Journal: bioRxiv

Article Title: Androgen receptor condensates as drug targets

doi: 10.1101/2022.08.18.504385

Figure Lengend Snippet: A) Structure of AR predicted with AlphaFold. The model is coloured by structure prediction confidence from high confidence (dark-blue) to low confidence (orange-yellow). The known AR domains are highlighted. B) Live-cell STED imaging of HEK293T cells transfected with the indicated AR constructs tagged with mEGFP. Cells were imaged after treatment with 10 nM DHT for four hours. Scale bar: 5 μm. Dashed line indicates the nuclear periphery. C) Intensity of the NMR resonances of the AR AD as a function of amino acid position, measured for the displayed AR AD concentrations. The position of Transactivation Unit 1 and 5 (Tau-1, Tau-5), and of the 23 FQNLF 27 motif are highlighted. Green circles indicate the positions of residues not assigned or not visible (NA/NV) in the NMR spectrum recorded at 25 μM, including residues in regions of low sequence complexity such as poly-glutamine (pQ), poly-proline (pP) and poly-glycine (pG) tracts. Yellow and orange circles represent the positions of tyrosine (Tyr) residues mutated to serine (Ser) in 8YtoS and 14YtoS, respectively; all residues Tyr were mutated to Ser in 22YtoS. D) Fluorescence microscopy images of 40 µM AR-AD in vitro droplets (WT* and Tyr to Ser mutants) at 1 M NaCl and room temperature. Scale bar: 10 μm. E) Schematic representation of the LCST phase diagram of the AR AD (WT) obtained by determining the cloud points of solutions of increasing NaCl concentration (left) and of how cloud point measurements under two different solution conditions (right), labeled as 1 and 2, allow ranking Tyr to Ser mutants in terms of their phase separate capacity. F) Determination of the cloud points of AR AD (WT* and Tyr to Ser mutants) under two different solution conditions, labeled as 1 and 2. G) Representative merged confocal images of 15 µM MED1-IDR (left column) and 5 µM RNAPII-CTD (right column) droplets obtained at 20 mM NaCl or 50 mM NaCl, respectively, and 10 % ficoll before and after addition of 1 µM AR AD (WT* or 22YtoS). Scale bar: 5 μm. H) Quantification of AR AD partitioning into MED1-IDR (top graph) and RNAPII-CTD droplets (bottom graph), by measuring AR AD fluorescence intensity in droplets. Boxes correspond to the mean and the quartiles of all droplets represented as coloured dots from three image replicates. **** p < 0.0001. I) Representative merged confocal images of MED1-IDR and RNAPII-CTD multiphasic droplets obtained in 125 mM NaCl and 10% ficoll with and without the addition of 1 µM AR AD (WT* or 22YtoS). Scale bar: 5 μm. J) Normalized intensity plot profile of droplet cross-sections from the images shown in panel I. See also .

Article Snippet: KpnI-KpnI fragment with that of the wild-type AR sequence from peGFP-C1-AR. mEGFP constructs: Monomeric EGFP was subcloned into vectors containing human AR (Addgene #29235) and AR-V7 (Addgene #86856) using Gibson assembly to create mEGFP-AR-FL and mEGFP-AR-V7 (referred to as ‘AD+DBD+NLS’ in , ) mammalian expression vectors.

Techniques: Imaging, Transfection, Construct, Sequencing, Fluorescence, Microscopy, In Vitro, Concentration Assay, Labeling

