Dynamic colocalization of BCL11A-XL and BCL6 within nuclear paraspeckles. (A) Colocalization of BCL11A isoforms with BCL6. Left Panel: COS-7 cells were transiently transfected with FLAG-epitope tagged BCL11A-XL and BCL6, and developed using antibodies reactive with the proteins (see Methods). Nuclei were counterstained using Hoescht 33342. Images were acquired by confocal microscopy. Right Panel: COS-7 cells were transiently transfected with GFP-BCL11A isoform fusion constructs and with a a FLAG-tagged BCL6 construct. Immunostaining was performed 24 hrs post-transfection using monoclonal anti-FLAG primary and rhodamine-conjugated rabbit-anti-mouse secondary antibodies, and then images were collected by confocal microscopy. Colocalization was determined by merging sequentially scanned images from the two channels. (B) Endogenously expressed BCL11A in BJAB B cells forms nuclear dots and colocalizes with BCL6 (see Methods). (C) BCL11A-XL resides within subnuclear domains adjacent to speckles. GFP-BCL11A-XL transfected COS-7 cells were immunostained using an anti-SC35 mAb and rhodamine-conjugated secondary. Confocal co-localization scans were performed as above. (D) Transcription inhibitors alter the localization of BCL11A. COS-7 cells were transiently transfected with pEGFP-BCL11A isoform constructs. At 24 hrs after transfection, Actinomycin D or 5,6-dichloro-1 – D-ribofuranosylbenzimidazole (DRB) was added to transfected cells. Cells were observed by fluorescence microscopy 6 hrs after treatment. Untreated COS-7 cells transfected with the pEGFP-BCL11A isoform constructs are shown as controls. (E) Identification of BCL11A-XL, nuclear dots as paraspeckles by virtue of co-staining with PSP1 and PSP2/nonO/p54nrb antibodies. Also shown in the middle panel is BCL11A-S, characteristically localizing to the cytoplasm, which unlike BCL11A-XL does not overlap with the nuclear paraspeckles. (F) DNase but not RNase treatment alters the localization of BCL11A. COS-7 cells were transiently transfected with pEGFP-BCL11A-XL. At 24 hrs after transfection, cells were treated with RNase (C), RNase and Actinomycin D (E), RNase and DRB (G), DNase (D), DNase and Actinomycin D (F), or DNase and DRB (H). Cells were observed via fluorescent microscopy 6 hrs after treatments. Untreated COS-7 cells transfected with pEGFP-BCL11A-XL are shown as controls (A) and (B). Hoescht 33342 staining of corresponding nuclei is shown to the right of each green fluorescence panel.