Transcription
1 Current Biology, Volume 22 Supplemental Information Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling Moe R. Mahjoub and Tim Stearns Supplemental Inventory 1. Supplemental Figures and Tables Figure S1, related to Figure 1 Figure S2, related to Figure 1 Tables S1A and S1B, related to Figure 1A 2. Supplemental Experimental Procedures 3. Supplemental References
2
3 Figure S1. Related to Figure 1 (A) Immunofluorescence images of stably-expressed Htr6-GFP (green) in mono- and bi-ciliated IMCD-3 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 5 µm. Graph (right panel) shows ratio of Htr6-GFP/glutamylated tubulin levels in cilia of mono-, and bi-ciliated cells. (B) Immunofluorescence images of ciliary PKHD-GFP (green) in mono-and bi-ciliated IMCD-3 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows ratio of PKHD-GFP/glutamylated tubulin levels in cilia of mono-, and biciliated cells. (C) Immunofluorescence images of PKHD-GFP (green) in mono-ciliated IMCD-3 cells, showing total cellular levels of the fusion protein. Cells were co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows distribution of total PKHD1-GFP signal intensity in cells with one and two cilia. (D) Immunofluorescence images of Htr6-GFP (green) in mono-ciliated IMCD-3 cells, showing total cellular levels of the fusion protein. Cells were co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) showing distribution of total Htr6- GFP signal intensity in cells with one and two cilia. (E) Distribution of ciliary levels of PKHD1-GFP (per unit length cilium) compared with total (whole-cell) PKHD1-GFP signal intensity in cells. (F) Distribution of ciliary levels of HTr6-GFP (per unit length cilium) compared with total (whole-cell) HTr6-GFP signal intensity in cells. For all graphs, N = 50 for each sample (from 2 independent experiments; *** indicates P < ).
4
5 Figure S2. Related to Figure 1 (A) Immunofluorescence images of endogenous Arl13b (green) in mono- and bi-ciliated RPE-1 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 10 µm. Graph (right panel) shows ratio of endogenous Arl13b/glutamylated tubulin levels in cilia of mono- and bi-ciliated cells. (B) Immunofluorescence images of endogenous Arl13b (green) in mono-ciliated RPE-1 cells, containing either 2 or >4 centrioles. Cells were co-stained with glutamylated tubulin (G.T., red). Scale bar, 2 µm. Graph (right panel) shows levels of endogenous Arl13b per unit length, in mono-ciliated wild-type and super-centrosomal cells (N = 25 for each sample). (C) Immunofluorescence images of endogenous IFT88 (green) in mono- and bi-ciliated RPE-1 cells, co-stained with glutamylated tubulin (G.T., red) and DNA (blue). Scale bar, 5 µm. Graph (right panel) shows ratio of IFT88/glutamylated tubulin levels in cilia of mono- and bi-ciliated cells. (D) Immunofluorescence images of endogenous Smo in mono-ciliated cells, compared to biciliated bi-nucleate MEFs generated by blocking cytokinesis. Cells were co-stained with glutamylated tubulin (G.T.) and DAPI. Scale bar, 10 µm. Graph (right panel) shows the ratio of Smo/glutamylated tubulin levels in these cells. For all graphs, N = 50 for each sample (from 2 independent experiments; *** indicates P < ).
6 Tables S1A and S1B. Related to Figure 1A (A) Frequency of ciliogenesis over time. Centriole amplification was achieved by briefly inducing expression of Plk4 in RPE-1 cells as described in the Methods section. Cells were then grown for 1, 2 or 3 additional days to allow centriole maturation. To induce ciliogenesis, cells were incubated with low-serum (0.5%) medium for 24 h, and the number of cilia per cell was determined for each time point (N = 200 for each sample, from 2 independent experiments). (B) Fraction of centrioles forming cilia over time. Super-ciliated cells were also analyzed on the indicated days for the number of centrioles per cell, the fraction of centrioles that form cilia per cell, and the fraction of mature centrioles per cell. (N = 50, from 2 independent experiments).
