Figure 4.
For figure legend see page 533.Figure 4 (See previous page). Restoration of Bax expression in Bax/Bak DKO MEFs promotes SIGRUNB in a caspase- and Bcl-xL-independent manner. Bax/Bak DKO MEFs were co-transfected with RFP-nucleolin and pEGFP or with RFP-nucleolin and GFP-Bax in the absence (A) or presence (B) of 20 μM Q-VD-OPH (QVD) or together with Bcl-xL (C). After 24 h, Hoechst 33342 was added to the culture medium and RFP-nucleolin, GFP-Bax and Hoechst fluorescence-expressing cells were monitored by live-cell time-lapse microscopy at 30-s intervals. Selected (still images from time lapse imaging) from each experiment are shown from the same field visualized separately for detection of RFP-nucleolin, GFP-Bax and Hoechst (A and C are from Vid. S4 and S5). Note that the bubbles (arrows) do not contain DNA. The dashed oval in (A) indicates the position of a DNA-lacking nuclear bubble (no Hoechst staining). Indicated time points (min:s) are expressed relative to the first image selected for presentation (t = 00:00) which corresponds to the image captured 44, 4 or 0 min into the recording (for A, B and C, respectively). Bar = 10 μm. (D) Quantification of the percentage of cells exhibiting nuclear bubbles (bubble), nuclear bubbles that ruptured (ruptured bubble) or that did not exhibit bubbles (no bubble) during recording. The number (n) of cells that were monitored for each treatment is indicated. Fisher's exact test of percentage of bubbles (ruptured bubble and bubble) revealed a significant difference (**p = 0.001) between GFP-Bax and pEGFP. The results shown are from 2, 3, 2, and 3 independent experiments for pEGFP, GFP-Bax, GFP-Bax+QVD and GFP-Bax+ Bcl-xL treatments respectively.