Figure 1.
Mcl-1, Bcl-2 and Bcl-xL expression and knockdown in human CRC cell lines.
(A) Western blot analyses of the colorectal cancer cell lines CaCo2, Colo205, SW480 and HT29. Basal expression levels of Bcl-xL, Mcl-1 and Bcl-2. Tubulin served as loading control. Cells are arranged with increasing tumorigenicity from left to right. Here, tumorigenicity indicates the ability of a cell line to metastasize in mice. The lowest tumorigenicity stands for local tumor growth lacking invasiveness; the highest tumorigenicity indicates the formation of distant organ metastasis (liver and/or lung). (B) Western blot analyses of HT29 (left) and SW480 (right) cells after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL 24, 48 and 72 h post transfection. Tubulin served as loading control. The Western blots presented are representative of three independent experiments.
Figure 2.
Viability, cell death and chemosensitization after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
(A) MTT-Assay of SW480 and HT29 cells after knockdown of Mcl-1, Bcl-2 and Bcl-xL. (B) Flow cytometric analyses and corresponding Western blots for cleavage of PARP 48 h after knockdown of Mcl-1, Bcl-2 and Bcl-xL in HT29 (left) and SW480 cells (right). (C) Flow cytometric analysis of HT29 (left) and SW480 cells (right) for pH2AX 48 h after knockdown of Mcl-1, Bcl-2 and Bcl-xL. 24 h Staurosporine treatment (1 µM) served as a positive control for cell death induction. (D) Flow cytometric analysis for DNA-fragmentation of HT29 cells after knockdown of Mcl-1, Bcl-2 and Bcl-xL followed by 48 h treatment with 30 µM oxaliplatin and 0,2% DMSO as a vehicle. Flow cytometry analysis was performed in triplicates. Bars represent mean ± SD. Assays are representative of at least three independent experiments. (oxa = oxaliplatin).
Figure 3.
Proliferation of SW480 and HT29 cells after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
SW480 and HT29 cells were transfected with siRNA against Mcl-1, Bcl-2 and Bcl-xL. 24 h post transfection, cells were pulsed with 20 µM BrdU and prepared for flow cytometry. (A) Representative original flow cytometry data with HT29 cells for BrdU positivity after staining with anti-BrdU antibody coupled to PerCP-CY5.5 fluorophore. (B) Flow cytometric analyses for BrdU incorporation in HT29 and (C) SW480 cells. (D) Total cell count of SW480 cells after knockdown of Mcl-1, Bcl-2 and Bcl-xL. Cells were seeded on 6 well plates, harvested and counted 24, 48 and 72 h post transfection. Values are expressed as means ± SD. Assays were run in triplicates (flow cytometry) and sextuplicates (cell counting). Assays are representative of at least three independent experiments. *p<0,05.
Figure 4.
Migration of SW480 and HT29 cells after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
SW480 and HT29 cells were transfected with siRNA against Mcl-1, Bcl-2 and Bcl-xL and grown as a monolayer. Gap distance was measured every 200 µm along the edge of the gap and gap closure calculated 24, 48 and 72 h post transfection. (A) Representative pictures captured after knockdown of Bcl-2 in HT29 cells (scale bar indicate magnification for all panels). (B) Gap closure kinetics of HT29 cells after knockdown of Mcl-1, Bcl-2 and Bcl-xL (left) and corresponding Western blots (right). (C) Gap closure kinetics of SW480 cells after knockdown of Mcl-1, Bcl-2 and Bcl-xL (left) and corresponding Western blots (right). Assays are representative of at least three independent experiments. Values are expressed as mean ± SD. (p-values for HT29: siMcl-1:<0,001; siBcl-2:<0,001; siBcl-xL: = 0,002. P-values for SW480: siMcl-1: = 0,0011; siBcl-2: = 0,0005; siBcl-xL: = 0,004).
Figure 5.
Migration of Mcl-1, Bcl-2 or Bcl-xL overexpressing SW480 cells.
SW480 cells were transfected with plasmids expressing human Mcl-1, Bcl-2 or Bcl-xL and grown as a monolayer. Gaps were generated and gap closure measured as described. (A) Representative pictures for SW480 cells overexpressing Bcl-2 (scale bar indicates magnification for all panels). (B) Gap closure kinetics of SW480 cells overexpressing Mcl-1, Bcl-2 and Bcl-xL (left) and corresponding Western blots (right). Assays are representative of at least three independent experiments. Values are expressed as mean ± SD. p-values: Mcl-1: = 0,0006; Bcl-2: = 0,0002; Bcl-xL:<0,0001.
Figure 6.
Invasion of SW480 cells after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
SW480 cells were seeded on 6 well plates and transfected as described. 24×105 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 indicates 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<0,01. ***p<0,001.
Figure 7.
Proliferation of HT29 cells in three dimensional scaffolds after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
HT29 cells were transfected with specific siRNA against Mcl-1, Bcl-2 or Bcl-xL. 24 h post transfection, cells were harvested and 1×106 cells were seeded on each scaffold. After 72 h, scaffolds were further processed for Western blotting or immunohistochemistry. (A) Representative pictures of scaffolds after siRNA mediated knockdown of Bcl-xL and corresponding Western blot. (B) Total cells in scaffolds were counted (n = 5 per group, lower left graph). Scaffolds were stained for Ki67 (representative pictures for knockdown of Mcl-1 and lower right graph, scale bar indicates magnification for all panels, n = 5 per group). Values are expressed as mean ± SD. Assays are representative of at least three independent experiments. *p<0,05.
Figure 8.
Migration of HT29 cells in three dimensional scaffolds after siRNA mediated knockdown of Mcl-1, Bcl-2 and Bcl-xL.
HT29 cells were transfected with specific siRNA against Mcl-1, Bcl-2 or Bcl-xL. 24 h post transfection, cells were harvested and 1×106 cells were seeded on each scaffold. (A) Haematoxylin and Eosin staining of transfected cells in scaffolds (representative pictures for knockdown of Bcl-2 in scaffolds, scale bar indicates magnification for both pictures). (B) Invasion depth was measured every 20 µm (n = 5 per group). Values are expressed as mean ± SD. Assays are representative of at least three independent experiments. *p<0,05.