7 Supplemental Results Generality of the Ciliary Dilution Phenotype in Superciliated Cells We addressed the generality and nature of the dilution phenotype in super-ciliated cells by examining the ciliary concentrations of two additional receptors that localize to cilia. The serotonin 6 (Htr6) protein is a G-protein-coupled receptor that localizes to the ciliary membrane in neurons [1], and in mouse kidney epithelial (IMCD) cells expressing it from an introduced construct [2]. IMCD-3 cells stably-expressing Htr6-GFP were transfected with Plk4 to produce super-ciliated cells, and the amount of Htr6-GFP per unit length of cilium was determined. As for Smo, the ciliary concentration of Htr6-GFP was reduced in bi-ciliated cells compared to mono-ciliated cells (Supplementary Figure S1A). The same dilution phenotype was also observed in IMCD-3 cells stably-expressing the ciliary-targeted cytoplasmic tail of the fibrocystin protein ( CTS PKHD1-GFP; Supplementary Figure S1B; [3]). The difference in ciliary concentration in mono- and bi-ciliated cells is not due to a difference in the expression level of the introduced protein, as quantification of whole-cell fluorescence values showed a similar range in both types of cells (Supplementary Figure S1C-D). Because there was substantial variation in expression, we could also address whether the ciliary dilution phenotype might be due simply to limiting amounts of the transported receptor protein. For both CTS PKHD1-GFP and Htr6-GFP, the amount of cilium-localized protein in mono-ciliated cells became saturated relative to the whole cell protein, and remained constant over a three-fold concentration range in the whole cell (Supplementary Figure S1E-F). Thus, the dilution of membrane-associated proteins in super-ciliated cells is true for several membrane proteins, and is not due to limitation of the total concentration of the transported protein. We next tested whether the super-ciliated dilution phenotype was restricted to membraneassociated proteins, or was common to other components of cilia. We examined the ciliary concentration of Arl13b, a small GTPase that localizes to the ciliary matrix (the compartment between the membrane and the axoneme) and is mutated in patients with ciliopathies [4]. Expression of Plk4 was induced in human retinal pigment epithelial (RPE-1) cells to generate super-ciliated cells, and the ciliary concentration of endogenous Arl13b was assessed. Similar to the membrane proteins, the ciliary concentration of Arl13b was reduced in bi-ciliated cells compared to mono-ciliated cells (Supplementary Figure S2A), demonstrating that proteins localized within the ciliary matrix may also be diluted in super-ciliated cells. We note that for each of the above proteins, Smo, Htr6, PKHD and Arl13b, the total amount of ciliary protein was similar in mono- and bi-ciliated cells, and that the dilution phenotype is due to partitioning of that protein over two cilia. To determine whether the ciliary dilution phenotype was due to the presence of extra cilia, rather than extra centrioles, we compared normal cells (containing 2 centrioles and one cilium) with cells bearing extra centrioles, but only one cilium. The ciliary concentration of endogenous Arl13b was identical in both cases (Supplementary Figure S2B), indicating that the dilution phenotype is due specifically to the presence of extra cilia. Finally, since super-ciliated cells assembled cilia of similar length to those in monociliated cells, we reasoned that components of the ciliary machinery might not display the dilution phenotype we have described for molecules involved in ciliary signaling. We examined the ciliary concentration of IFT88, a conserved component of the IFT machinery that is essential for the assembly of cilia from Chlamydomonas to mammals [5]. Consistent with this hypothesis,
8 the ciliary concentration of IFT88 was equal in mono- and bi-ciliated cells (Supplementary Figure S2C). In the above experiments the number of cilia per cell was manipulated while keeping ploidy constant. We considered the possibility that the ratio of cilia to genome might determine the amount of transported signaling molecules, and thus examined cells in which both cilium number and ploidy were increased two-fold by blocking cytokinesis. NIH3T3 cells were enriched in S-phase by a single thymidine block for 16 h, released and allowed to progress to mitosis. The cells were treated with cytochalasin B for 1.5 h to block cytokinesis, and released into low-serum medium to induce ciliogenesis, then treated with Shh and assessed for ciliary Smo as described above. In this case, bi-nucleate, bi-ciliated cells were compared with mononucleate, mono-ciliated cells in the same population, which had presumably not undergone mitosis during the cytochalasin treatment. Remarkably, the bi-nucleate bi-ciliated cells displayed the same Smo dilution phenotype with respect to the mono-nucleate mono-ciliated cells (Supplementary Figure S2D). This suggests that the mechanism determining ciliary protein levels assesses the number of cilia per cell, rather than cilia per unit genome. Supplemental Experimental Procedures Cell Culture and Media Cultured mammalian cells including MEFs, NIH3T3, NIH3T3::Gli-GFP (gift from James Chen, Stanford, CA), RPE-1::Tet-Plk4 (gift from Bryan Tsou, Sloan-Kettering, NY), Tsc2 +/+ and Tsc2 - /- MEFs (gift from Elizabeth Henske, Harvard Medical School, MA) were grown in DMEM with 10% FBS (Invitrogen). IMCD-3, IMCD-3::Htr6-GFP and IMCD-3:: CTSPKHD1-GFP (gift from Max Nachury, Stanford, CA) cells were grown in DMEM/F12 medium with 10% FBS. Cilium formation was induced by incubating cells in low-serum medium (0.5% serum) for 24 h. All media were supplemented with 100 U/ml penicillin, and 100 µg/ml streptomycin (Invitrogen). For spheroid formation assays, IMCD-3 cells were trypsinized, washed with PBS and resuspended in DMEM/F12 supplemented with 2% FBS and 2% growth factor-deleted Matrigel (BD Bioscience). Lab-Tek chamber slides (8-well) were treated with 40 µl of 100% Matrigel and incubated at 37 C for 15 min to allow