Extended Data Fig. 2: Microscopy validation and quantification analysis.
From: Formin-mediated nuclear actin at androgen receptors promotes transcription
a 3D-volume rendering of DAAM2 IF signal for homozygous wt and heterozygous mutant cells, showing DAAM2 distribution throughout the cell. b Polystyrene beads (Bangs Laboratories, IN, USA) with 200 nm diameter labelled with Dragon Green (Exc: 480/Em: 520) or Flash Red (Exc: 660/Em: 690) were premixed (2:1 ratio) and subsequently mixed with viscous Mowiol resin for embedding. Z-stacks of beads were acquired with 0.091 µm z-distance in lattice-SIM mode similar to the acquisition of droplets in nuclei which are shown at the top of the figure. Droplets and beads were analyzed for colocalizing volume (CoLo). AR-/DAAM2 droplet pairs are closer than the Rayleigh criterion. In contrast, no beads were observed that missed the Rayleigh criterion. An additional comparison of lattice-SIM illumination and laser-epi illumination is shown in the bottom panel. c For comparing the association of beads and droplets a colocalization analysis was performed similar to Extended Data Fig. 2d. 15 bead pairs with differential fluorescence and maximal proximity in the lateral- or z-plane were selected for analysis and compared to representative 15 droplet pairs in the same size of DHT treated cells from Fig. 2f, j; Extended Data Fig. 2d. Droplets have an increased colocalizing volume indicating closer association. d Quantification of colocalizing volume between DAAM2 and AR of detected colocalization events in Fig. 2j. e Quantification showing percentage of DAAM2 droplets colocalizing with AR droplets in comparison to total nuclear DAAM2 droplets. f Immunofluorescence of endogenous AR (magenta) and RNA Polymerase-II (Ser2P, cyan) in control scrotal cells, treated with EtOH or DHT as well as addition of Hexanediol or Swinholide to DHT-treated cells. Area of zooms are indicated above (upper panel); lower panels displays magnified areas. Colocalization between AR and RNA Pol-II Ser2P is indicated by white asterisks. Scale bar overview: 3 µm; zoom: 0.3 µm. All images are shown as maximum intensity projections.