Journal: bioRxiv

Article Title: Androgen receptor condensates as drug targets

doi: 10.1101/2022.08.18.504385

Figure Lengend Snippet: A) Live-cell confocal imaging of the indicated mEGFP construct transfected into HEK293T after treatment with vehicle or 10 nM DHT for four hours. Scale bar: 3 µm. Dashed lines indicate nuclear periphery. B) Quantification of confocal data in . Y-axis indicates the standard deviation, and x-axis indicates the mean intensity of pixels in the corresponding nucleus. Each dot represents measurements from an individual cell, and lines represent standard regression fits to the corresponding data spread (N = 2). C) Distribution of aromatic (Histidine, Phenylalanine, Tryptophan, Tyrosine) and Tyrosine residues along the AR AD sequence, clustered using a 9 amino acid window, where the shaded areas correspond to those represented in . D) Average intensity ratio of the NMR resonances of the AR AD at the tested protein concentrations (57.5, 100.8, 122.5 and 155.0 μM) relative to their intensity at 25 μM grouped by amino acid type. E) Fluorescence microscopy images of in vitro AR AD (WT*) concentration-dependent condensation obtained in AR AD buffer (20 mM NaP, 1 mM TCEP pH 7.4) with 150 mM NaCl and 10% ficoll, where ca 1 % of AR-AD molecules were labeled with the dye Dylight 405. Scale bar: 10 µm. F) AR AD WT* liquid character in vitro by FRAP. Top panel: confocal microscopy images of WT* AR AD droplets labeled with Alexa-647, in 150 mM NaCl and 10 % ficoll before and after photobleaching in FRAP experiment. Scale bar 5 µm. Lower panel: average relative fluorescence intensity curve of WT* AR AD droplets as a function of time following photobleaching. Error bars represent s.d. of n=10 droplets. G) (Left) microcopy images of in vitro droplets formed by the indicated proteins. The signal of the AR AD channel and merged channel are shown. AR AD proteins were used in five-fold higher concentrations than . Scale bar: 1 µm. (Right) the representative droplet’s cross section intensity profile.

Article Snippet: KpnI-KpnI fragment with that of the wild-type AR sequence from peGFP-C1-AR. mEGFP constructs: Monomeric EGFP was subcloned into vectors containing human AR (Addgene #29235) and AR-V7 (Addgene #86856) using Gibson assembly to create mEGFP-AR-FL and mEGFP-AR-V7 (referred to as ‘AD+DBD+NLS’ in , ) mammalian expression vectors.

Techniques: Imaging, Construct, Transfection, Standard Deviation, Sequencing, Fluorescence, Microscopy, In Vitro, Concentration Assay, Labeling, Confocal Microscopy

Journal: bioRxiv

Article Title: Lysophosphatidic acid provokes fibroblast chemotaxis through combinatorial regulation of myosin II

doi: 10.1101/355610

Figure Lengend Snippet: A. Total internal reflection fluorescence (TIRF) microscopy images of RBD-GFP localization upon uniform stimulation of cells with either PDGF or LPA. Analysis of fluorescent image intensity (mean±s.e.m.) shows significant increase in RBD-GFP signal after LPA stimulation but PDGF stimulation did not produce an increase in RBD-GFP intensity B. Line scans of RBD-GFP (Rho) and (C1) 2 -GFP (DAG) intensity at the leading edge of chemotaxing cells. C. Intensity maps of RBD-GFP and (C1) 2 -GFP expression at the periphery of cells in an LPA gradient over time. D. Histograms showing the cumulative intracellular RHO (n=15) and DAG (n=17) distribution in cells in an LPA gradient.

Article Snippet: Tagged tandem C1 domains of PKCδ, (C1) 2 -GFP was a gift from Tobias Meyer (Addgene plasmid #21212) and RBD-GFP from Burridge lab. Plasmids were transiently transfected into IA32 fibroblasts using Lipofectamine 2000 reagent.

Techniques: Fluorescence, Microscopy, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A and C) HEK293T cells stably expressing the indicated TRIM21 mutants (EV = empty vector) were transfected with WT His-ubiquitin and infected with AdV5 ± 9C12 or 9C12(H433A). Ubiquitinated proteins were isolated by denaturing His-pulldown using Ni-NTA beads in 6M Guanidine buffer before immunoblot analysis with anti-TRIM21 antibody. (B) A co-crystal structure of TRIM21 RING:Ube2N∼ubiquitin complex (PDB: 6S53) showing the tri-anionic anchor motif (E12, E13 and D21) and second site residues (R67 and N71). (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from HEK293T cells overexpressing the indicated ubiquitin mutant at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Plasmid Preparation, Transfection, Ubiquitin Proteomics, Infection, Isolation, Western Blot, Mutagenesis

