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Fig. 5. Silencing of Nesprin-2 Giant in human keratinocytes affects nuclear shape and Nesprin-2 C-terminal-isoform localization. (A) Indirect immunofluorescence analysis of control and Nesprin-2-Giant-silenced HaCaT cells using Nesprin-2-Giant-specific antibodies (K20-478). Nuclei are stained by DAPI. Arrows indicate the NE phenotypes observed. Scale bar: 10 µm. (B) Histogram representing a statistical evaluation of specific NE phenotypes in control and Nesprin-2-Giant knockdown HaCaT cells (300 cells counted for each experiment). Error bars denote s.e.m. (C) Control and Nesprin-2-Giant knockdown cells were subjected to indirect immunofluorescence analysis using Nesprin-2-Giant-specific (K20-478) and Nesprin-2 polyclonal (pAbK1) antibodies; the latter also detects the C-terminal isoforms. Note that the pAbK1 pattern is affected or absent in Nesprin-2-Giant-silenced cells (asterisks). Nuclei are stained by DAPI. Scale bar: 10 µm. Confocal images are shown. (D) Immunoblot analysis of Nesprin-2-Giant-silenced human keratinocytes and control cells. Note the loss of the 800-kDa Giant isoform (arrowhead) in Nesprin-2-Giant-silenced human keratinocytes. Silencing of the Nesprin-2-Giant isoform also results in a downregulation of the smaller isoforms (^*) detected by pAbK1. Actin is shown for control.

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