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. 2004 Dec;24(23):10223–10235. doi: 10.1128/MCB.24.23.10223-10235.2004

FIG. 3.

FIG. 3.

FIG. 3.

FIG. 3.

Nuclear distribution of Pnn is altered in response to RNAi-mediated CtBP1 knockdown. (A) s-HeLa cells were transiently cotransfected with a targeting vector expressing short hairpin RNA (shRNA) directed against CtBP1 mRNA coding region and the GFP vector to identify the transfected cells (rows 2 and 4) or the GFP vector alone as a control (rows 1 and 3). Cells were immunostained with mouse anti-CtBP1 antibody (panels 1b and 2b) or 143 antibody against the endogenous Pnn (panels 3b and 4b). Panels 1c to 4c show merged DAPI nuclear stain (blue), GFP (green), and Pnn or CtBP (red) images. Arrowheads indicate the transfected cells. Bar, 10 μm. (B) Rows 1 and 2 show HCETs that were transiently cotransfected with a targeting vector expressing short hairpin RNA (shRNA) directed against CtBP1 mRNA coding region and GFP vector to identify the trasfected cells. Cells were immunostained with mouse anti-CtBP1 antibody (panel 1b) and 143 antibody to the endogenous Pnn (panel 2b). Cells that received CtBP1 shRNAi vector exhibited drastic Pnn redistribution from nuclear speckles. Rows 3 and 4 show HCETs that were transiently cotransfected with a targeting vector expressing short hairpin RNA (shRNA) directed against Pnn mRNA coding region and a GFP vector to identify the transfected cells. Cells were immunostained with 143 antibody to the endogenous Pnn (panel 3b) or mouse anti-CtBP1 antibody (panel 4b). Panels 1c to 4c show merged DAPI nuclear stain (blue), GFP (green), and Pnn or CtBP (red) images. In contrast to the CtBP1 RNAi-dependent Pnn relocalization, RNAi-mediated Pnn knockdown did not result in visible CtBP1 redistribution. Arrowheads indicate the transfected cells. Bar, 10 μm. (C) HCETs were transiently cotransfected with a targeting vector expressing short hairpin RNA (shRNA) directed against CtBP1 mRNA coding region and GFP vector to identify the transfected cells. Cells were immunostained with the following antibodies: anti-Pnn (1b), anti-SR proteins (2b), anti-sm proteins (3b), anti-NuMa (4b), and anti-PCNA (5b). Panels 1c to 5c show merged DAPI nuclear stain (blue), GFP (green), and Pnn, SR, sm, NuMa, and PCNA (red) images. Arrowheads indicate the transfected cells. Bar, 10 μm.