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: Denaturing TRIM21-His pulldown from HEK293T cells stably expressing TRIM21-His and infected with AdV5 ± 9C12 or 9C12(H433A) which does not bind TRIM21. Cells were lysed in buffer containing 4M urea and where indicated, the Nickel beads were treated with the deubiquitinase USP2 before boiling and immunoblotting with anti-TRIM21 antibody.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Stable Transfection, Expressing, Infection, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Sequence of TRIM21 with RING domain in gray, B Box in red, coiled-coil in cyan, L2 linker helices in orange & green and PRYSPRY in magenta. (B) SEC-MALS chromatograms of TRIM21 CC (129-235) loaded at concentrations of 15 (black), 5 (green), 0.55 (blue) and 0.25 mg/ml (red) are shown, in which the refractive index is indicated by the solid lines while the molar mass evaluated from the light scattering analysis is indicated with the corresponding coloured dotted lines. (C) Using a MicroCal iTC200 calorimeter, 140 µM TRIM21 coiled-coil was added in 2 µl injections into buffer at 25 °C. Integrated heats were then fit to a dimer dissociation model reveal an enthalpy of 65 kcal/mol and Kd of 7 µM. (D-F) SAXS data on TRIM21 constructs. (D) SAXS data were collected at the beam line P12 of the EMBL at the Petra-III storage ring (DESY, Hamburg). Protein expression, purification, data analysis and collection are in Supplementary Information. Data statistics are shown in Table 1. (D) Concentration-normalised scattering plots and DAM fits for CC235 (cyan, χ = 1.00), MBP-CC235 (green, χ = 1.31) and RBCC (purple, χ = 1.05). (E) The linear Guinier regions from (D). (F) Derived P( r ) curves. The CC235 P( r ) curve is consistent with an elongated rod, while the two peaks observed for MBP-CC235 and RBCC are consistent with ‘dumbbell’-shaped molecules. (G-I). SAXS data on TRIM21:Fc complex. (G) Scattering plot with DAM fit (red line, 1.00), TRIM21:Fc atomic model fit (blue, χ = 1.04), and apo-TRIM21 atomic model fit (brown dashed, χ = 2.00). (H) The linear Guinier regions from (G). (I) Derived P( r ) curves.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Sequencing, Refractive Index, Construct, Expressing, Purification, Concentration Assay, Derivative Assay

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Dummy atom model (DAM) structures of TRIM21 calculated from SAXS data shown in . Overlay of reconstructions of constructs comprising TRIM21 residues 1-235 (RBCC, purple spheres) and TRIM21 residues 129-235 (CC235, blue spheres). A structural model of the TRIM21 RBCC region based on atomic models of the TRIM21 RING (yellow) and B Box (orange) domains and TRIM25 coiled-coil domain (cyan) using PDBs 5OLM and 4CFG respectively. (B) SAXS-derived model of TRIM21:Fc complex. Averaged and filtered DAM’s (white and green spheres, respectively) overlaid with an atomic model of the TRIM21:Fc complex. The RING, B Box and coiled-coil regions are as above. The PRYSPRY (magenta) and IgG Fc (grey) domains are taken directly from PDB 2IWG. (C) Crystal structure of TRIM21 RING dimer showing the important residues M10 and M72 at the dimer interface. (D) Immunoblot of TRIM21 following denaturing His-ubiquitin pulldown from TRIM21 lentivector reconstituted HEK293T cells overexpressing WT His-ubiquitin at 30 min post infection with AdV5 ± 9C12 or 9C12(H433A). (E) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (F) in vitro ubiquitination assay using the indicated TRIM21 constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (G) Immunoblot analysis of ubiquitin discharge from Ube2N by the indicated TRIM21 constructs over a course of 20 minutes. (H) Quantification of the Ube2N∼Ub band in G relative to time point 0 in each reaction. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Derivative Assay, Western Blot, Ubiquitin Proteomics, Infection, Electroporation, In Vitro

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic illustration of multimeric TRIM21 assembly on the surface of a virus-antibody complex. (B) Remaining infectivity of AdV5-GFP particles on HEK293T cells following incubation with saturating concentrations of 9C12. For 1/e neutralization, approximately 12% of antibodies must bear an intact TRIM21 binding site which is present on 9C12(WT) but lacking in 9C12(H433A). This equates to 24 out of the maximum ∼200 antibodies bound to AdV5 at saturation. (C, E, J and L) Neutralisation of AdV5 by 9C12 in lentivector reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D and F) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (G) In vitro ubiquitination assay using the indicated TRIM21 RING-linker-RING (RLR) constructs and Ube2N. Samples were taken at the indicated timepoints and analysed by immunoblotting with anti-ubiquitin antibody. (H) Trim-away of IKKα in TRIM21 lentivector reconstituted HEK293T cell lines by electroporation of anti-IKKα IgG (anti-IKKα). Cell lysates were immunoblotted for the indicated proteins. (I) Crystal structure of TRIM21-RING-B-box (PDB: 5OLM) showing residue S80 at situated at the RING:B Box interface. (K) Structure of the hydrophobic core at the TRIM21 RING:Ube2N interface (PDB: 6S53). See also Figures S3 and S4

