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Fig. 7. RNAi knockdown of BAF and lamin A. (A) Western blotting analysis of HeLa cells after siRNA treatment for BAF or lamin A. (B) Number of cells showing core localization after knockdown of BAF or lamin A. Loss of BAF abolished core localization of both emerin and lamin A. (C) Representative image data which was analyzed to generate the results shown in B. Scale bars: 10 µm. (D) Western blotting analysis of HeLa cells stably expressing GFP-BAF after siRNA treatment for BAF, lamin A or emerin. (E) Electron microscopy images of HeLa cells subjected to siRNA treatment for control luciferase (a) and BAF (c). The respective insets (b,d) are fluorescence images of the same specimen: magenta represents chromosomes and green represents BAF. The arrows show the BAF core (a,b), and the arrowheads indicate no accumulation of BAF (c,d). Scale bars: 2 µm (a,c), 10 µm (b,d). (F) Time-lapse images of living cells stably expressing GFP-BAF are shown for control RNAi (luciferase RNAi), lamin A RNAi, and emerin RNAi. Numbers on the top represent the time in minutes after the metaphase-anaphase transition. Numbers on the left or the bottom represent the lifetime of the core region. Scale bars: 10 µm. (G) HeLa cells were subjected to siRNA treatment for control luciferase, BAF, lamin A and emerin, as indicated on the left. After siRNA treatment, cells expressing GFP-BAF were fixed with formaldehyde and observed (GFP-BAF, labeled at the top); cells without GFP-BAF were fixed with methanol and endogenous proteins (emerin, lamin A and lamin B as indicated) were detected by indirect immunofluorescence with specific antibodies. Scale bar: 10 µm.

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