Microscopy validation of proteins with a high SPK-CEN ratio. (A) Nuclear speckle localization (SC35, red) of MFAP1 or ZNF207 (green) in U2OS after immunostaining of methanol-fixed cells (but not after formaldehyde fixation; see Fig. S2 D). DNA (DAPI), blue. (B and C) Nuclear speckle (SC35 immunostaining, red) localization of GFP-MFAP1 and GFP-PRPF38A (GFP-PR38A) after formaldehyde fixation at lower transgene expression levels (upper panel). At higher expression levels (lower panel), GFP-MFAP1 and GFP-PR38A increasingly form droplet-like bodies outside but frequently adjacent to nuclear speckles. (C) Arrowhead points to nucleus with lower GFP-PR38A expression and only nuclear speckle staining; arrow points to nucleus with high GFP-PR38A expression. Insets are magnifications of boxed areas. (D) Colocalization of tagRFP-MFAP1 (red) and GFP-PRPF38A (green) in nuclear speckles (SON immunostaining, magenta) and bodies outside of speckles in cells fixed with formaldehyde. (E) GFP-ZNF207 (green) localizes in nuclear speckles (Cherry-SON, red) in live cells (but not after formaldehyde fixation; see Fig. S2 E). Scale bars: main panel, 10 µm; inset, 2 µm.