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Virus, Infection, Incubation, Neutralization, Binding Assay, Expressing, Activation Assay, Stable Transfection, In Vitro, Ubiquitin Proteomics, Construct, Western Blot, Electroporation, Residue

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Upper panel: TRIM5α (rhesus) residues involved in mediating the three layers of box-box interactions (PDB:5EIA). Lower panel: model of TRIM21 B box domain structure (PDB:5OLM) onto the trimeric box structure of TRIM5α (PDB:5EIA). Residues that could mediate B box: B box interactions are highlighted. (B and C) Neutralisation of AdV5 by 9C12 in lentivector-reconstituted HEK293T cells expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SEM. (D) AdV5-9C12 immune complex-induced NF-kB activation in HEK293T cells stably expressing the indicated TRIM21 mutants. Data normalised to the virus only condition and presented as the mean ± SD. (E) Neutralisation of AdV5 by 9C12 in HEK293T cells stably expressing TRIM21-ΔBox construct with or without MG132 (20 µM). Data normalised to the virus only condition and presented as the mean ± SD.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Virus, Activation Assay, Stable Transfection, Construct

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of myc-mEGFP constructs. (B-D) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with either control IgG (9C12), ant-Myc (9E10) or anti-GFP (polyclonal) antibodies. 8 hours post-electroporation cellular GFP fluorescence was imaged (B) and quantified (C) using the IncuCyte system, or total GFP protein levels analysed by immunoblotting (D) cell extracts with the indicated antibodies. Scale bar 100 µm. (E) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding the indicated myc-mEGFP constructs together with control IgG or increasing concentrations of anti-Myc antibody and GFP fluorescence quantified 8 hours later. (F) HEK293T-mCherry-TRIM21 cells were electroporated with mRNA encoding 2myc-mEGFP together with the indicated antibodies and GFP fluorescence quantified 8 hours later. (G-I) The indicated antibodies (50 nM) either alone, or mixed with GFP protein (100 nM), were analysed by mass photometry. (J) RPE-1 cells expressing mEGFP were electroporated with the indicated antibodies and cell extracts blotted 3 hours later for the indicated proteins. See also

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Electroporation, Fluorescence, Western Blot, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: RPE-1 TRIM21 KO cells expressing membrane-localised GFP (mem-mEGFP) were electroporated with PBS, control IgG or the indicated anti-GFP antibodies. Cells were fixed 3 hours post-electroporation and stained with alexa 647-conjugated anti-IgG secondary antibodies and imaged by confocal microscopy. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Membrane, Control, Electroporation, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of light-induced clustering of TRIM21. (B-C) Drosophila S2 cells expressing the indicated constructs were incubated with or without MG132 and RFP fluorescence quantified by live imaging. Time shows minutes (min) from onset of blue light exposure. Scale bar 5 µm. Pseudo-coloured kymographs show fluorescence intensity in regions defined by red dotted lines. Graph shows mean fluorescence intensity (± SD) of RFP-CRY2-TRIM21 (n = 23) and RFP-CRY2-TRIM21+MG132 (n = 20) normalised for the respective controls (RFP-CRY2 (n=16) and RFP-CRY2 + MG132 (n=15)). (D-G) RPE-1 cells expressing the indicated constructs together with mem-mEGFP were incubated with or without MG132 and blue light for 3 hours prior to immunoblotting for the indicated proteins. See also Movie S2

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Expressing, Construct, Incubation, Fluorescence, Imaging, Western Blot

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Catalysis of unanchored ubiquitin chains by TRIM21 RING (R), RING-Box (RB), RING-Box-Coiled-Coil (RBCC) and full length MBP-tagged TRIM21 (FL-hT21). (B-C) Catalysis of ubiquitin discharge from ubiquitin-conjugated Ube2N by RING (R), RING Box (RB), RING Box coiled-coiled (RBCC) and full length MBP-tagged TRIM21 (FL-hT21).

