JCS03418 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Silencing of DRP1 rescues the increased radical production within SenP shRNA-treated cells. (A) COS7 cells stably expressing shRNA to knock down SenP5 were transfected with either non-targeting N-T, or DRP1 targeted siRNA, as described in Materials and methods. Whole cell extracts were separated by SDS-PAGE and probed for DRP1 and the control mitochondrial matrix chaperone HSP60. The same knock-down of DRP1 was achieved in stable shRNA vector control cells (not shown). (B) Same as in Fig. 7D, but in cells silenced or not (with non-targeting siRNA) with DRP1 dsRNA. Each value represents the mean ± s.d. of two independent aliquots, *P<0.001; **P<0.01.

• Movie 1 -

  Movie 1. Morphology of SenP5 silenced mitochondria. Time-lapse video microscopy of SenP5-silenced cells labeled with 100 nM MitofluorRed633. Images were taken every 2 seconds with a Polychrome IV monochrometer from TillPhotonics and Cy5 filters and played back at 10 frames/second. A number of fission events are evident within this short video.

• Movie 2 -

  Movie 2. Reconstructed stack of mitochondria from SenP5 silenced cells stained with Tom20. 3D video reconstructions of a serial Z-stack of a region of mitochondria within a cell silenced for SenP5 and stained with anti-cytochrome c.

• Movie 3A
• Movie 3B -

  Movie 3. SUMO:YFP is recruited in puncta at sites of mitochondrial fission in SenP5-silenced cells. Time-lapse confocal video microscopy showing a cropped region of (A) control (48 frames) or (B) SenP5-silenced cells (8 frames) transfected with SUMO-1:YFP and pOCT:CFP. Images were taken every 2 seconds with the Olympus FV1000 confocal microscope with the 514 Argon (SUMO:YFP, shown green) and 440 solid state diode (OCT:CFP, shown white) lasers and played back at 2 frames per second. In both cases the SUMO-1:YFP is visualized at sites of active fission, but there are additional SUMO-1 puncta in the SenP5-silenced cells.