the Matrigel to solidify. Approximately 5,000 cells/well were layered over the bed of Matrigel and grown at 37 C for 4 days to form spheroids. Electron Microscopy For TEM analysis, RPE-1::Tet-Plk4 cells were grown on Aclar strips, and centriole amplification/ciliogenesis was induced as described above. Cells were fixed with PBS containing 4% paraformaldehyde and 2% glutaraldehyde for 5 h, post fixed in 1% osmium tetroxide (Electron Microscopy Systems) for 1 h at room temperature, washed 3X with filtered water, then en-bloc stained for 2 h at room temperature (or incubated at 4 o C overnight). The cells were then dehydrated in a series of 50%, 70%, and 95% ethanol washes for 5 minutes each at 4 o C, allowed to rise to room temperature, washed twice with 100% ethanol, followed by incubation in acetonitrile for 15 min. Samples were infiltrated with a 1:1 mixture of acetonitrile:embed-812 resin (Electron Microscopy Systems), followed by 1:2 mixture, then EMbed-812, with each incubation lasting 2 h. Polymerization was achieved by incubating the samples at 65 o C overnight. 90 nm thin-sections were collected on formvar/carbon coated slot grids or 100 mesh
9 Cu grids (Electron Microscopy Systems), contrast stained for 30 seconds in 1:1 saturated uranyl acetate (~7.7%) to 100% acetone followed by 30 seconds of staining in 0.2% lead citrate. Grids were imaged in a JEOL JEM-1400 TEM at 120 kv and images taken using a Gatan Orius digital camera. Supplemental References 1. Hamon, M., Doucet, E., Lefevre, K., Miquel, M.C., Lanfumey, L., Insausti, R., Frechilla, D., Del Rio, J., and Verge, D. (1999). Antibodies and antisense oligonucleotide for probing the distribution and putative functions of central 5-HT6 receptors. Neuropsychopharmacology 21, 68S-76S. 2. Berbari, N.F., Johnson, A.D., Lewis, J.S., Askwith, C.C., and Mykytyn, K. (2008). Identification of ciliary localization sequences within the third intracellular loop of G protein-coupled receptors. Mol Biol Cell 19, Follit, J.A., Li, L., Vucica, Y., and Pazour, G.J. (2010). The cytoplasmic tail of fibrocystin contains a ciliary targeting sequence. J Cell Biol 188, Lim, Y.S., Chua, C.E., and Tang, B.L. (2011). Rabs and other small GTPases in ciliary transport. Biol Cell 103, Pazour, G.J., Dickert, B.L., Vucica, Y., Seeley, E.S., Rosenbaum, J.L., Witman, G.B., and Cole, D.G. (2000). Chlamydomonas IFT88 and its mouse homologue, polycystic kidney disease gene tg737, are required for assembly of cilia and flagella. J Cell Biol 151,
Report. Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary Cilium Signaling
Current Biology 22, 1628 1634, September 11, 2012 ª2012 Elsevier Ltd All rights reserved http://dx.doi.org/10.1016/j.cub.2012.06.057 Supernumerary Centrosomes Nucleate Extra Cilia and Compromise Primary
More informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb2988 Supplementary Figure 1 Kif7 L130P encodes a stable protein that does not localize to cilia tips. (a) Immunoblot with KIF7 antibody in cell lysates of wild-type, Kif7 L130P and Kif7
More informationSUPPLEMENTARY INFORMATION
DOI: 1.138/ncb222 / b. WB anti- WB anti- ulin Mitotic index (%) 14 1 6 2 T (h) 32 48-1 1 2 3 4 6-1 4 16 22 28 3 33 e. 6 4 2 Time (min) 1-6- 11-1 > 1 % cells Figure S1 depletion leads to mitotic defects
More informationPhosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling
Developmental Cell Supplemental Information Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling Francesc R. Garcia-Gonzalo, Siew Cheng Phua, Elle C. Roberson, Galo Garcia
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION doi:10.1038/nature12107 Supplementary Figure 1. CLARITY preserves GFP and TdTomato signals. (a) 3D rendering of a 1mm-thick Thy1-EGFP M line mouse brain block processed by CLARITY
More informationSupplementary Figure 1
Supplementary Figure 1 AAV-GFP injection in the MEC of the mouse brain C57Bl/6 mice at 4 months of age were injected with AAV-GFP into the MEC and sacrificed at 7 days post injection (dpi). (a) Brains
More informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb3076 Supplementary Figure 1 btrcp targets Cep68 for degradation during mitosis. a) Cep68 immunofluorescence in interphase and metaphase. U-2OS cells were transfected with control sirna
More informationFixation methods can differentially affect ciliary protein immunolabeling
DOI 10.1186/s13630-017-0045-9 Cilia METHODOLOGY Open Access Fixation methods can differentially affect ciliary protein immunolabeling Kiet Hua 1 and Russell J. Ferland 1,2* Abstract Background: Primary
More informationSupplementary Figure S1
Supplementary Figure S1 Supplementary Figure S1. PARP localization patterns using GFP-PARP and PARP-specific antibody libraries GFP-PARP localization in non-fixed (A) and formaldehyde fixed (B) GFP-PARPx
More informationNature Medicine doi: /nm.2860
Supplemental Figure Legends Supplemental Figure 1: Hypomorphic expression of IFT88 results in olfactory signaling proteins no longer localizing to the ciliary layer. (a) ACIII localizes to the cilia and
More informationSupplementary Information
Supplementary Information CEP41 is mutated in Joubert syndrome and is required for tubulin glutamylation at the cilium Ji Eun Lee, Jennifer L. Silhavy, Maha S. Zaki, Jana Schroth, Stephanie L. Bielas,
More informationSupplementary figures
Supplementary figures Supplementary Figure 1. B cells stimulated with pokeweed mitogen display normal mitotic figures but not cells infected with B95-8. The figures show cells stimulated with pokeweed
More informationA Role for Intraflagellar Transport Proteins in Mitosis: A Dissertation
University of Massachusetts Medical School escholarship@umms GSBS Dissertations and Theses Graduate School of Biomedical Sciences 6-18-2013 A Role for Intraflagellar Transport Proteins in Mitosis: A Dissertation
More information* * A3027. A4623 e A3507 A3507 A3507
a c L A327 d e A37 A37 A37 Supplementary Figure 1. Clinical manifestations of individuals with mutations. (a) Renal ultrasound of right kidney in A327 reveals small renal cysts, loss of corticomedullary
More informationSupplementary Figure 1.