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Ubiquitin Proteomics

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Schematic of TRIM21, nanobody-Fc and TRIM21-nanobody chimeric constructs. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with mRNA encoding the indicated constructs. 16 hours later cell extracts were immunoblotted for the indicated proteins. (C-D) NIH3T3-Caveolin-1-GFP and (F-G) RPE-1H2B-mEGFP-FKBP cells were electroporated with water (control) or mRNAs encoding the indicated TRIM21-nanobody chimeric constructs and GFP fluorescence was imaged (C and F) and quantified (D and G) using the IncuCyte system. Time shows hours:minutes (h:min) post-electroporation. Scale bar 50 µm (C) and 30 µm (F). (E) NIH3T3-Caveolin-1-GFP cells and (H) RPE-1-H2B-mEGFP-FKBP cells were electroporated with mRNA encoding mCherry-tagged versions of the indicated constructs and GFP and mCherry fluorescence quantified using the IncuCyte system. TRIM21-nanobody chimera expression levels (mCherry fluorescence) are plotted against GFP fluorescence. See also and Movie S2.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Construct, Control, Fluorescence, Electroporation, Expressing

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A-D) NIH3T3-Caveolin-1-GFP (A and B) and RPE-1-H2B-mEGFP-FKBP (C and D) cells were electroporated with mRNA encoding mCherry-T21R-vhhGFP4, incubated with either DMSO (control) or MG132 and imaged (A and C) and GFP fluorescence quantified (B and D) with the IncuCyte system. Scale bar 20 µm. (E and F) NIH3T3 cells (E) and RPE-1 cells (F) were electroporated with mRNA encoding mCherry-tagged versions of the indicated TRIM21-nanobody constructs and mCherry fluorescence quantified using the IncuCyte system. (G and H) NIH3T3-Caveolin-1-GFP cells were electroporated with the indicated concentrations of T21R-vhhGFP4 (G) or T21RB-vhhGFP4 (H) proteins and GFP fluorescence quantified with the IncuCyte system. (I) Data from G and H plotted as protein concentration against GFP fluorescence at 4 hours post-electroporation.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Incubation, Control, Fluorescence, Construct, Protein Concentration, Electroporation

Journal: bioRxiv

Article Title: Substrate-induced clustering activates Trim-Away of pathogens and proteins

doi: 10.1101/2020.07.28.225359

Figure Lengend Snippet: (A) Bacterially-expressed 6His-T21R-vhhGFP4 was purified using a two-step protocol of NiNTA-followed by size exclusion-chromatography and analysed by Coomassie blue staining of SDS-PAGE. (B) NIH3T3-Caveolin-1-GFP cells were electroporated with PBS (control) or the indicated concentrations of T21R-vhhGFP4 protein and GFP fluorescence was quantified using the IncuCyte system. (C) H2B-GFP primary MEFs were electroporated with PBS (control) or T21R-vhhGFP4 in the form or mRNA or protein and GFP fluorescence quantified using the IncuCyte system. Time shows hours (h) post-electroporation. (D-G) Drosophila S2 cells expressing GFP-aPKC and (D, E) vhhGFP4-RFP-CRY2-TRIM21 or (F, G) a LARIAT module that includes RFP-CRY2 fused with vhhGFP4 and which enables clustering in the absence of TRIM21. (E, G) Graphs show mean ± SD fluorescence intensity quantified by live imaging (n = 30 in E and n=24 in G). Time shows minutes (min) from onset of blue light exposure. Scale bar 10 µm.

Article Snippet: To generate optogenetic constructs for S2 cell expression, vhhGFP4 , mRFP , mouse TRIM21 (Addgene #105516) and CRY2Clust ( ) coding sequences were inserted into heat-shock inducible Drosophila Gateway expression vectors (Life Technologies) to generate pHR-CRY2Clust-TRIM21, pHR-CRY2Clust and pH-vhhGFP4-RFP-CRY2Clust-TRIM21.

