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. 2014 Oct 30;5(6):527–541. doi: 10.4161/19491034.2014.970105

Figure 6.

Figure 6.

Bax KASH induces generation and rupture of nuclear bubbles, which are inhibited by the L63E and Δα5/6 mutations. (A) FLAG-Bax KASH is associated with the NE. Bax/Bak DKO MEFs were transfected with FLAG-Bax KASH and 24 h later were stained with anti-FLAG antibody, Mab414 antibody (to detect NPC) and Hoechst 33258 dye (to detect the nuclei). Thereafter, the cells were visualized by confocal fluorescence microscopy. Presented results are from a representative experiment (out of at least 3 independent experiments). Bar = 10 μm. (B) Bax/Bak DKO MEFs were co-transfected with RFP-nucleolin together with GFP-mini-nesprin-2G (mini-nesp2G) or FLAG-Bax KASH. Hoechst 33342 was added to the culture medium 24 h later, and RFP-nucleolin and Hoechst fluorescence was monitored by time-lapse microscopy. The presented results are quantification of the percentage of cells exhibiting bubbles, bubbles that ruptured (ruptured bubble), or no bubbles (no bubble) during the recording period (30-s intervals) out of all recorded RFP-nucleolin-expressing cells. Fisher's exact test of percentage of bubbles (ruptured bubble and bubble) revealed significant differences (**p = 0.0003) between GFP-mini-nesprin-2G and FLAG-Bax KASH. (C) Bax/Bak DKO MEFs were co-transfected with RFP-nucleolin together with GFP-mini-nesprin-2G (mini-nesp2G), FLAG-Bax KASH or the indicated FLAG-Bax KASH mutants. The cells were fixed and stained with Hoechst 33258 dye and anti-FLAG (to detect Bax KASH-and Bax KASH mutant-expressing cells) 24h later. RFP-nucleolin distribution in the nucleus, bubbles and cytosol in the transfected cells was determined microscopically. The results are expressed as the percentage of RFP-nucleolin in the nuclei, bubbles and cytosol for each treatment out of the respective RFP-nucleolin-expressing cells (n = 500 cells; 5 independent experiments). Pearson Chi-square test revealed significant differences (p < 0.0001) in the percentage of nuclear, bubble and cytosolic distribution of RFP-nucleolin between GFP-mini-nesprin-2G and FLAG-Bax KASH, or between FLAG-Bax KASH and each of FLAG-Bax KASH mutants.