Supplementary Figure 1. Visualization of endoplasmic reticulum-mitochondria interaction by in situ proximity ligation assay. A) Illustration of targeted proteins in mitochondria (M), endoplasmic reticulum
More informationSUPPLEMENTARY MATERIAL. Sample preparation for light microscopy
SUPPLEMENTARY MATERIAL Sample preparation for light microscopy To characterize the granulocytes and melanomacrophage centers, cross sections were prepared for light microscopy, as described in Material
More informationMechanistic Studies of Pentamidine Analogs on Leishmania donovani Promastigotes
Mechanistic Studies of Pentamidine Analogs on Leishmania donovani Promastigotes Undergraduate Honors Thesis The Ohio State University, College of Pharmacy Division of Medicinal Chemistry and Pharmacognosy
More informationPolycystic kidney disease (PKD) is one of the most common
Fast Track NIMA-Related Kinases Defective in Murine Models of Polycystic Kidney Diseases Localize to Primary Cilia and Centrosomes Moe R. Mahjoub, Melissa L. Trapp, and Lynne M. Quarmby Department of Molecular
More informationSupplemental Materials. STK16 regulates actin dynamics to control Golgi organization and cell cycle
Supplemental Materials STK16 regulates actin dynamics to control Golgi organization and cell cycle Juanjuan Liu 1,2,3, Xingxing Yang 1,3, Binhua Li 1, Junjun Wang 1,2, Wenchao Wang 1, Jing Liu 1, Qingsong
More informationSupplementary information. The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic. Spindle Orientation
Supplementary information The Light Intermediate Chain 2 Subpopulation of Dynein Regulates Mitotic Spindle Orientation Running title: Dynein LICs distribute mitotic functions. Sagar Mahale a, d, *, Megha
More informationWDR62 is associated with the spindle pole and mutated in human microcephaly
WDR62 is associated with the spindle pole and mutated in human microcephaly Adeline K. Nicholas, Maryam Khurshid, Julie Désir, Ofélia P. Carvalho, James J. Cox, Gemma Thornton, Rizwana Kausar, Muhammad
More informationSupplementary Figure Legends Supplementary Figure S1. Aurora-A is essential for SAC establishment in early mitosis. (a-c) RPE cells were treated with DMSO (a), MLN8237 (b) or BI2536 (c) for Two hours.
More information(A) PCR primers (arrows) designed to distinguish wild type (P1+P2), targeted (P1+P2) and excised (P1+P3)14-
1 Supplemental Figure Legends Figure S1. Mammary tumors of ErbB2 KI mice with 14-3-3σ ablation have elevated ErbB2 transcript levels and cell proliferation (A) PCR primers (arrows) designed to distinguish
More informationSupplementary Figure S1. Venn diagram analysis of mrna microarray data and mirna target analysis. (a) Western blot analysis of T lymphoblasts (CLS)
Supplementary Figure S1. Venn diagram analysis of mrna microarray data and mirna target analysis. (a) Western blot analysis of T lymphoblasts (CLS) and their exosomes (EXO) in resting (REST) and activated
More informationSUPPLEMENTARY LEGENDS...
TABLE OF CONTENTS SUPPLEMENTARY LEGENDS... 2 11 MOVIE S1... 2 FIGURE S1 LEGEND... 3 FIGURE S2 LEGEND... 4 FIGURE S3 LEGEND... 5 FIGURE S4 LEGEND... 6 FIGURE S5 LEGEND... 7 FIGURE S6 LEGEND... 8 FIGURE
More informationSUPPLEMENTAL EXPERIMENTAL PROCEDURES
SUPPLEMENTAL EXPERIMENTAL PROCEDURES Crystal violet assay Cells were seeded in 24-well plates and cultured in media supplemented with % FBS for 7 days. Media were then removed, plates were briefly washed
More informationSupporting Information
Supporting Information The Effects of Spacer Length and Composition on Aptamer-Mediated Cell-Specific Targeting with Nanoscale PEGylated Liposomal Doxorubicin Hang Xing +, [a] Ji Li +, [a] Weidong Xu,
More informationThe clathrin adaptor Numb regulates intestinal cholesterol. absorption through dynamic interaction with NPC1L1
The clathrin adaptor Numb regulates intestinal cholesterol absorption through dynamic interaction with NPC1L1 Pei-Shan Li 1, Zhen-Yan Fu 1,2, Ying-Yu Zhang 1, Jin-Hui Zhang 1, Chen-Qi Xu 1, Yi-Tong Ma
More informationSUPPLEMENTARY INFORMATION
sirna pool: Control Tetherin -HA-GFP HA-Tetherin -Tubulin Supplementary Figure S1. Knockdown of HA-tagged tetherin expression by tetherin specific sirnas. HeLa cells were cotransfected with plasmids expressing
More informationgenome edited transient transfection, CMV promoter
Supplementary Figure 1. In the absence of new protein translation, overexpressed caveolin-1-gfp is degraded faster than caveolin-1-gfp expressed from the endogenous caveolin 1 locus % loss of total caveolin-1-gfp
More informationSupplementary Figure 1. Gene schematics of hyls-1, gasr-8 and k10g6.4, and TEM analysis of TFs in WT and hyls-1 cilia. (a) Gene structure of hyls-1,
Supplementary Figure 1. Gene schematics of hyls-1, gasr-8 and k10g6.4, and TEM analysis of TFs in WT and hyls-1 cilia. (a) Gene structure of hyls-1, gasr-8 and k10g6.4 based on WormBase (http://wormbase.org),
More informationProgrammed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration
Programmed necrosis, not apoptosis, is a key mediator of cell loss and DAMP-mediated inflammation in dsrna-induced retinal degeneration The Harvard community has made this article openly available. Please
More informationSUPPLEMENT. Materials and methods
SUPPLEMENT Materials and methods Cell culture and reagents Cell media and reagents were from Invitrogen unless otherwise indicated. Antibiotics and Tet-certified serum were from Clontech. In experiments
More informationLow Cell Binding Property of LIPIDURE -COAT
Technical Note_1 ver.1 Low Cell Binding Property of LIPIDURE -COAT 1. LIPIDURE -COAT MULTI DISH A-6MD (Cat. No. 51011617) 2. Cell; NIH 3T3 (Fibroblast, mouse) 1. 10 %CS-DMEM; DMEM (Dulbecco's Modified
More informationSupplemental Information. Autophagy in Oncogenic K-Ras. Promotes Basal Extrusion. of Epithelial Cells by Degrading S1P. Current Biology, Volume 24
Current Biology, Volume 24 Supplemental Information Autophagy in Oncogenic K-Ras Promotes Basal Extrusion of Epithelial Cells by Degrading S1P Gloria Slattum, Yapeng Gu, Roger Sabbadini, and Jody Rosenblatt
More informationImpact of hyper-o-glcnacylation on apoptosis and NF-κB activity SUPPLEMENTARY METHODS
SUPPLEMENTARY METHODS 3D culture and cell proliferation- MiaPaCa-2 cell culture in 3D was performed as described previously (1). Briefly, 8-well glass chamber slides were evenly coated with 50 µl/well
More informationSupplementary Information for. Shi and King, Chromosome Nondisjunction Yields Tetraploid Rather than Aneuploid Cells in Human Cell Lines.