Techniques: Purification, Size-exclusion Chromatography, Staining, SDS Page, Control, Fluorescence, Electroporation, Expressing, Imaging

Journal: Cells

Article Title: GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells

doi: 10.3390/cells13040316

Figure Lengend Snippet: GFP-GOLPH3 localizes to mitochondrial fission sites after the recruitment of YFP-DRP1. HeLa cells grown in 35 mm glass-bottom culture dishes were co-transfected to express GFP-GOLPH3 and the mitochondrial fission protein YFP-DRP1. After 16 h, cells were incubated with MitoTracker TM Deep Red FM for 20 min at 37 °C, followed by the transfer of culture dishes to a microscope heating stage for time-lapse live-cell imaging. Images were acquired every ~0.87 s. ( A ) Representative imaged cell showing the cytoplasmic distribution of the labeled mitochondrial network (MitoTracker Deep Red; gray channel), GFP-GOLPH3 (green channel), and YFP-DRP1 (red channel). The fourth image depicts the superposition of the gray, green, and red channels (Merge). The boundary of the cell and nucleus (N) is marked with pale blue dashed lines. Bar, 10 μm. ( B ) Magnification of time-lapse images acquired at the indicated times of the cell region highlighted with an orange dashed line in the images shown in ( A ). The yellow arrows depict a mitochondrial fission event; the green solid arrows depict the recruitment and displacement of a GFP-GOLPH3 vesicle at the site of the mitochondrial fission highlighted by yellow arrows; and the red solid arrows depict the recruitment and displacement of YFP-DRP1 at the site of the mitochondrial fission highlighted by the yellow arrow. The green and red open arrows depict the initial position of the respective solid arrows. Bar, 2 μm.

Article Snippet: The pEYFP-C1-DRP1 mammalian expression construct encoding human full-length DRP1 (YFP-DRP1) (Addgene, Watertown, MA, USA; cat # 45160) was a generous gift from Verónica Eisner (Pontificia Universidad Católica de Chile, Santiago, Chile).

Techniques: Transfection, Incubation, Microscopy, Live Cell Imaging, Labeling

Journal: Cells

Article Title: GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells

doi: 10.3390/cells13040316

Figure Lengend Snippet: GFP-GOLPH3 localizes to mitochondrial fission sites after the recruitment of YFP-DRP1 in MDA-MB-231 cells. MDA-MB-231 cells grown in 35 mm glass-bottom culture dishes were co-transfected to express GFP-GOLPH3 and YFP-DRP1. After 16 h, cells were incubated with MitoTracker TM Deep Red FM for 20 min at 37 °C, followed by the transfer of culture dishes to a microscope heating stage for time-lapse live-cell imaging. Images were acquired every ~0.86 s. ( A ) Representative imaged cell showing the cytoplasmic distribution of the labeled mitochondrial network (MitoTracker Deep Red; gray channel), GFP-GOLPH3 (green channel), and YFP-DRP1 (red channel). The fourth image depicts the superposition of the gray, green, and red channels (Merge). The boundary of the cell and nucleus (N) is marked with pale blue dashed lines. Bar, 10 μm. ( B ) Magnification of time-lapse images acquired at the indicated times of the cell region highlighted with an orange dashed line in the images shown in ( A ). The yellow arrows depict a mitochondrial fission event; the green arrows depict the recruitment and displacement of a GFP-GOLPH3 vesicle at the site of the mitochondrial fission highlighted by yellow arrows; and the red arrows depict the recruitment and displacement of YFP-DRP1 at the site of the mitochondrial fission highlighted by yellow arrows. Bar, 2 μm.

Article Snippet: The pEYFP-C1-DRP1 mammalian expression construct encoding human full-length DRP1 (YFP-DRP1) (Addgene, Watertown, MA, USA; cat # 45160) was a generous gift from Verónica Eisner (Pontificia Universidad Católica de Chile, Santiago, Chile).

Techniques: Transfection, Incubation, Microscopy, Live Cell Imaging, Labeling

Journal: Cells

Article Title: GOLPH3 Participates in Mitochondrial Fission and Is Necessary to Sustain Bioenergetic Function in MDA-MB-231 Breast Cancer Cells

doi: 10.3390/cells13040316

Figure Lengend Snippet: The depletion of GOLPH3 in MDA-MB-231 cells modifies the levels of mitochondrial fusion and fission proteins. ( A , C ) Detergent-soluble extracts were prepared from the indicated cells grown in 6-well plates, and the samples were processed by SDS-PAGE and immunoblot analysis using antibodies to detect the proteins indicated on the right involved in mitochondrial fusion ( A ) or mitochondrial fission ( C ). The immunoblot signal of anti-β-actin was used as the loading control. The position of molecular mass markers is indicated on the left. ( B , D ) Quantification of the respective immunoblot signal as shown in A and C. In C and D, pDRP1 denotes DRP1 phosphorylated at serine 616. Bars represent the mean ± SEM (n = 3). * p < 0.05; ** p < 0.01; ns: not statistically significant.

