Experiments were repeated at least three times. hours. Error bars show SD. Scale pub = 10 m. Quantification at right demonstrates Wnt5a reduces the area of nucleoli. Image J software was used to compare the total part of AgNOR staining in equal numbers of cells. Error bars show SD. *P 0.05; (n = 3). (d) AgNOR staining of BT549 and MCF7 cells stably expressing exogenous Wnt5a. Level pub = 10 m. Quantification demonstrates cells expressing exogenous Wnt5a have a reduced nucleolar area. Error bars show SD. (BT549, *P 0.05) (n = 3). (e) MTT assay demonstrates BT549/Wnt5a cells proliferate more slowly Cilastatin sodium than BT549 vector control cells. Viable cell numbers were determined by MTT assay over successive days. Results Cilastatin sodium demonstrated are from 3 self-employed experiments in which data points were acquired in quadruplicate. *P 0.05 (n = 3).(TIF) pgen.1006217.s001.tif (2.4M) GUID:?A38C1490-1CCC-4AFC-8BFE-5AB45658C784 S2 Fig: DVL2 and DVL3 are excluded from nucleoli. (a) Immunofluorescence and confocal microscopy using antibodies to DVL1 (green) and Fibrillarin (reddish) merged with DNA (blue) in MCF7 cells and MCF7 cells stably expressing Wnt5a treated with ActD at 1000 ng/mL for 4 hours. (b) Immunofluorescence and confocal microscopy using antibodies to DVL2 (reddish) and UBF (green) in MCF7 cells and MCF7 cells stably expressing Wnt5a treated with vehicle or ActD at 40 ng/ml for 4 hours. Level pub, 10m (n = 3). Immunofluorescence and confocal microscopy using antibodies to DVL3 Rabbit Polyclonal to OR52E5 (green) and Fibrillarin (reddish) in MCF7 cells and MCF7 cells stably expressing Wnt5a treated with vehicle or ActD at 40 ng/ml for 4 hours. Level pub, 10m (n = 3).(TIF) pgen.1006217.s002.tif (7.0M) GUID:?EDF12EAF-2B93-4337-B1AB-B8196FB54930 S3 Fig: Nucleolar localization of DVL1. (a) Immunofluorescence with rabbit polyclonal antibody Cilastatin sodium for DVL1 (reddish) merged with DNA (blue) in MCF7 and MDA-MB-231 breast cancer cells. Level pub, 10 m. (n = 3). (b) Exogenous DVL1 ectopically indicated in Rat2 cells localizes to the nucleolus. Immunofluorescence of DVL1 (green) and Fibrillarin (reddish) and their co-localization (yellow, right) in Rat2 cells transduced having a FLAG-tagged DVL1 retrovirus (Rat2/DVL1) or control vector (Rat2/Ctrl). Exogenous DVL1 in Rat/DVL1 fibroblast cells was also recognized with FLAG antibody (green) and co-localized with Fibrillarin (reddish). Scale pub, 10 m. (n = 3). (c) Immuno-gold transmission electron micrographs of MCF7 cells stably expressing Wnt5a (MCF7/Wnt5a) and MDA-MB-231 cell nuclei, showing DVL1 within nucleoli (arrows). Small arrowheads in the top panel point to the nuclear envelope. Level pub, 500nm. All experiments were performed at least three times (n = 3), except immuno-EM which was performed twice. (d) Immunoblots of lysates of MCF7 cells stably expressing Wnt5a (MCF7/Wnt5a) display unaltered levels of DVL1, SIRT7 and UBF manifestation with respect to control MCF7 cells Cilastatin sodium (Ctrl) not expressing Wnt5a. GAPDH and Actin were used as loading settings. (n = 3).(TIF) pgen.1006217.s003.tif (4.7M) GUID:?20AB2752-DD3F-40EA-96E1-27A93940AEC3 S4 Fig: Knockdown of endogenous DVL1 does not affect DVL2 or DVL3 levels. (a) European blots showing specific reduction of DVL1 protein levels, but no switch in DVL2 or DVL3, in BT549 and MCF7 cells transduced with DVL1 shRNA (shDVL1) compared to non-silencing shRNA (Ctrl). Tubulin and GAPDH served as loading settings (n = 3). (b) Nucleofection of MCF7 cells with siRNA oligonucleotides reduces DVL1 RNA levels (top) and causes up-regulation of 47S pre-rRNA manifestation (bottom), confirming results acquired with shRNA-mediated silencing of DVL1 in Fig 4b. Error bars show SD (n = 3).(TIF) pgen.1006217.s004.tif (399K) GUID:?B64FB7B4-B1F6-4FEF-B488-71778617878F Data Availability StatementAll relevant data are within the paper and its Supporting Information documents. Abstract Ribosome biogenesis is essential for cell growth and proliferation and is commonly elevated in malignancy. Accordingly, several oncogene and tumor suppressor signaling pathways target rRNA synthesis. In breast tumor, non-canonical Wnt signaling by Wnt5a has been reported to antagonize tumor growth. Here, we display that Wnt5a rapidly represses rDNA gene transcription in breast tumor cells and produces a chromatin state with reduced transcription of rDNA by RNA polymerase I (Pol I). These effects were specifically dependent on Dishevelled1 (DVL1), which accumulates in nucleolar organizer areas (NORs) and binds to rDNA regions of the.