Supplementary Information for Shi and King, Chromosome Nondisjunction Yields Tetraploid Rather than Aneuploid Cells in Human Cell Lines Contains Supplementary Methods Supplementary Figures 1-7 Supplementary
More informationELLEN ROTER DIRKSEN. From the Cancer Research Institute, University of California San Francisco, San Francisco, California 94143
CILIOGENESIS IN THE MOUSE OVIDUCT A Scanning Electron Microscope Study ELLEN ROTER DIRKSEN. From the Cancer Research Institute, University of California San Francisco, San Francisco, California 94143 INTRODUCTION
More informationSupplementary Figure 1 Induction of cellular senescence and isolation of exosome. a to c, Pre-senescent primary normal human diploid fibroblasts
Supplementary Figure 1 Induction of cellular senescence and isolation of exosome. a to c, Pre-senescent primary normal human diploid fibroblasts (TIG-3 cells) were rendered senescent by either serial passage
More informationSupporting Information
Supporting Information Kim et al. 10.1073/pnas.0912180106 SI Materials and Methods DNA Constructs. The N-terminally-tagged mouse Gli2 expression construct, pcefl/3 HA-Gli2, was constructed by ligating
More information(a) Schematic diagram of the FS mutation of UVRAG in exon 8 containing the highly instable
Supplementary Figure 1. Frameshift (FS) mutation in UVRAG. (a) Schematic diagram of the FS mutation of UVRAG in exon 8 containing the highly instable A 10 DNA repeat, generating a premature stop codon
More informationProteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival
Supplementary Information for Proteomic profiling of small-molecule inhibitors reveals dispensability of MTH1 for cancer cell survival Tatsuro Kawamura 1, Makoto Kawatani 1, Makoto Muroi, Yasumitsu Kondoh,
More informationSUPPLEMENTARY INFORMATION
Supplementary Figures Supplementary Figure S1. Binding of full-length OGT and deletion mutants to PIP strips (Echelon Biosciences). Supplementary Figure S2. Binding of the OGT (919-1036) fragments with
More informationSupplemental Data Figure S1 Effect of TS2/4 and R6.5 antibodies on the kinetics of CD16.NK-92-mediated specific lysis of SKBR-3 target cells.
Supplemental Data Figure S1. Effect of TS2/4 and R6.5 antibodies on the kinetics of CD16.NK-92-mediated specific lysis of SKBR-3 target cells. (A) Specific lysis of IFN-γ-treated SKBR-3 cells in the absence
More informationCells and reagents. Synaptopodin knockdown (1) and dynamin knockdown (2)
Supplemental Methods Cells and reagents. Synaptopodin knockdown (1) and dynamin knockdown (2) podocytes were cultured as described previously. Staurosporine, angiotensin II and actinomycin D were all obtained
More informationLongitudinal tracking of single live cancer cells to understand cell cycle effects of the
Longitudinal tracking of single live cancer cells to understand cell cycle effects of the nuclear export inhibitor, selinexor Joshua M. Marcus 1, Russell T. Burke 1, John A. DeSisto 1, Yosef Landesman
More informationOncolytic Adenovirus Complexes Coated with Lipids and Calcium Phosphate for Cancer Gene Therapy
Oncolytic Adenovirus Complexes Coated with Lipids and Calcium Phosphate for Cancer Gene Therapy Jianhua Chen, Pei Gao, Sujing Yuan, Rongxin Li, Aimin Ni, Liang Chu, Li Ding, Ying Sun, Xin-Yuan Liu, Yourong
More informationModeling lymphangiogenesis in a three-dimensional culture system
Modeling lymphangiogenesis in a three-dimensional culture system Françoise Bruyère, Laurence Melen-Lamalle, Silvia Blacher, Guy Roland, Marc Thiry, Lieve Moons, Francis Frankenne, Peter Carmeliet, Kari
More informationValidation & Assay Performance Summary
Validation & Assay Performance Summary LanthaScreen IGF-1R GripTite Cells Cat. no. K1834 Modification Detected: Phosphorylation of Multiple Tyr Residues on IGF-1R LanthaScreen Cellular Assay Validation
More informationNature Medicine: doi: /nm.4322
1 2 3 4 5 6 7 8 9 10 11 Supplementary Figure 1. Predicted RNA structure of 3 UTR and sequence alignment of deleted nucleotides. (a) Predicted RNA secondary structure of ZIKV 3 UTR. The stem-loop structure
More informationEssential Medium, containing 10% fetal bovine serum, 100 U/ml penicillin and 100 µg/ml streptomycin. Huvec were cultured in
Supplemental data Methods Cell culture media formulations A-431 and U-87 MG cells were maintained in Dulbecco s Modified Eagle s Medium. FaDu cells were cultured in Eagle's Minimum Essential Medium, containing
More informationa" b" 2N c" d" e" f" !!Aurora!A!!!CP110!
