Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: Relative luciferase activity in U2OS Flp/Trx cells expressing GFP-FAM83A, GFP-FAM83B, GFP-FAM83C, GFP-FAM83D, GFP-FAM83E, GFP-FAM83F, GFP-FAM83G, GFP-FAM83H, and GFP only treated with either L-CM or Wnt3A-CM for 24 h. TOPflash and FOPflash luciferase activity presented as relative light units normalised to Renilla expression, the transfection control plasmid. Data presented as scatter graph illustrating individual data points with an overlay of the mean ± SD. Expression of GFP-FAM83A, GFP-FAM83B, GFP-FAM83C, GFP-FAM83D, GFP-FAM83E, GFP-FAM83F, GFP-FAM83G, GFP-FAM83H, and GFP only in U2OS Flp/Trx cells was induced by a treatment with 20 ng/ml doxycycline for 24 h. Statistical significance was determined using a Student’s unpaired t test and comparing cell lines as denoted on graph. Statistically significant P -values are denoted by asterisks (**** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05).

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Luciferase, Activity Assay, Expressing, Transfection, Control, Plasmid Preparation

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: (A) Cartoon of FAM83F protein illustrating the location of the CK1 binding and farnesylation motifs. Cartoons of the GFP-tagged FAM83F point mutants expressed in (B, C, D) are also indicated. (B) Lysates from U2OS Flp/Trx cells expressing GFP, GFP-FAM83A, GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , or GFP-FAM83F F284A/F288A were subjected to immunoprecipitation with GFP trap beads. Input lysates and GFP IPs were resolved by SDS–PAGE and subjected to Western blotting with the indicated antibodies. (C) Representative wide-field immunofluorescence microscopy images of U2OS Flp/Trx cells expressing GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , or GFP-FAM83F F284A/F288A , labelled with antibodies recognising GFP (far left panels, green), CK1α (second row of panels from left, magenta), and DAPI (third row of panels from left, blue). Overlay of GFP, CK1α, and DAPI images as a merge is shown on the right. Immunofluorescence images captured with a 60× objective. Scale bar represents 10 μm. (D) Relative luciferase activity in U2OS Flp/Trx cells expressing GFP, GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , or GFP-FAM83F F284A/F288A treated with either L- or Wnt3A-conditioned medium for 6 h. Luciferase activity is presented as TOPflash luciferase normalised to FOPflash luciferase and Renilla expression, the transfection control plasmid. Data presented as scatter graph illustrating individual data points with an overlay of the mean ± SD. Expression of GFP, GFP-FAM83A, GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , and GFP-FAM83F F284A/F288A in U2OS Flp/Trx cells was induced by a treatment with 20 ng/ml doxycycline for 24 h. Statistical analysis of (C) was completed using a Student’s unpaired t test and comparing cell lines as denoted on graph. Statistically significant P -values are denoted by asterisks (**** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05). Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Binding Assay, Expressing, Immunoprecipitation, SDS Page, Western Blot, Immunofluorescence, Microscopy, Luciferase, Activity Assay, Transfection, Control, Plasmid Preparation

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: (A) Representative widefield immunofluorescence microscopy images of U2OS Flp/Trx cells expressing GFP only, GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , or GFP-FAM83F F284A/F288A , labelled with antibodies recognising GFP (far left panels, green), β-catenin (second row of panels from left, magenta) and DAPI (third row of panels from left, blue). Overlay of GFP, β-catenin, and DAPI images as a merge is shown on the right. Cells were treated with either L- or Wnt3A–conditioned media for 6 h. Immunofluorescence images captured with a 20× objective. Scale bar represents 10 μm. (B) Cytoplasmic and nuclear extracts from U2OS Flp/Trx cells expressing GFP only, GFP-FAM83F, GFP-FAM83F C497A , GFP-FAM83F D250A , or GFP-FAM83F F284A/F288A following treatment with either L- or Wnt3A–conditioned media for 6 h, were resolved by SDS–PAGE and subjected to Western blotting with indicated antibodies. The specificity of cytoplasmic and nuclear lysates was determined by Western blotting with the following subcellular compartment-specific antibodies: α-tubulin (cytoplasmic) and Lamin A/C (nuclear). Expression of GFP tagged proteins in U2OS Flp/Trx cells was induced by a treatment with 20 ng/ml doxycycline for 24 h. Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Immunofluorescence, Microscopy, Expressing, SDS Page, Western Blot

