Figure 1.
CRC cell growth, viability and death under BH3-mimetic treatment.
(A and B) Flow cytometric analysis for DNA-fragmentation as an indicator for apoptotic death in four CRC cell lines; 48 h treatment with ABT-737 or Obatoclax. (C) MTT assay of HT29 cells after 72 h of Obatoclax and ABT-737 treatment. (P-values: Oba 0.25 µM: 0.003; Oba 0.05 µM: 0.004; ABT-737 5 µM: 0.589) (D) Representative Western blot for cleaved PARP after 24 h of Obatoclax treatment. Tubulin served as loading control. 2 µM treatment with Staurosporine for 24 h served as a positive control for cell death induction. Assays were performed in triplicates. Bars represent mean ± SD. Assays are representative of at least three independent experiments. Oba = Obatoclax, STS = Staurosporine.
Figure 2.
Western blot analysis for E-Cadherin and antiapoptotic Bcl-2 proteins in CRC cells treated with Obatoclax.
(A) Western blotting for E-Cadherin in four CRC cell lines after 24 h treatment with Obatoclax in escalating doses (left). Corresponding densitometric analysis relative to untreated controls and adjusted to Tubulin as loading control. (B) Western blotting for Mcl-1, Bcl-2 and Bcl-xL in HT29 cells (left) and SW480 cells (right) after 24 h treatment with Obatoclax. Tubulin served as a loading control. Western blots are representative for at least three blots from independent experiments.
Figure 3.
Migration of CRC cells treated with Obatoclax.
(A) Representative pictures of wound healing scratch assays of vehicle (upper) and Obatoclax (lower) treated HT29 cells. Scale bar apply for all pictures. (B) Gap closure of HT29 cells after 48 h treatment with Obatoclax. (C) Gap closure of CaCo2 cells treated 48 h with Obatoclax. Assays were performed in triplicates. Bars represent mean ± SD. Assays are representative of at least three independent experiments. *p<0.05, ***p<0.001. Oba = Obatoclax.
Figure 4.
Migration of CRC cells treated with Obatoclax for 7 days in 3D scaffolds.
(A) Representative pictures of HT29 cells in scaffolds after 7 days treatment with Obatoclax (Hematoxylin and Eosin staining. Scale bar applies for both pictures) (B) Corresponding analysis of invasion depth in scaffolds. Assays were performed in triplicates. Bars represent mean ± SD. Assays are representative of at least three independent experiments. ***p<0.001. Oba = Obatoclax.
Figure 5.
Invasion of SW480 cells treated with Obatoclax for 72 h in Matrigel coated Boyden chambers.
SW480 cells were seeded into the upper chamber of a transwell. 48 h after seeding, nuclei on the lower surface were visualized by Hoechst staining. (A) Representative pictures of lower insert surface after Hoechst staining (scale bar indicate magnification for all panels). (B) Five fields of view per insert were counted. N = 5 per group. Values are expressed as mean ± SD. Assays are representative of at least three independent experiments. ***p<0.001. Oba = Obatoclax, ctrl = control.
Figure 6.
Migration of HT29 cells overexpressing antiapoptotic Bcl-2 proteins and treated with Obatoclax.
(A–D) Gap closure of wound healing migration assays of HT29 cells treated with Obatoclax. (A) Vector and Mcl-1 transfected cells (B) vector and Bcl-xL transfected cells and (D) vector and Bcl-2 transfected cells. (C) Representative pictures of wound healing of vector and Mcl-1 transfected cells treated with Obatoclax. Values are expressed as mean ± SD. Assays are representative of at least three independent experiments. ***p<0.001. Vec = vector.
Figure 7.
Cell cycle and proliferation analysis of CRC cells treated with Obatoclax.
(A) Representative flow cytomeric analysis for DNA content in HT29 cells treated with 0.25 µM Obatoclax. 2N = diploid cells in G1-phase, 4N = tetraploid cells in G2-Phase. (B) Graphical analysis of cell cycle phase distribution corresponding to (A). Values are expressed as mean ± SD. ***p<0.001. (C) Rel. mRNA levels of Cyclin D1, p21 and p27 in HT29 and CaCo 2 cells after 24 h treatment with Obatoclax. mRNA levels were quantified by qrT-PCR and normalized to GAPDH as housekeeping gene. Assays are representative of at least three independent experiments. (D) Representative Western blots for Cyclin D1, p21, and p27 in HT29 and CaCo2 cells treated with Obatoclax for 24 h. Tubulin served as a loading control. Oba = Obatoclax, ctrl = control.