DLD1/Reference a" 2N 2N 2N/DLD1 2N/ /DLD1 c" d" e" f" TargetID 2N.AVG_Sig 2N.Det Pval.AVG_Sig.Det Pval Diff Pval DiffScore SYMBOL ILMN_26396 18.35238 0.0080058 44.81118 0.0021834 0.000323 34.90542 KRTHA4
More informationSupplementary Information POLO-LIKE KINASE 1 FACILITATES LOSS OF PTEN-INDUCED PROSTATE CANCER FORMATION
Supplementary Information POLO-LIKE KINASE 1 FACILITATES LOSS OF PTEN-INDUCED PROSTATE CANCER FORMATION X. Shawn Liu 1, 3, Bing Song 2, 3, Bennett D. Elzey 3, 4, Timothy L. Ratliff 3, 4, Stephen F. Konieczny
More informationBone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by
Nakano et al. Supplementary information 1. Supplementary Figure 2. Methods 3. References Bone marrow-derived mesenchymal stem cells improve diabetes-induced cognitive impairment by exosome transfer into
More informationSUPPLEMENTARY INFORMATION
DOI:.38/ncb2822 a MTC02 FAO cells EEA1 b +/+ MEFs /DAPI -/- MEFs /DAPI -/- MEFs //DAPI c HEK 293 cells WCE N M C P AKT TBC1D7 Lamin A/C EEA1 VDAC d HeLa cells WCE N M C P AKT Lamin A/C EEA1 VDAC Figure
More informationSupplementary Figure 1 Expression of Crb3 in mouse sciatic nerve: biochemical analysis (a) Schematic of Crb3 isoforms, ERLI and CLPI, indicating the
Supplementary Figure 1 Expression of Crb3 in mouse sciatic nerve: biochemical analysis (a) Schematic of Crb3 isoforms, ERLI and CLPI, indicating the location of the transmembrane (TM), FRM binding (FB)
More informationSupplementary Figures
Inhibition of Pulmonary Anti Bacterial Defense by IFN γ During Recovery from Influenza Infection By Keer Sun and Dennis W. Metzger Supplementary Figures d a Ly6G Percentage survival f 1 75 5 1 25 1 5 1
More informationSupplementary Figure 1: si-craf but not si-braf sensitizes tumor cells to radiation.
Supplementary Figure 1: si-craf but not si-braf sensitizes tumor cells to radiation. (a) Embryonic fibroblasts isolated from wildtype (WT), BRAF -/-, or CRAF -/- mice were irradiated (6 Gy) and DNA damage
More informationT H E J O U R N A L O F C E L L B I O L O G Y
T H E J O U R N A L O F C E L L B I O L O G Y Supplemental material Krenn et al., http://www.jcb.org/cgi/content/full/jcb.201110013/dc1 Figure S1. Levels of expressed proteins and demonstration that C-terminal
More informationhexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This
SUPPLEMENTAL FIGURE LEGEND Fig. S1. Generation and characterization of. (A) Coomassie staining of soluble hexahistidine tagged GRP78 devoid of the KDEL motif (GRP78-His) on SDS-PAGE. This protein was expressed
More informationSupplementary Figures
Supplementary Figures Supplementary Figure 1 Increased ABHD5 expression in human colon cancer associated macrophages. (a) Murine peritoneal macrophages were treated with regular culture medium (Ctrl) or
More informationNature Structural and Molecular Biology: doi: /nsmb Supplementary Figure 1
Supplementary Figure 1 Mutational analysis of the SA2-Scc1 interaction in vitro and in human cells. (a) Autoradiograph (top) and Coomassie stained gel (bottom) of 35 S-labeled Myc-SA2 proteins (input)
More informationSupplementary Figure 1: GFAP positive nerves in patients with adenocarcinoma of
SUPPLEMENTARY FIGURES AND MOVIE LEGENDS Supplementary Figure 1: GFAP positive nerves in patients with adenocarcinoma of the pancreas. (A) Images of nerves stained for GFAP (green), S100 (red) and DAPI
More informationSUPPLEMENTARY INFORMATION. Supplementary Figures S1-S9. Supplementary Methods
SUPPLEMENTARY INFORMATION SUMO1 modification of PTEN regulates tumorigenesis by controlling its association with the plasma membrane Jian Huang 1,2#, Jie Yan 1,2#, Jian Zhang 3#, Shiguo Zhu 1, Yanli Wang
More informationsupplementary information
DOI: 10.1038/ncb2133 Figure S1 Actomyosin organisation in human squamous cell carcinoma. (a) Three examples of actomyosin organisation around the edges of squamous cell carcinoma biopsies are shown. Myosin
More informationInstructions. Fuse-It-Color. Overview. Specifications
Membrane fusion is a novel and highly superior method for incorporating various molecules and particles into mammalian cells. Cargo-specific liposomal carriers are able to attach and rapidly fuse with
More informationFigure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or
Figure S1. Western blot analysis of clathrin RNA interference in human DCs Human immature DCs were transfected with 100 nm Clathrin SMARTpool or control nontargeting sirnas. At 90 hr after transfection,
More informationSupplementary Materials for
www.sciencesignaling.org/cgi/content/full/6/283/ra57/dc1 Supplementary Materials for JNK3 Couples the Neuronal Stress Response to Inhibition of Secretory Trafficking Guang Yang,* Xun Zhou, Jingyan Zhu,
