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: Irreversible cell cycle exit associated with senescence is mediated by constitutive MYC degradation

doi: 10.1016/j.celrep.2023.113079

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: CSII-pEF1a-H2B-mTurquoise, CSII-pEF1a-DHB (aa994–1087)-mVenus, CSII-pEF1a-mCherry-Geminin (aa1–110) were described previously., CSII-pEF1a-DHB (aa994–1087)-mCherry was described previously. pUltra-puro-RTTA2 was a gift from Yildirim Dogan and Kitai Kim (Addgene plasmid #58750).

Techniques: Recombinant, Luminex, Staining, Control, shRNA, Software, Imaging