MFAP1 KD increases nuclear speckle size and changes nuclear distribution of nuclear speckle proteins after transcriptional inhibition. (A) Western blot showing (top) KD of endogenous MFAP1 protein after siRNA treatment against MFAP1 (siMFAP1) versus control siRNA (siCTRL) in U2OS cells. Tubulin (bottom) is the loading control. (B) SON immunostaining (green) in U2OS cells transfected with siCTRL or siMFAP1. DNA (DAPI) is shown in blue. Boxed areas are magnified on the right. Box plots show nuclear speckle sizes after siMFAP1 versus siCTRL (n = 80 from three independent experiments t test; ***, P < 0.0001). (C) Schematic depicting increased nuclear speckle size with MFAP1 KD followed by rounding of nuclear speckles after transcriptional inhibition but with redistribution to nucleolar periphery. (D and E) SON (green) and nucleophosmin (Nuc; red) immunostaining with DNA (DAPI, blue) counterstaining of U2OS cells transfected with siCTRL (top) or siMFAP1 (bottom) 48 h before DRB treatment for 2 h (D) or α-amanitin (α-ama) overnight treatment (E). Boxed areas in D are magnified on the right. (F) Video stills (Video 1) showing nuclear speckles after addition of DRB to inhibit transcription in MFAP1 KD (siMFAP1) U2OS cells (top of each panel, time in minutes). Arrows (top) indicate nuclear speckle accumulating around nucleolar periphery. Bottom arrows show a larger nuclear speckle merging with nucleolar periphery. Nuclear speckles were visualized with GFP-ZNF207. Scale bars: main panel, 10 µm; inset, 5 µm.