michael 445 davidsons  (Addgene inc)

Journal: bioRxiv

Article Title: CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

doi: 10.1101/2022.04.05.487175

Figure Lengend Snippet: a , Co-immunoprecipitation assay showing association of Pyo-KSR1 with endogenous TOLLIP in 293T cells. Quantitation of three experiments is shown on the right. b , Live cell imaging shows perinuclear localization of GFP-tagged TOLLIP in A549 lung ADC cells and pan-cytoplasmic distribution in a non-transformed bronchial epithelial cell line, HBEC. Relative nuclear proximity index (RNPI) measures perinuclear clustering of fluorescent signals in each cell (see Methods). Values are averages for A549 cells (n=12) and HBEC cells (n=12). c , IF staining of A549 cells demonstrates co-localization of TOLLIP with KSR1, CK2α, and RAB11A but not p-ERK. d , TOLLIP-EGFP shows partial co-localization with KSR1-mCherry and nearly complete overlap with CK2α-mCherry and RAB11A-tagRFP in A549 cells. e , Proximity ligation assays (PLA) were used to confirm the juxtaposition of TOLLIP with KSR1, CK2α, and KSR1 but not p-ERK in A549 cells. Bottom panel: PLA signal quantification, expressed as positive signals/cell. f , Fluorescently labeled KSR1, CK2α, TOLLIP and RAB11A are present on vesicles embedded within the Sec61β-GFP positive ER network in A549 cells. Scale bars, 10 μm. Statistical significance for quantitation of immunoprecipitation and RNPI was calculated using Student’s t test; * p ≤ 0.05.

Article Snippet: The lentiviral pUltra-hot vector was a gift from Malcolm Moore (Addgene plasmid #24130; http://n2t.net/addgene:24130 ; RRID: Addgene_24130). mEmerald tagged human RAB11A and RFP tagged human RAB11A were cloned into the Ultra-hot vector using Age1 and BamH1 cloning sites.

Techniques: Co-Immunoprecipitation Assay, Quantitation Assay, Live Cell Imaging, Transformation Assay, Staining, Ligation, Labeling, Immunoprecipitation

Journal: bioRxiv

Article Title: CK2 signaling from TOLLIP-dependent perinuclear endosomes is an essential feature of KRAS mutant cancers

doi: 10.1101/2022.04.05.487175

Figure Lengend Snippet: a , TOLLIP depletion in A549 cells causes pan-cytoplasmic dispersal of KSR1, CK2α and RAB11A. b , TOLLIP ablation impairs proliferation of A549 and PANC1 tumor cells. c , TOLLIP-depleted A549 cells display increased senescence (SA-βGal positivity) and apoptosis (cleaved CASPASE-3). d , TOLLIP silencing does not affect the growth rate of HBEC cells. e , f , RAB11A knockdown results in more uniform cytoplasmic distributions of TOLLIP, KSR1 and CK2α. Two-way ANOVA was used to analyze growth curves. Student’s t-test was performed for SA-βGal assay; * p ≤ 0.05.

Article Snippet: The lentiviral pUltra-hot vector was a gift from Malcolm Moore (Addgene plasmid #24130; http://n2t.net/addgene:24130 ; RRID: Addgene_24130). mEmerald tagged human RAB11A and RFP tagged human RAB11A were cloned into the Ultra-hot vector using Age1 and BamH1 cloning sites.

