stable u2os flp  (DSMZ)

Journal: Molecular cancer research : MCR

Article Title: Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin-Independent Targets by Proteomics

doi: 10.1158/1541-7786.MCR-18-1154

Figure Lengend Snippet: MINK1 binds to and is negatively regulated by APC. A, co-IP of MINK1 with full-length, endogenous APC in HeLa and U2OS cells. B, co-IP of MINK1 with C-terminally truncated APC in Colo320 and SW480 colorectal cancer cells; both cell lines lack the WT allele. C, Changes in MINK1 proteins levels in response to siRNA-mediated depletion of APC and/or β-catenin measured by WB. Shown are means and SD relative to control samples from four independent transfections. Significance relative to control determined by two-way ANOVA followed by Dunnett multiple comparison test (*, P < 0.05; **, P < 0.01).

Article Snippet: U2OS Flp-In T-Rex MINK1-GFP/GFP Stable cell lines with tetracycline/doxycycline-inducible expression of MINK1-GFP/GFP-MINK1 and GFP, respectively, were generated using the Flp-In T-Rex System according to the manufacturer’s instructions (Thermo Fisher Scientific) by transfecting U2OS Flp-In T-Rex host cells with pcDNA5 FRT/TO C-GFP, pcDNA5 FRT/TO MINK1-GFP, or pcDNA5 FRT/TO GFP-MINK1, respectively, and pOG44, a constitutive Flp recombinase expression plasmid.

Techniques: Co-Immunoprecipitation Assay, Control, Transfection, Comparison

Journal: Molecular cancer research : MCR

Article Title: Identification of Endogenous Adenomatous Polyposis Coli Interaction Partners and β-Catenin-Independent Targets by Proteomics

doi: 10.1158/1541-7786.MCR-18-1154

Figure Lengend Snippet: MINK1 localizes to cell-cell junctions and its overexpression enhances cell adhesion. A, mRNA expression of MINK1, CTNNB1, and AXIN2 measured by qRT-PCR 48 and 72 hours after siRNA transfection. Indicated are mean expression levels relative to ACTB expression with SD from four independent transfections. Significance determined by one-way ANOVA followed by Dunnett multiple comparison test (*, P < 0.05; **, P < 0.01; ***, P < 0.001). Note, the same HeLa AXIN2 mRNA quantification data are also shown in Supplementary Fig. S4D. B, MINK1 protein levels in HeLa cells after treatment with neddylation inhibitor MLN4924 [3 mmol/L] as measured by WB. Shown are relative mean signals normalized to DMSO-treated samples with SD from three independent experiments. Significance determined by one-way ANOVA, **, P < 0.01. C, Live imaging of HeLa SEC-C cells expressing endogenously mNeonGreen-tagged MINK1. Scale bars, 10 μm. D, Adhesion assay with U2OS cells overexpressing MINK1-GFPand GFP, respectively. Adhesion to collagen matrix after 1 hour was quantified by staining of firmly attached cells with Crystal Violet. Indicated is mean absorbance with SD of independent experiments with two different GFP/MINK1-GFP clones and eight technical replicates/condition. Significance determined by two-way ANOVA followed by Sidak multiple comparison test (***, P < 0.0003). E, MTT proliferation assay in Colo320 cells treated with siRNA against β-catenin or MINK1. Shown is the mean absorbance from triplicate measurements. Significance relative to control determined by one-way ANOVA followed by Dunnett multiple comparison test (*, P < 0.05; **, P < 0.01). n.s., not significant.

