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A) Fluorescence microscopy images of preassembled equimolar condensates showing reciprocal recruitment of HOTAIR 1-360 and AR LBD . Upper: DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after addition of 25 μM <t>Cy3-labeled</t> HOTAIR 1-360 . Lower: DyLight488-labeled AR NTD and Cy3-labeled HOTAIR 1-360 droplets after addition of 20 μM Cy5-labeled AR LBD . An equimolar mixture of Cy5-labeled AR LBD and Cy3-labeled HOTAIR 1-360 (25 μM each) did not undergo LLPS. Scale bar, 10 μm. Representative of three biological replicates. B) Quantification of Cy3-labeled HOTAIR 1-360 enrichment in preassembled AR NTD -HOTAIR 1-360 droplets before and after the addition of 20 μM Cy5-labeled AR LBD . C) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of 25 μM Cy3-labeled HOTAIR 1-360 . D) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of an equivalent amount of PolyU-20nt. For B-D, enrichment was calculated from raw-image fluorescence measurements. Boxes indicate quartiles and the mean; dots represent individual droplets across three biological replicates. Representative images are shown with identical linear scaling within each channel across conditions. E-G) Fluorescence microscopy images from three biological replicates of preassembled equimolar DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after the addition of E) 400 μM SLNCR1-20nt, F) HOTAIR-20nt, or G) PolyU-20nt. Scale bar, 10 μm. The corresponding normalized line-scan intensity profiles are shown.
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A) Fluorescence microscopy images of preassembled equimolar condensates showing reciprocal recruitment of HOTAIR 1-360 and AR LBD . Upper: DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after addition of 25 μM Cy3-labeled HOTAIR 1-360 . Lower: DyLight488-labeled AR NTD and Cy3-labeled HOTAIR 1-360 droplets after addition of 20 μM Cy5-labeled AR LBD . An equimolar mixture of Cy5-labeled AR LBD and Cy3-labeled HOTAIR 1-360 (25 μM each) did not undergo LLPS. Scale bar, 10 μm. Representative of three biological replicates. B) Quantification of Cy3-labeled HOTAIR 1-360 enrichment in preassembled AR NTD -HOTAIR 1-360 droplets before and after the addition of 20 μM Cy5-labeled AR LBD . C) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of 25 μM Cy3-labeled HOTAIR 1-360 . D) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of an equivalent amount of PolyU-20nt. For B-D, enrichment was calculated from raw-image fluorescence measurements. Boxes indicate quartiles and the mean; dots represent individual droplets across three biological replicates. Representative images are shown with identical linear scaling within each channel across conditions. E-G) Fluorescence microscopy images from three biological replicates of preassembled equimolar DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after the addition of E) 400 μM SLNCR1-20nt, F) HOTAIR-20nt, or G) PolyU-20nt. Scale bar, 10 μm. The corresponding normalized line-scan intensity profiles are shown.

Journal: bioRxiv

Article Title: Binding of long non-coding RNAs within the androgen receptor N-terminal tail modulates interdomain communication and liquid-liquid phase separation

doi: 10.64898/2026.05.28.728402

Figure Lengend Snippet: A) Fluorescence microscopy images of preassembled equimolar condensates showing reciprocal recruitment of HOTAIR 1-360 and AR LBD . Upper: DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after addition of 25 μM Cy3-labeled HOTAIR 1-360 . Lower: DyLight488-labeled AR NTD and Cy3-labeled HOTAIR 1-360 droplets after addition of 20 μM Cy5-labeled AR LBD . An equimolar mixture of Cy5-labeled AR LBD and Cy3-labeled HOTAIR 1-360 (25 μM each) did not undergo LLPS. Scale bar, 10 μm. Representative of three biological replicates. B) Quantification of Cy3-labeled HOTAIR 1-360 enrichment in preassembled AR NTD -HOTAIR 1-360 droplets before and after the addition of 20 μM Cy5-labeled AR LBD . C) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of 25 μM Cy3-labeled HOTAIR 1-360 . D) Quantification of Cy5-labeled AR LBD enrichment in preassembled AR NTD -AR LBD droplets before and after the addition of an equivalent amount of PolyU-20nt. For B-D, enrichment was calculated from raw-image fluorescence measurements. Boxes indicate quartiles and the mean; dots represent individual droplets across three biological replicates. Representative images are shown with identical linear scaling within each channel across conditions. E-G) Fluorescence microscopy images from three biological replicates of preassembled equimolar DyLight488-labeled AR NTD and Cy5-labeled AR LBD droplets after the addition of E) 400 μM SLNCR1-20nt, F) HOTAIR-20nt, or G) PolyU-20nt. Scale bar, 10 μm. The corresponding normalized line-scan intensity profiles are shown.

Article Snippet: PolyU and purified HOTAIR 1-360 were 3′-end labeled with Cy5 or Cy3 using T4 RNA ligase and Cy5- or Cy3-labeled cytidine-5′-phosphate-3′-(6-aminohexyl)phosphate (triethylammonium salt) purchased from Jena Bioscience.

Techniques: Fluorescence, Microscopy, Labeling