FIG. 4.
Nuclear morphology defects in hMOF-depleted cells. (A) Knockdown of hMSL3 by RNA interference in HeLa cells 4 days after transfection of two independent hMSL3 siRNAs. The Western blot was probed with antibodies against hMSL3 (top panel), hMOF (middle panel), and β-tubulin (bottom panel). (B) Spectrum of nuclear defects in HeLa and HepG2 cells. Panels show hMOF-depleted cells, hMSL3-depleted cells, and control cells 4 days after transfection of siRNA. Nuclei were stained with Hoechst 33342. (C) Still images from a movie of a dividing hMOF-depleted cell with chromatin labeled with H2B-GFP from early prophase (top left) to G1 (bottom right). Arrows indicate lobes appearingin the late telophase. (D) Confocal images of hMOF-depleted and control cells. Top left, confocal slice of mAB414-stained nuclei. Bar, 5 μm. Lamin B1 and lamin A/C (middle left and bottom left, respectively) are stacks of multiple confocal slices. (E) Ultrastructure of a lobulated nucleus of an hMOF-depleted cell. Upper panel: ultrathin section from the substrate side of the nucleus. The inset shows the corresponding LSM optical section of the GFP signal (inverted grayscale) from the same nucleus. The arrow points to the fold shown in detail in the lower panel. Note that the arrowed structure of this fold is also visible in the LSM image. Bar, 5 μm. Lower panel, consecutive ultrathin sections of the nuclear fold, starting with the bottommost section at the left. The inset shows an enlarged view of the front of the fold to show vesicles and filaments in this region. Bar, 1 μm.