Article Snippet: The pEYFP-C1-DRP1 mammalian expression construct encoding human full-length DRP1 (YFP-DRP1) (Addgene, Watertown, MA, USA; cat # 45160) was a generous gift from Verónica Eisner (Pontificia Universidad Católica de Chile, Santiago, Chile).

Techniques: SDS Page, Western Blot, Control

Journal: eLife

Article Title: Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1

doi: 10.7554/elife.17896

Figure Lengend Snippet: Figure 4. MUL1 and PARKIN have redundant functions in OXPHOS-induced mitophagy. (A) Quantification of red-only puncta in cells grown in acetoacetate-containing medium. Presence (+) or absence (-) of Pink1, Parkin, or Mul1 is indicated. Error bars indicate SD of three biological replicates, p=0.015 (Pink1), p=0.0011 (Parkin-/- Mulan shRNA) (Student’s t-test). (B) Mitophagy in wild-type and mutant cells. Cells stably expressing Cox8-EGFP- mCherry were grown in acetoacetate-containing medium and imaged by fluorescence microscopy. (C) Co-localization of LC3B with mitophagy intermediates. Wild-type and mutant cells were retrovirally transduced with mTurquoise2-LC3B, grown in acetoacetate-containing medium and imaged by fluorescence microscopy. Examples of LC3B co-localization with mitophagy intermediates are indicated by arrows. (D) Accumulation of polyubiquitinated proteins in mitochondria. Cells were grown in the indicated medium, and mitochondria were isolated by differential centrifugation. Mitochondrial lysates were analyzed by Western blot for pan-Ubiquitin. HSP60 is a loading control. (E) Quantification of polyubiquitinated proteins in mitochondria. Three independent experiments were quantified by densitometry and averages are shown. Ubiquitin level was normalized to HSP60. Error bars indicate SD, p=0.0003 (WT Glu vs. Ac), p=0.0011 (Pink1-/-), p=0.0016 (Parkin-/- Mulan shRNA), p=0.0206 (Parkin-/-) (Student’s t-test). DOI: 10.7554/eLife.17896.008 The following figure supplement is available for figure 4:

Article Snippet: To clone mTurquoise2 fusion proteins, mTurquoise2 was amplified from pmTurquoise2-Mito (Addgene plasmid # 36208, Dorus Gadella, [Goedhart et al., 2012]).

Techniques: shRNA, Mutagenesis, Stable Transfection, Expressing, Fluorescence, Microscopy, Transduction, Isolation, Centrifugation, Western Blot, Ubiquitin Proteomics, Control

Journal: Cancer Research

Article Title: Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels

doi: 10.1158/0008-5472.can-08-0634

Figure Lengend Snippet: Figure 2. Pim kinases bind to and directly phosphorylate p27Kip1. A, alignment of the amino acid sequence of the Pim consensus sequence with the primary sequences of human p27Kip1 around T157 and T198 residues. Identical residues are denoted by white letters on black background (left). HEK293T, K562, and 22Rv1 cells were transfected with empty pHM6 alone () or pHM6 encoding wt-Pim1S (wt) or KD-Pim1S (KD) together with pFLAG-CMV-2 encoding p27Kip1. After transfection for 24 h, the cells were harvested and lysed. The whole cell lysates were electrophoresed and immunoblotted with the indicated antibodies (right). B, HEK293T cells were transfected with empty pc5FLAG alone () or pc5FLAG encoding PIm1S (+) together with pHM6-p27Kip1. After transfection for 24 h, the cells were lysed. The FLAG-tag protein was immunoprecipitated from the cell lysates. The immunoprecipitated proteins (IP) and the cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies (left). The nuclear fraction of K562 and 22Rv1 cells were incubated with protein A agarose that had been conjugated with normal rabbit IgG (IgG), rabbit anti-p27Kip1 antibody (p27Kip1), or rabbit anti-FoxO3a antibody (FoxO3a). The immunoprecipitated proteins were subjected to immunoblot analysis with the indicated antibodies (right). C, HEK293T cells were transfected with empty pHM6 alone () or pHM6-Pim1S (+) together with empty pFLAG-CMV-2 () or pFLAG-CMV-2 encoding wt-p27kip1 or the indicated p27Kip1 mutants. After transfection for 24 h, the cell lysates were electrophoresed and immunoblotted with the indicated antibodies. D, purified recombinant wt-p27Kip1 (lane 1), S10A-p27Kip1 (lane 2), T157A-p27Kip1 (lane 3), T187A-p27Kip1 (lane 4), T198A-p27Kip1 (lane 5), and GST (lane 6) proteins were incubated with GST-tagged recombinant Pim1S for 30 min at 30jC in the presence of [g-32P]ATP. The reaction samples were electrophoresed and stained with CBB (bottom). After staining, the levels of incorporated radioactivity were visualized with Typhoon 9410 (top).