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: Cytoplasmic, nuclear and membrane lysates from U2OS Flp/Trx cells expressing GFP only, GFP-FAM83F, GFP-FAM83F C497A or GFP-FAM83F F284A/F288A were resolved by SDS–PAGE and subjected to Western blotting with indicated antibodies. The specificity of subcellular fractions was determined by Western blotting with the following subcellular compartment-specific antibodies: GAPDH (cytoplasmic), Lamin A/C (nuclear) and Na/K ATPase (membrane). Densitometry of cytoplasmic β-catenin and GAPDH was performed and quantification is expressed as a fold-change compared with U2OS Flp/Trx cells expressing GFP only. Expression of GFP tagged proteins in U2OS Flp/Trx cells was induced by a treatment with 20 ng/ml doxycycline for 24 h. Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Membrane, Expressing, SDS Page, Western Blot

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: (A) Cell lysates from A-172, A549, NMuMG, SK-OV-3, K-562, MDA-MB-468, U2OS, PC-3, ARPE-19, SH-SY5Y, HUH7, THP-1, and HCT116 cells were resolved by SDS–PAGE and subjected to Western blotting with indicated antibodies. (A, B) Table indicating the cell of origin for all the cell lines used in (A). Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: SDS Page, Western Blot

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: (A) Cell lysates from U2OS wild-type and U2OS FAM83F −/− cells were subjected to immunoprecipitation with anti-FAM83F antibody. FAM83F IPs were resolved by SDS–PAGE and subjected to Western blotting with FAM83F antibody. (B) Relative luciferase activity in U2OS wild-type and U2OS FAM83F −/− cells treated with either L-CM or Wnt3A-CM for 6 h. Luciferase activity presented as TOPflash luciferase normalised to FOPflash luciferase and Renilla expression, the transfection control plasmid. Data presented as scatter graph illustrating individual data points with an overlay of the mean ± SD. (C) qRT-PCR was performed using cDNA from U2OS wild-type and U2OS FAM83F −/− cell lines following treatment with L-CM or Wnt3A-CM with or without 0.5 μM CHIR99021 for 6 h, and primers for Axin2 and GAPDH genes. Axin2 mRNA expression was normalised to GAPDH mRNA expression and represented as fold change compared with L-CM–treated cells. Data presented as scatter graph illustrating individual data points with an overlay of the mean ± SD. Statistical analysis of (B) and (C) was completed using a Student’s unpaired t test and comparing cell lines as denoted on graphs. Statistically significant P -values are denoted by asterisks (**** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05). Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Luciferase, Activity Assay, Expressing, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR

Journal: Life Science Alliance

Article Title: FAM83F regulates canonical Wnt signalling through an interaction with CK1α

doi: 10.26508/lsa.202000805

Figure Lengend Snippet: (A) Cytoplasmic, nuclear, and membrane lysates from U2OS Flp/Trx (GFP only, GFP-FAM83F, and GFP-FAM83F C497A ) cells were resolved by SDS–PAGE and subjected to Western blotting with the indicated antibodies. The specificity of cytoplasmic, nuclear and membrane compartment lysates were determined by Western blotting with the following subcellular compartment-specific antibodies: α-tubulin (cytoplasmic), Lamin A/C (nuclear), Na/K ATPase (membrane). (B) Lysates from U2OS Flp/Trx (GFP only, GFP-FAM83F and GFP-FAM83F C497A ) cells following treatment with L-CM or Wnt3A-CM for 6 h were resolved by SDS–PAGE and subjected to Western blotting with the indicated antibodies. (C) Densitometry of p-β-catenin (S45) protein abundance from (B) normalised to GAPDH protein abundance and represented as fold change compared to U2OS Flp/Trx cells expressing GFP only. Data presented as scatter graph illustrating individual data points with an overlay of the mean ± SD. Statistical significance was determined using a Student’s unpaired t test and comparing cell lines as denoted on graphs. Statistically significant P -values are denoted by asterisks (**** < 0.0001, *** < 0.001, ** < 0.01, * < 0.05). Expression of GFP, GFP-FAM83F, and GFP-FAM83F C497A in U2OS Flp/Trx cells was induced by a treatment with 20 ng/ml doxycycline for 24 h. Source data are available for this figure.

