Extended Data Fig. 3: DAAM2 is required for nuclear actin assembly at the AR.
From: Formin-mediated nuclear actin at androgen receptors promotes transcription
a ELYRA7 SIM-burst mode timelapse video of NIH3T3 cell, stably expressing the nuclear actin chromobody-mCherry transfected with AR-GFP (magenta) displaying nuclear actin tails at the AR after DHT-induced signaling for 16 h; areas of zoom are indicated. b Images of NIH3T3 cells stably expressing nAC-mcherry (green) and transfected with AR-GFP (magenta). Cells were treated with si-ctrl or siDAAM2 for 72 h and imaged after DHT addition for 16 h showing inhibition of actin-positive AR droplet formation; area of zooms are indicated; zoom 2 is indicated in Fig. 4d. c Western blot showing DAAM2 knockdown efficiency after 72 h. d SIM-burst mode timelapse video of LNCaP cells transfected with AR-GFP (magenta), DAAM2-mScarlet (orange) and nAC-SNAP (green) and stimulated with DHT for 16 h, showing DAAM2 mediated nuclear actin assembly at bigger AR droplet indicated by white asterisks. e Western blot of PSA reporter gene lysates in LNCaP cells showing expression of transfected NLS-BFP-R62D-myc and NLS-BFP-myc using anti myc antibody. Histone H3 serves as loading control. f Western blot of PSA reporter gene lysates in LNCaP cells showing expression of transfected NLS-Arpin, NLS-d.n.Arp2 and Exportin-6 using anti mCherry, anti RFP antibodies. GAPDH serves as loading control. g,h Representative Western blot displaying knockdown efficiency of DAAM2 and DIAPH3 in LNCaP cells 48 h after siRNA transfection using anti DAAM2 and anti DIAPH3 antibodies. Tubulin serves as loading control. i Representative Western blot showing test-expression of si-RNA resistant wt and mutant DAAM2-mScarlet constructs used for PSA gene reporter assay, detected with anti RFP antibody.