Article Snippet: In some experiments, cell lysates were incubated with normal mouse IgG-conjugated agarose (Santa Cruz Biotechnology) or protein A agarose that had been conjugated with normal rabbit IgG.

Techniques: Sequencing, Transfection, FLAG-tag, Immunoprecipitation, Western Blot, Incubation, Purification, Recombinant, Staining, Radioactivity

Journal: Cancer Research

Article Title: Pim Kinases Promote Cell Cycle Progression by Phosphorylating and Down-regulating p27Kip1 at the Transcriptional and Posttranscriptional Levels

doi: 10.1158/0008-5472.can-08-0634

Figure Lengend Snippet: Figure 3. Pim-mediated p27Kip1 phosphorylation promotes its binding to 14-3-3 and nuclear export. A, HEK293T cells were transfected with empty pHM6 (, lanes 1 and 2) or pHM6-wt-Pim1S (+, lanes 3–8) together with empty pFLAG-CMV-2 (, lane 1) or pFLAG-CMV-2 encoding wt-p27kip1 (wt, lanes 2 and 3) or the indicated p27Kip1 mutants. After transfection for 24 h, the cells were harvested, lysed, and immunoprecipitated with an anti-FLAG agarose, as described in Materials and Methods. The immunoprecipitants (IP) and the cell lysates (Input) were subjected to immunoblot analysis with the indicated antibodies. B, HT1080 cells were transfected with pHM6-wt-Pim1S together with pFLAG-CMV-2 encoding wt-p27Kip1 (wt), T157A-p27Kip1 (T157A), T198A-p27Kip1 (T198A), or T157A/ T198A-p27Kip1 (T157A/T198A). After transfection for 24 h, HA-tagged Pim1S proteins were detected by staining with an anti-HA antibody following Oregon green–conjugated anti-rat antibody incubation. The FLAG-tagged p27Kip1 proteins were detected by staining with an anti-FLAG antibody, following treatment with Alexa Flour 568–conjugated anti-rabbit antibody. Nuclei were detected by staining with Hoechst 33342. The cells were washed and then visualized using a fluorescence microscope equipped with a CCD camera (top). The percentage of the cells that exhibited cytoplasmic p27Kip1 was determined by counting 200 transfectants that expressed both Pim1 and p27Kip1 protein in each cell (bottom). C, HEK293T cells were transfected with empty pc5FLAG alone () or pc5FLAG encoding PIm1S (+) together with pFLAG-CMV-2 encoding p27Kip1 (+). After transfection for 24 h, the cells were harvested. The cytoplasmic and nuclear fractions were separated, electrophoresed, and immunoblotted with the indicated antibodies. D, HEK293T cells were cotransfected with empty pHM6 alone () or pHM6 encoding wt-, T157A- or T198A-p27Kip1 together with pc5FLAG encoding none () or wt-Pim1S (+). After transfection for 36 h, the cells were stained with propidium iodide and analyzed with a flow cytometer.

Article Snippet: In some experiments, cell lysates were incubated with normal mouse IgG-conjugated agarose (Santa Cruz Biotechnology) or protein A agarose that had been conjugated with normal rabbit IgG.

Techniques: Phospho-proteomics, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Staining, Incubation, Fluorescence, Microscopy, Flow Cytometry