Article Snippet: U2OS (HTB-96; ATCC), HCT116 (CCL-247; ATCC), DLD-1 (CCL-221; ATCC), mouse fibroblast L-cells that stably overexpress Wnt3A (CRL-2647; ATCC), mouse fibroblast L cells (CRL-2648; ATCC), Flp-In T-Rex U2OS (which were created using Flp-In T-Rex Core kit [K650001; Thermo Fisher Scientific] and have been previously reported ( )), Flp-In T-Rex HEK293 (R78007; Thermo Fisher Scientific) and HaCaT (obtained from Joan Massague’s lab at Memorial Sloan Kettering Cancer Centre, not commercially available but can be provided on request) ( ) cells were maintained in DMEM (Gibco) containing 10% FCS (Hyclone), 100 U/ml penicillin (Lonza), 100 mg/ml streptomycin (Lonza), and 2 mM L-glutamine (Lonza).

Techniques: Membrane, SDS Page, Western Blot, Quantitative Proteomics, Expressing

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

Journal: Nature Communications

Article Title: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II

doi: 10.1038/s41467-020-15903-8

Figure Lengend Snippet: a Outline of a new IP method to isolate RNAPIIo and associated proteins from mock-treated or UV-irradiated (20 J/m 2 ) U2OS (FRT) cells. b Endogenous RNAPII Co-IPs on WT cells stained for the indicated TCR proteins. Note that it is not possible to stain for all these proteins on one membrane. This panel is a composite of several representative Co-IPs. See Supplementary Fig. for each individual Co-IP. c Endogenous RNAPII Co-IP followed by slot blot analysis of CPDs d Western blot analysis of CSA, CSB, and UVSSA knockout cells complemented with inducible GFP-tagged versions of these proteins ( n = 2). See Supplementary Fig. for validation of knockouts by sequencing. e Clonogenic Illudin S survival of WT, CSA, CSB, and UVSSA knockout and rescue cell lines. Each symbol represents the mean of an independent experiment ( n = 2 for all except for WT in UVSSA-KO figure which is n = 3) each experiment contains two or three technical replicates. Endogenous RNAPII Co-IP on f WT, CSA, CSB, and UVSSA knockout cells, g CSB-KO stably expressing GFP-CSB, and h WT, XPC, CSA, CSB, and UVSSA knockout cells. The asterisk in e indicates the heavy chain of the RNAPII antibody. At least two independent replicates of each IP experiment were performed obtaining similar results. Source data are provided as a file.

Article Snippet: To generate stable knockouts, U2OS Flp-In/T-REx cells were co-transfected with pLV-U6g-PPB encoding a guide RNA from the LUMC/Sigma-Aldrich sgRNA library (see Supplementary Table for plasmids, Supplementary Table for sgRNA sequences) together with an expression vector encoding Cas9-2A-GFP (pX458; Addgene #48138) using lipofectamine 2000 (Invitrogen).

Techniques: Irradiation, Staining, Membrane, Co-Immunoprecipitation Assay, Dot Blot, Western Blot, Knock-Out, Biomarker Discovery, Sequencing, Stable Transfection, Expressing

Journal: Nature Communications

Article Title: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II

doi: 10.1038/s41467-020-15903-8

Figure Lengend Snippet: a Outline of the chromatin-tethering approach in U2OS 2-6-3 cells. b A schematic representation of CSB and its deletion mutants. c Recruitment of CSA-GFP to the LacO array upon tethering of the indicated mCherry-LacR fusion proteins (scale bar = 5 µm). See Supplementary Figs. and for a full overview of all tested mutants. d Quantification of CSA-GFP and mCherry-LacR-CSB co-localization at the LacO array. Each symbol represents the mean of an independent experiment ( n = 2 for all except LacR-NLS and LacR-CSB WT which is n = 8, >50 cells collected per experiment). e Sequence alignment of CSB orthologues. See Supplementary Fig. for additional alignments. Source data are provided as a file.

Article Snippet: To generate stable knockouts, U2OS Flp-In/T-REx cells were co-transfected with pLV-U6g-PPB encoding a guide RNA from the LUMC/Sigma-Aldrich sgRNA library (see Supplementary Table for plasmids, Supplementary Table for sgRNA sequences) together with an expression vector encoding Cas9-2A-GFP (pX458; Addgene #48138) using lipofectamine 2000 (Invitrogen).

