Figure 1.
The expression of caspase-2 in various cancer cell lines.
A, Human XP-C cells were UV irradiated at 10 J/m2, and further cultured for the indicated time periods. Whole cell lysates were prepared and subjected to Western blot to detect casp-2L, casp-2S and cleaved casp-2L simultaneously with the anti-casp-2 antibody. B, C. The expression of casp-2L and casp-2S was determined by Western blot analysis of protein extracts of the cervical cancer cell line (HeLa), ovarian cancer cell lines (A2780, CP70, CDDP, 2008, 2008C13, PEO1, PEO4 and SKOV3), lung cancer cell lines (A549, H460, and H1299), colorectal cancer cell lines (HCT116p53+/+ and HCT116p53−/−), and glioblastoma cell lines (Gli36, U87MG, and T98G) with the anti-casp-2 antibody used in A. Tubulin was detected to serve as loading control.
Figure 2.
Casp-2S inhibits cisplatin-induced apoptosis.
Casp-2S was either over-expressed in A2780 and 2008 cells (A), or knocked down in CDDP cells and 2008C13 cells (B) for 48 h. Cells were treated with cisplatin for another 48 h, the cleaved PARP and cleaved casp-3 were detected in these cells by using Western blotting with anti-cleaved PARP and anti-cleaved casp-3 antibodies. Meanwhile, the expression of casp-2L and casp-2S was also detected. Tubulin was blotted to serve as a loading control.
Figure 3.
Casp-2S inhibits cisplatin-induced phosphatidylserine externalization.
Casp-2S was over-expressed in A2780 cells (A), or knocked down in CDDP cells (B) for 48 h. Cells were then treated with cisplatin for another 24 and 48 h, and the phosphatidylserine externalization was detected with Annexin V staining by using Flow cytometry. n = 3, bar: SD, *: p<0.05 compared to the cisplatin-treated vector or control siRNA transfected cells at the same time point.
Figure 4.
Casp-2S interacts with cytoskeleton proteins.
A, Casp-2S was purified from HeLa cells stably transfected with the FLAG-tagged casp-2S using an anti-FLAG affinity gel. Purified proteins were separated by SDS-PAGE and stained with Coomassie blue. The differentially expressed bands in the HeLa-casp-2S cells were identified by mass-spectrometry. B, FLAG-tagged casp-2S was immunoprecipitated from the HeLa-Casp-2S cells and Fodrin was detected using Western blotting with the anti-Fodrin antibody. C, D, Fodrin was immunoprecipitated from the HeLa-Casp-2S cells (C) or CDDP cells (D) with an anti-Fodrin antibody, and the existence of casp-2S was detected with anti-casp-2S antibody.
Figure 5.
Casp-2S inhibits cisplatin-induced Fodrin cleavage.
Casp-2S was either over-expressed in A2780 cells and 2008 cells (A), or knocked down in CDDP cells and 2008C13 cells (B) for 48 h as in Fig. 2. Cells were treated with cisplatin for another 48 h, and the cleavage of Fodrin was detected with the anti-Fodrin antibody. Tubulin was detected as a loading control.
Figure 6.
Casp-2S and cleavage of Fodrin mainly occur in cytoplasm.
A, HeLa cells were UV irradiated at 20 J/m2, and further cultured for 4 h. Cytoplasm (C) and nuclei (N) were separated and the Fodrin cleavage in protein extracts was detected with an anti-Fodrin antibody. Tubulin and PARP were used to indicate the cytoplasm and nuclei fraction, respectively. B. CDDP cells were fractionated into cytoplasm and nuclei, the endogenous casp-2L and casp-2S were detected simultaneously with the anti-casp-2 antibody. C, Two clones of casp-2S stably expressed HeLa cells were fractionated to the cytoplasm and nuclei, casp-2L and FLAG-tagged casp-2S were detected with the anti-casp-2 and the anti-FLAG antibodies, respectively. D, HCT116 p53−/− and HCT116 p53+/+ cells were transiently transfected with FLAG-tagged casp-2S, UV irradiated at various doses, and further cultured for 4 h. The cytoplasm and nuclei were separated, the Fodrin cleavage, casp-2L, and FLAG-tagged casp-2S were detected with anti-Fodrin, anti-casp-2 and anti-FLAG antibodies, respectively.
Figure 7.
Caps-2S inhibits cisplatin-induced membrane blebbing.
A, CDDP cells were transfected with casp-2S siRNA or control siRNA for 48 h, and treated with cisplatin for 48 h. The apoptotic bleb and dead cells were stained separately as described in Materials and Methods. B, The number of apoptotic blebbing cells were counted and plotted. n = 50, bar: SD, *: p<0.05 compared to siControl.