Fig. 9. Downregulation of eIF3k promotes the sequestration of active caspase 3 in K8/K18-residing cytoplasmic inclusions. (A) HeLa cells stably expressing eIF3k-specific siRNA (si eIF3k) or control siRNA (si control) as described in Fig. 5 were irradiated with UV and then cultivated for 7 hours. Cells were fixed, double stained with antibody to active caspase 3 and M30 antibody, and examined by confocal microscopy. (Top) The projection view of stacked confocal images, which were created using 19 (for control-siRNA-expressing cells) or 20 (for eIF3k-siRNA-expressing cells) overlaying 0.42-µm z-sections is shown (see Movies 1, 2 in supplementary material for 3D-reconstructed images). (Bottom) The images of a single z-section are shown. Scale bar: 5 µm. (B) Measurement of the correlation coefficient of the M30 (green) and active caspase 3 (red) images shown in A. Confocal images from one section were used for colocalization analysis with the LSM510 software. A total of 14 randomly selected cells in each group were analyzed. (C) Measurement of the percentage of M30-positive dots in a cell with active-caspase-3 signal. M30 dots with a diameter greater than 0.2 µm were counted and the projection view of 3D images from ten randomly selected cells in each group was analyzed. Data shown are mean ± s.d. (D) Interaction of active caspase 3 with K18. HeLa cells irradiated with UV at 0.015 J/cm² were lysed for pull-down analysis with GST, GST-K18 (K18), and GST-K18 head-domain deletion mutant (H). Bound proteins and lysates of HeLa cells with or without UV irradiation were analyzed by western blot with antibody to active caspase 3. The relative amounts of various GST fusion proteins used for pull-down analysis are shown on the bottom panel. (E) Downregulation of eIF3k increases the association of active caspase 3 with K18. HeLa cells expressing eIF3k siRNA or control siRNA were irradiated with UV at 0.015 J/cm² and then cultivated for 12 hours. Cells were lysed with Empigen lysis buffer and lysates were used for immunoprecipitation with anti-K18 antibody. The immunoprecipitates and cell lysates were resolved by SDS-PAGE and then analyzed by western blot with antibodies as indicated. The positions of the19 kDa and 17 kDa fragments of the active caspase 3 as well as the full-length protein (48 kDa) and caspase-3-cleaved K18 (26 kDa) are indicated.