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: STAR protocols

Article Title: Detection of thermal shift in cellular Keap1 by protein-protein interaction inhibitors using immunoblot- and fluorescence microplate-based assays.

doi: 10.1016/j.xpro.2022.101265

Figure Lengend Snippet: Figure 3. An example of data obtained with Keap1-Glow CETSA (Protocol B) showing that PRL-295 alters the thermostability of Keap1-mCherry, but not free mCherry measured at the same temperature range, in U2OS cell lysates Temperature-dependent aggregation curves in lysates from U2OS cells stably expressing Keap1-mCherry (left) or mCherry (right) pre-treated for 1 h with 15 mM of PRL-295 (red) or the same volume of vehicle (DMSO, blue) are displayed as mCherry fluorescence intensities that remained after removal of precipitated material from lysates heated to the indicated temperatures, normalized to that of vehicle-treated sample heated to 39C (dots). The smoothing lines that represent local polynomial regressions fitted to the data are added to enhance visualization. Data from Dayalan Naidu et al., 2021.

Article Snippet: Stored at 20 C–25 C. any N/A Experimental models: Cell lines HL-60 cells ATCC CCL-240 Doxycycline (Dox)-inducible Keap1mCherry U2OS (Flp-In T-Rex ) cell line (KC-U2OS) Dayalan Naidu et al., (2021) N/A Doxycycline (Dox)-inducible mCherry U2OS (Flp-In T-Rex ) cell line (C-U2OS) Dayalan Naidu et al., (2021) N/A Software and algorithms GraphPad Prism 8.0 GraphPad https://www.graphpad.com/ scientific-software/prism/ Image Studio Lite Software LI-COR N/A SoftMax Pro.

Techniques: Stable Transfection, Expressing