Techniques:

Journal: Cell reports

Article Title: Nanoscale Architecture of the Cortical Actin Cytoskeleton in Embryonic Stem Cells.

doi: 10.1016/j.celrep.2019.06.089

Figure Lengend Snippet: Figure 7. Mechanisms Regulating the Actin Cortex Architecture and Mechanics in mESCs (A) STORM images of mESCs overexpressing mDia1 CA-GFP (top) and WAVE2::mEmerald (bottom). Center: enlarged left insets. Right: filament orientation maps of the insets. Color bar, orientation angle. (B) Recruitment of myosin II into the cortex upon SMIFH2-induced network density increase. MyosinIIA::EGFP, green; LifeAct::mCherry, magenta. (C) Possible mechanisms for aster formation and cortical generation. 1a–4a: F-actin filaments in the mESC actin cortex (1a) could undergo mechanical or spontaneous breakage (2a), exposing free barbed ends that become tipped with ATP-actin (yellow) upon polymerization (3a) and, in turn, recruit additional factors, such as Arp2/3 (green), CP (purple), and formin (blue) (4a). 1b–3b: CP may displace formin from the barbed end (1b). The ATP-actin-enriched barbed end (2b) could promote recruitment of Arp2/3, CP, and formin, resulting in aster formation (3b). Asters likely depend on the balance among Arp2/3, CP, and formin (1c–3c). Although most filaments are sequestered by CP (1c and 2c), occasional filaments (presumably formin-bound) may be able to outgrow the aster zone (3c) to populate the rest of the cortex. (D) Phase diagram of the mESC actin cortex. Horizontal axis, network topological complexity control via formin and Arp2/3; vertical axis, filament extension control via formin and CP. The mESC actin cortex (center) results from a fine-tuned balance of Arp2/3, formin, and CP, with asters as transient intermediates. (legend continued on next page)

Article Snippet: N/A mEmerald-MyosinIIB-C-18 Addgene Addgene #54192,RRID:Addgene_54192 mEmerald-Integrin-Linked-Kinase Addgene Addgene #54126,RRID:Addgene_54126 mCherry-Lifeact Addgene Addgene #54491,RRID:Addgene_54491 mEmerald-Lifeact Addgene Addgene #54148,RRID:Addgene_54148 mEmerald-Capping Protein B Addgene Addgene #54027,RRID:Addgene_54027 mEmerald-Coronin1B Addgene Addgene #54049,RRID:Addgene_54049 mCherry-Arp2 Addgene Addgene #54979,RRID:Addgene_54979 mEmerald-ARPp34 Addgene Addgene #53997,RRID:Addgene_53997 mEmerald-FilaminA-N-9 Addgene Addgene #54098,RRID:Addgene_54098 mEmerald-WAVE2 Gift from the laboratory of Michael W. Davidson (The Florida State University) N/A mEmerald-WASH1 Gift from the laboratory of Michael W. Davidson (The Florida State University) N/A mEmerald-Rab5a-7 Addgene Addgene #54243,RRID:Addgene_54243 mEmerald-Rab7a-7 Addgene Addgene #54244,RRID:Addgene_54244 mEmerald-Rab11a-7 Addgene Addgene #54245,RRID:Addgene_54245 GFP-mDia1 CA Addgene Addgene #45583,RRID:Addgene_45583 EGFP-FP4-mito Gift from the laboratory of Alpha Yap (The University of Queensland, Australia) Scott et al., 2006 EGFP-AP4-mito Gift from the laboratory of Alpha Yap (The University of Queensland, Australia) Scott et al., 2006 Experimental Models: Cell Lines E14TG2a (E14) a gift from Dr. Cheng Gee Koh, Nanyang Technological University, Singapore, purchased from ATCC Cat#: ATCC CRL-1821, RRID:CVCL_C320 Oligonucleotides Oligonucleotides for siRNA Dharmacon see STAR Methods Software and Algorithms Fiji (ImageJ) NIH https://fiji.sc/, RRID:SCR_002285 GraphPad Prism 6 GraphPad Software, Inc. RRID:SCR_002798 OriginPro OriginLab RRID:SCR_014212 MATLAB Mathworks RRID:SCR_001622 IDL Harris Geospatial Solutions http://www.harrisgeospatial.com/ ProductsServices/IDL.aspx Aster Analyzer 1.0 The authors https://github.com/KanchanawongLab/ AsterAnalyzer Meshwork Analyzer 1.0 The authors https://github.com/KanchanawongLab/ MeshworkAnalyzer

Techniques: Control