Article Snippet: U2OS Flp-In T-Rex MINK1-GFP/GFP Stable cell lines with tetracycline/doxycycline-inducible expression of MINK1-GFP/GFP-MINK1 and GFP, respectively, were generated using the Flp-In T-Rex System according to the manufacturer’s instructions (Thermo Fisher Scientific) by transfecting U2OS Flp-In T-Rex host cells with pcDNA5 FRT/TO C-GFP, pcDNA5 FRT/TO MINK1-GFP, or pcDNA5 FRT/TO GFP-MINK1, respectively, and pOG44, a constitutive Flp recombinase expression plasmid.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, Transfection, Comparison, Imaging, Cell Adhesion Assay, Staining, Clone Assay, Proliferation Assay, Control

Journal: Communications Biology

Article Title: Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

doi: 10.1038/s42003-026-09832-3

Figure Lengend Snippet: a In vitro ADP-ribosylation assay. Recombinant His 6 -PARP15cat or His 6 -PARP15cat-H559Y proteins were incubated with radioactively labeled 32 P-NAD + for 30 min at 30 °C. Proteins were separated by SDS-PAGE and visualized by Coomassie blue (CB) staining. The incorporated radioactive label was visualized by exposure to X-ray films ( 32 P). b The expression of PARP15.1 fusion proteins in U2OS-tdTomato-G3BP1/GFP-PARP15 or U2OS-tdTomato-G3BP1/GFP-PARP15-H559Y was induced by increasing doxycycline concentrations as indicated. Protein expression was evaluated by immunoblotting 24 h post-induction. PARP15 fusion proteins were visualized using a GFP antibody. γ-Tubulin was detected as loading control. c U2OS-tdTomato-G3BP1/GFP-PARP15 or U2OS-tdTomato-G3BP1-GFP-PARP15-H559Y were treated with 0.5 µg/ml doxycycline for 16 h to induce expression of GFP-PARP15.1 variants. Cells were fixed, and PARP15.1 localization investigated by confocal microscopy. PARP15.1 and its catalytically inactive mutant (H559Y) are stained in green, G3BP1 in magenta. Nuclei were visualized by Hoechst staining (blue). Scale bar, 10 µM.

Article Snippet: U2OS-tdTomato-G3BP1 (provided by Paul Taylor, St. Jude Children’s Hospital, Memphis, USA) , U2OS-tdTomato-G3BP1/EGFPm-PARP15 (this study), U2OS (originally obtained from DSMZ, ACC 785) and stable U2OS Flp-In T-Rex (provided by Dorothee Dormann, Johannes Gutenberg University, Mainz, Germany), stable HeLa Flp-In T-REx cells (originally provided by Steven Taylor, the University of Manchester, Manchester, UK), as well as HEK293 cells (originally obtained from DSMZ, ACC 305) were cultivated in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37 °C in 5% CO 2 .

Techniques: In Vitro, Recombinant, Incubation, Labeling, SDS Page, Staining, Expressing, Western Blot, Control, Confocal Microscopy, Mutagenesis

Journal: Communications Biology

Article Title: Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

doi: 10.1038/s42003-026-09832-3

Figure Lengend Snippet: a Schematic representation of the assay illustrating foci formation of MARylated PARP15.1, which is reversed upon hydrolase activity of a macrodomain (created in BioRender. Verheugd, P. (2026) https://BioRender.com/zm1a7k2 ). b Schematic representation of constructs designed to generate cell lines stably expressing the indicated fusion proteins. The integration of a T2A site between GFP-PARP15 and the 3xFLAG-NLS-NB GFP (a GFP-specific nanobody) enables the simultaneous expression of both fusion proteins in equimolar ratios. Additionally, the inclusion of a Gateway cassette downstream of the sequence encoding the NB GFP facilitates recombination of various macrodomains or their respective mutants for analysis. GFP green fluorescent protein, MD macrodomain, NLS nuclear localization signal, NB nanobody. c The indicated constructs were transiently expressed in HEK293 cells. Protein expression was analyzed by immunoblotting. GFP-PARP15.1 was detected using a specific PARP15 antibody, 3x-FLAG-NLS-NB GFP fusion proteins were visualized by an anti-FLAG antibody. γ-Tubulin served as loading control. d U2OS cells were transiently transfected with constructs as shown in ( b ) expressing GFP-PARP15.1 and the indicated NB GFP , either without the MD, with the SARS-CoV-2 MD, the CHIKV MD, or its catalytically inactive mutant (V33E). Cells were fixed, nuclei visualized by Hoechst staining (blue) and localization of GFP-PARP15.1 (green) and 3xFLAG-NLS-NB GFP variants (red) determined by confocal microscopy.

