JCS023911 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. PARP cleavage and zVAD-fmk inhibition of PARP cleavage. (A) PARP cleavage after TNFα treatment. HeLa cells were treated with 50 ng/ml TNFα for 6 hours, harvested and western blotted with a PARP antibody. (B) PARP cleavage induced by STS was inhibited by broad based caspase inhibitor zVAD-fmk. Cells were treated for the indicated times in the presence of 50 µM zVAD-fmk, where indicated, harvested and western blotted with a PARP antibody.

• Supplemental Figure S2 -

  Fig. S2. Treatment of cells with latrunculin A disrupts cell cytoskeleton. HeLa cells were treated with 5 µM latrunculin A (LA) for 1 hour, and the cells were observed under a phase microscope. Similarly to the STS-treated cells, LA-treated cells rounded up, indicating disruption of the cytoskeleton.

• Supplemental Figure S3 -

  Fig. S3. Reduction of CAP1 level in CAP1-knockdown cells as detected by immunofluorescence. HeLa WT cells and CAP1 knockdown cells (S3-2) were labeled with mitotracker, cells were fixed and stained with anti-CAP1 antibody as in Fig. 2 and detected using an Alexa Fluor 350 goat anti-mouse IgG (H+L). The cells were observed for mitochondria staining and CAP1 level. CAP1 staining in S3-2 cells is significantly reduced.

• Supplemental Figure S4 -

  Fig. S4. Translocation of Bak to mitochondria after STS treatment. NIH 3T3 cells were treated with STS for 3 hours to stimulate apoptosis. Cells were incubated with mitotracker, fixed, stained with Rabbit anti-Bak (Sigma) and Goat anti-rabbit Alexa Fluor 488. Cells were observed under confocal microscopy.

• Supplemental Figure S5 -

  Fig. S5. Knockdown of cofilin did not prevent CAP1 translocation to mitochondria upon STS stimulation. HeLa cells grown on 100 mm plates were transfected with cofilin shRNA or a scrambled control using lipofectamine. 72 hours after transfection, the cells were treated with 1 µM STS for 2 hours. The mitochondrial and cytosol fractions were collected and western blotted to determine CAP1 translocation to mitochondria. The cofilin knockdown efficiency was typically 50-75% in our experiments.

• Supplemental Figure S6 -

  Fig. S6. Treatment with STS does not affect CAP1 association with actin. 293T cells were transfected with a plasmid expressing a 6xHis tagged CAP1. After 24 hours, the cells were treated with 1 µM STS for 2 hours as indicated, harvested and the fusion protein was purified using nickel beads. After elution with a buffer containing imidazole, the samples were resolved on a SDS gel and stained using the Colloidal Blue Stain Kit (Invitrogen). The (His)CAP1 and actin bands are indicated with arrow and arrowheads, respectively.

• Supplemental Figure S7 -

  Fig. S7. Reduction of cytochrome c release to cytosol in CAP1-knockdown cells detected by immunofluorescence. HeLa WT or CAP1 KD (S3-2) cells were incubated with mitotracker for 1 hour followed by treatment with 100 µM etoposide for 24 hours. Cells were fixed, and stained with cytochrome c antibody and goat anti-rabbit Alexa Fluor 488. Cells were observed under fluorescence microscopy.