megfp c1 construct  (Addgene inc)

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