Article Snippet: U2OS-tdTomato-G3BP1 (provided by Paul Taylor, St. Jude Children’s Hospital, Memphis, USA) , U2OS-tdTomato-G3BP1/EGFPm-PARP15 (this study), U2OS (originally obtained from DSMZ, ACC 785) and stable U2OS Flp-In T-Rex (provided by Dorothee Dormann, Johannes Gutenberg University, Mainz, Germany), stable HeLa Flp-In T-REx cells (originally provided by Steven Taylor, the University of Manchester, Manchester, UK), as well as HEK293 cells (originally obtained from DSMZ, ACC 305) were cultivated in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37 °C in 5% CO 2 .

Techniques: Activity Assay, Construct, Stable Transfection, Expressing, Sequencing, Western Blot, Control, Transfection, Mutagenesis, Staining, Confocal Microscopy

Journal: Communications Biology

Article Title: Screening assay to monitor mono-ADP-ribosylhydrolase activity of viral macrodomains in cells

doi: 10.1038/s42003-026-09832-3

Figure Lengend Snippet: a Stable U2OS Flp-In TREx cells were induced by 0.5 µg/ml doxycycline for the expression of GFP-PARP15.1 (wt, -HY, -KR). The cells were treated subsequently with DMSO or OUL232 as indicated. Cell lysates were separated and analyzed for protein abundance via immunoblotting. b Representative images from confocal microscopy indicating the localization pattern of GFP-PARP15.1 and mutant variants (magenta) in the stable U2OS Flp-In TREx upon doxycycline-induced expression. Nuclei were stained using SpyDNA, applied one hour prior to live-cell imaging (blue). c Stable HeLa Flp-In T-REx cells were induced for expression of GFP-PARP15.1-KR and the indicated 3xFLAG-NB GFP -macrodomain fusion proteins by 1 µg/ml doxycycline overnight. One hour prior to live-cell imaging by confocal microscopy, nuclei were stained using SpyDNA. Representative images of each cell line are shown with GFP-PARP15.1-KR (magenta), SpyDNA (blue) and merged. d Images obtained from confocal microscopy were analyzed using the CellProfiler pipeline. The data were plotted with ggplot in R to indicate percentages of cells showing either nuclear foci (blue), uniformly distributed, spread signal (black), or both (yellow) in the presence of the respective macrodomain variant. e Stable HeLa Flp-In T-REx cells were induced for expression of GFP-PARP15.1-KR and the SARS-CoV2 MD wt and subsequently left untreated, treated with DMSO or the indicated inhibitors overnight. Representative images of each condition are shown with GFP-PARP15.1-KR (magenta), SpyDNA (blue) and merged. f As in ( d ), but here upon treatment with the indicated inhibitors.

Article Snippet: U2OS-tdTomato-G3BP1 (provided by Paul Taylor, St. Jude Children’s Hospital, Memphis, USA) , U2OS-tdTomato-G3BP1/EGFPm-PARP15 (this study), U2OS (originally obtained from DSMZ, ACC 785) and stable U2OS Flp-In T-Rex (provided by Dorothee Dormann, Johannes Gutenberg University, Mainz, Germany), stable HeLa Flp-In T-REx cells (originally provided by Steven Taylor, the University of Manchester, Manchester, UK), as well as HEK293 cells (originally obtained from DSMZ, ACC 305) were cultivated in DMEM supplemented with 10% heat-inactivated fetal calf serum (FCS) at 37 °C in 5% CO 2 .

Techniques: Expressing, Quantitative Proteomics, Western Blot, Confocal Microscopy, Mutagenesis, Staining, Live Cell Imaging, Variant Assay