More informationSupplemental Information. 3D-CLEM Reveals that a Major Portion. of Mitotic Chromosomes Is Not Chromatin
Molecular Cell, Volume 64 Supplemental Information 3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin Daniel G. Booth, Alison J. Beckett, Oscar Molina, Itaru Samejima, Hiroshi
More informationVEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization
Cell Reports, Volume 9 Supplemental Information VEGFR2-Mediated Vascular Dilation as a Mechanism of VEGF-Induced Anemia and Bone Marrow Cell Mobilization Sharon Lim, Yin Zhang, Danfang Zhang, Fang Chen,
More informationSUPPLEMENTARY INFORMATION
DOI:.38/ncb3399 a b c d FSP DAPI 5mm mm 5mm 5mm e Correspond to melanoma in-situ Figure a DCT FSP- f MITF mm mm MlanaA melanoma in-situ DCT 5mm FSP- mm mm mm mm mm g melanoma in-situ MITF MlanaA mm mm
More informationwell for 2 h at rt. Each dot represents an individual mouse and bar is the mean ±
Supplementary data: Control DC Blimp-1 ko DC 8 6 4 2-2 IL-1β p=.5 medium 8 6 4 2 IL-2 Medium p=.16 8 6 4 2 IL-6 medium p=.3 5 4 3 2 1-1 medium IL-1 n.s. 25 2 15 1 5 IL-12(p7) p=.15 5 IFNγ p=.65 4 3 2 1
More informationFigure S1. (A) SDS-PAGE separation of GST-fusion proteins purified from E.coli BL21 strain is shown. An equal amount of GST-tag control, LRRK2 LRR
Figure S1. (A) SDS-PAGE separation of GST-fusion proteins purified from E.coli BL21 strain is shown. An equal amount of GST-tag control, LRRK2 LRR and LRRK2 WD40 GST fusion proteins (5 µg) were loaded
More informationLPS LPS P6 - + Supplementary Fig. 1.
P6 LPS - - - + + + - LPS + + - - P6 + Supplementary Fig. 1. Pharmacological inhibition of the JAK/STAT blocks LPS-induced HMGB1 nuclear translocation. RAW 267.4 cells were stimulated with LPS in the absence
More informationOptimization of the Fuse-It-mRNA Protocol for L929 Cells in the µ-plate 24 Well
Optimization of the Fuse-It-mRNA Protocol for L929 Cells in the µ-plate 24 Well 1. General Information... 1 2. Background... 1 3. Material and Equipment Required... 2 4. Experimental Procedure and Results...
More informationMesenchymal Stem Cells Reshape and Provoke Proliferation of Articular. State Key Laboratory of Bioreactor Engineering, East China University of
Mesenchymal Stem Cells Reshape and Provoke Proliferation of Articular Chondrocytes by Paracrine Secretion Lei Xu, Yuxi Wu, Zhimiao Xiong, Yan Zhou, Zhaoyang Ye *, Wen-Song Tan * State Key Laboratory of
More informationSUPPLEMENTARY FIGURES
SUPPLEMENTARY FIGURES 1 2 3 4 SUPPLEMENTARY TABLES Supplementary Table S1. Brain Tumors used in the study Code Tumor Classification Age Gender HuTuP51 Glioblastoma 57 Male HuTuP52 Glioblastoma 53 Male
More informationMetal swap between Zn 7 metallothionein 3 and amyloid β Cu protects against amyloid β toxicity
Metal swap between Zn 7 metallothionein 3 and amyloid β Cu protects against amyloid β toxicity Supplementary Information Gabriele Meloni 1, Vanessa Sonois 2,3, Tamara Delaine 2, Luc Guilloreau 2, Audrey
More informationTubulointerstitial Renal Disease. Anna Vinnikova, MD Division of Nephrology
Tubulointerstitial Renal Disease Anna Vinnikova, MD Division of Nephrology Part I: Cystic Renal Disease www.pathguy.com Simple cysts Simple cysts May be multiple Usually 1 5cm, may be bigger Translucent,
More informationSupplemental Information. The Hedgehog Pathway Effector Smoothened. Exhibits Signaling Competency in the Absence. of Ciliary Accumulation
Chemistry & Biology, Volume 21 Supplemental Information The edgehog Pathway Effector Exhibits Signaling Competency in the Absence of Ciliary Accumulation Chih-Wei Fan, Baozhi Chen, Irene Franco, Jianming
More information(a) Significant biological processes (upper panel) and disease biomarkers (lower panel)
Supplementary Figure 1. Functional enrichment analyses of secretomic proteins. (a) Significant biological processes (upper panel) and disease biomarkers (lower panel) 2 involved by hrab37-mediated secretory
More informationSupplementary Information for. Inhibitors of Hedgehog Acyltransferase Block Sonic Hedgehog Signaling. Marilyn D. Resh 1,2,6*
Supplementary Information for Inhibitors of Hedgehog Acyltransferase Block Sonic Hedgehog Signaling Elissaveta Petrova 1,5, Jessica Rios-Esteves 1,2, Ouathek Ouerfelli 3, J. Fraser Glickman 4, and Marilyn
More informationSUPPLEMENTARY FIGURES AND TABLES
SUPPLEMENTARY FIGURES AND TABLES Supplementary Figure S1: CaSR expression in neuroblastoma models. A. Proteins were isolated from three neuroblastoma cell lines and from the liver metastasis of a MYCN-non
More informationSupplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice.