Techniques: Sequencing

Journal: Nature Communications

Article Title: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II

doi: 10.1038/s41467-020-15903-8

Figure Lengend Snippet: a A schematic representation of CSB and the CSB ΔCIM mutant. b Western blot analysis of U2OS (FRT) and CSB-KO complemented with either GFP-CSB WT or GFP-CSB ΔCIM ( n = 2). c Co-IP of GFP-CSB WT and GFP-CSB ΔCIM on the combined soluble and chromatin fraction. d Endogenous RNAPII Co-IP in GFP-CSB WT and GFP-CSB ΔCIM cell lines. See also Supplementary Fig. for additional Co-IP data. e Clonogenic Illudin S survival of WT and CSB-KO cell lines and the GFP-tagged CSB rescue cell lines. Each symbol represents the mean of an independent experiment ( n = 2), each of which is based on two technical replicates. Note that the same survival data for WT, CSB-KO and CSB-KO + GFP-CSB is also shown in Fig. . f In vitro ubiquitylation of recombinant Xenopus laevis (xl) and Homo sapiens (hs) CSB variants with recombinant xlCRL4 CSA , E1, E2, ubiquitin, and ATP. At indicated times, in vitro ubiquitylation reactions were stopped and blotted with anti-FLAG (top three panels) or anti-xlCSA (bottom panel) antibodies. See also Supplementary Fig. . g Immobilized recombinant CSB variants were incubated with Xenopus laevis nucleoplasmic extract (NPE), recovered, and blotted with anti-FLAG (top panel) or anti-xlCSA (bottom panel) antibody. At least two independent replicates of each IP and in vitro ubiquitylation experiment were performed obtaining similar results. Source data are provided as a file.

Article Snippet: To generate stable knockouts, U2OS Flp-In/T-REx cells were co-transfected with pLV-U6g-PPB encoding a guide RNA from the LUMC/Sigma-Aldrich sgRNA library (see Supplementary Table for plasmids, Supplementary Table for sgRNA sequences) together with an expression vector encoding Cas9-2A-GFP (pX458; Addgene #48138) using lipofectamine 2000 (Invitrogen).

Techniques: Mutagenesis, Western Blot, Co-Immunoprecipitation Assay, In Vitro, Recombinant, Ubiquitin Proteomics, Incubation

Journal: Nature Communications

Article Title: The cooperative action of CSB, CSA, and UVSSA target TFIIH to DNA damage-stalled RNA polymerase II

doi: 10.1038/s41467-020-15903-8

Figure Lengend Snippet: a A schematic representation of UVSSA WT and deletion mutants. The CSA-interacting region (CIR) and TFIIH-interacting region (TIR) are indicated. b Recruitment of CSA-GFP and TFIIH (p89) to the LacO array upon tethering of the indicated mCherry-LacR fusion proteins (scale bar = 5 µm). c Quantification of CSA-GFP and endogenous TFIIH (p89) co-localization at the LacO array. Each symbol represents the mean of an independent experiment ( n = 2, >50 cells collected per experiment). d Western blot analysis of U2OS (FRT) and UVSSA-KO cells complemented with GFP-UVSSA WT , GFP-UVSSA ΔCIR , and GFP-UVSSA ΔTIR ( n = 2). e Co-IP of GFP-UVSSA WT , GFP-UVSSA ΔCIR , and GFP-UVSSA ΔTIR . f Endogenous RNAPIIo Co-IP in GFP-UVSSA WT , GFP-UVSSA ΔCIR , and GFP-UVSSA ΔTIR cell lines. At least two independent replicates of each IP experiment were performed obtaining similar results. See also Supplementary Fig. for additional Co-IP data. Source data are provided as a file.

Article Snippet: To generate stable knockouts, U2OS Flp-In/T-REx cells were co-transfected with pLV-U6g-PPB encoding a guide RNA from the LUMC/Sigma-Aldrich sgRNA library (see Supplementary Table for plasmids, Supplementary Table for sgRNA sequences) together with an expression vector encoding Cas9-2A-GFP (pX458; Addgene #48138) using lipofectamine 2000 (Invitrogen).

Techniques: Western Blot, Co-Immunoprecipitation Assay