Supplementary Figures: Supplementary Figure S I: Effects of D4F on body weight and serum lipids in apoe -/- mice. Male apoe -/- mice were fed a high-fat diet for 8 weeks, and given PBS (model group) or
More informationSupplementary Data. Different volumes of ethanol or calcium solution were slowly added through one of four
Supplementary Data METHODS Liposome preparation Different volumes of ethanol or calcium solution were slowly added through one of four methods: Method I, no ethanol or calcium solution; Method II, exactly
More informationInfluenza virus exploits tunneling nanotubes for cell-to-cell spread
Supplementary Information Influenza virus exploits tunneling nanotubes for cell-to-cell spread Amrita Kumar 1, Jin Hyang Kim 1, Priya Ranjan 1, Maureen G. Metcalfe 2, Weiping Cao 1, Margarita Mishina 1,
More informationklp-18 (RNAi) Control. supplementary information. starting strain: AV335 [emb-27(g48); GFP::histone; GFP::tubulin] bleach
DOI: 10.1038/ncb1891 A. starting strain: AV335 [emb-27(g48); GFP::histone; GFP::tubulin] bleach embryos let hatch overnight transfer to RNAi plates; incubate 5 days at 15 C RNAi food L1 worms adult worms
More informationSUPPLEMENTARY INFORMATION
DOI: 10.1038/ncb2294 Figure S1 Localization and function of cell wall polysaccharides in root hair cells. (a) Spinning-disk confocal sections of seven day-old A. thaliana seedlings stained with 0.1% S4B
More informationSupplementary Figure 1. Validation of astrocytes. Primary astrocytes were
Supplementary Figure 1. Validation of astrocytes. Primary astrocytes were separated from the glial cultures using a mild trypsinization protocol. Anti-glial fibrillary acidic protein (GFAP) immunofluorescent
More information(A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and a
Supplementary figure legends Supplementary Figure 1. Expression of Shh signaling components in a panel of gastric cancer. (A) RT-PCR for components of the Shh/Gli pathway in normal fetus cell (MRC-5) and
More informationSupporting Information
Supporting Information ou et al..73/pnas.08791112 dd Thymidine Release & transfection dd Thymidine Release dd MG132 Fix and IF -14 h 0 h 8 h 24 h 34 h 36 h siontrol simps1-1 simps1-1 simps1-1 simps1-2
More informationAnalysis of small RNAs from Drosophila Schneider cells using the Small RNA assay on the Agilent 2100 bioanalyzer. Application Note
Analysis of small RNAs from Drosophila Schneider cells using the Small RNA assay on the Agilent 2100 bioanalyzer Application Note Odile Sismeiro, Jean-Yves Coppée, Christophe Antoniewski, and Hélène Thomassin
More informationSUPPLEMENTARY FIGURE LEGENDS
SUPPLEMENTARY FIGURE LEGENDS Supplemental FIG. 1. Localization of myosin Vb in cultured neurons varies with maturation stage. A and B, localization of myosin Vb in cultured hippocampal neurons. A, in DIV
More informationFIG S1 Replication rates of S. suis strain 10, 10Δsly, and 10cpsΔEF on mono- and virus pre-infected porcine PCLS.
A strain 10 10cps EF B strain 10 H1N1 + strain 10 10cps EF H1N1 + 10cps EF 10 8 10 sly 10 7 H3N2 + strain 10 H3N2 + 10cps EF CFU/ml media 10 7 10 6 10 5 10 4 CFU/ml media 10 6 10 5 10 4 10 3 0 2 4 8 12
More informationT H E J O U R N A L O F C E L L B I O L O G Y
Supplemental material Jewell et al., http://www.jcb.org/cgi/content/full/jcb.201007176/dc1 T H E J O U R N A L O F C E L L B I O L O G Y Figure S1. IR Munc18c association is independent of IRS-1. (A and
More informationSpatial Control of Primary Ciliogenesis by Subdistal Appendages Alters Sensation-Associated Properties of Cilia
Article Spatial Control of Primary Ciliogenesis by Subdistal Appendages Alters Sensation-Associated Properties of Cilia Graphical Abstract Authors Gregory Mazo, Nadine Soplop, Won-Jing Wang, Kunihiro Uryu,
More informationSUPPLEMENTARY INFORMATION
SUPPLEMENTARY INFORMATION Advances in pancreatic islet monolayer culture on glass surfaces enable superresolution microscopy and insights into beta cell ciliogenesis and proliferation Edward A. Phelps,
More informationSupporting Information For
Supporting Information For MicroRNA-Catalyzed Cancer Therapeutics Based on DNA-Programmed Nanoparticle Complex Xucheng Luo, 1 Zhi Li, 1 Ganglin Wang, 1 Xuewen He, 2,3 Xiaoqin Shen, 1 Quanhong Sun, 1 Li
More informationAdditional methods appearing in the supplement are described in the Experimental Procedures section of the manuscript.
Supplemental Materials: I. Supplemental Methods II. Supplemental Figure Legends III. Supplemental Figures Supplemental Methods Cell Culture and Transfections for Wild Type and JNK1-/-,JNK2-/- MEFs: The
More informationSupplementary Figure 1: Neuregulin 1 increases the growth of mammary organoids compared to EGF. (a) Mammary epithelial cells were freshly isolated,
1 2 3 4 5 6 7 8 9 10 Supplementary Figure 1: Neuregulin 1 increases the growth of mammary organoids compared to EGF. (a) Mammary epithelial cells were freshly isolated, embedded in matrigel and exposed
More information