Figure - PMC

Skip to main content

View full-text article in PMC

. Author manuscript; available in PMC: 2012 Aug 8.

Published in final edited form as: Ann Surg Oncol. 2011 Nov 16;19(Suppl 3):634–645. doi: 10.1245/s10434-011-2142-0

FIG. 2 — Transient ZEB1 knockdown suppresses cellular proliferation and liquid and soft agar colony formation in MPM cell lines. (a) Western blots of ZEB1, E-cadherin, and vimentin in ACC-MESO-1 and H2052 transfected with ZEB1 knockdown siRNA or control oligos. Actin was used as a loading control. (b) Cell proliferation (WST-1) assay for ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were transfected with 3 different siRNA oligos targeting ZEB1 or control oligos. Cells were counted 48 h after transfection, and 1000 cells were plated in 96-well plates. Absorbance values were determined 96 h after transfection. Results are from 3 independent experiments with 8 replicates each and shown as mean ± SD. Values of cells transfected with control oligos are set as 100%. (c) Liquid colony formation assay of ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were counted 48 h after transfection, and 500 to 1000 cells were plated in 6-well plates in triplicate, cultured for 2 weeks, and stained with methylene blue. Colonies >2 mm were counted. Results are from 3 independent experiments and shown as mean ± SD. Colony numbers of cells transfected with control oligos are set as 100%. (d) Soft agar colony formation assay for ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were counted 48 h after transfection, and 1000 cells were suspended in 0.37% SeaKem GTG Agarose (Lonza, Rockland, ME) in triplicate 12-well plates. About 2 weeks later, colonies (>50 cells) were counted. Results from 3 independent experiments are shown as mean ± SD. Colony numbers of cells transfected with control oligos are set as 100%. (e) qRT-PCR analysis of ZEB1, E-cadherin, and vimentin in Y-MESO-8A and Y-MESO-29 transfected with ZEB1 knockdown siRNA or control oligos. (f) Liquid colony formation assay of Y-MESO-8A and Y-MESO-29 cells transfected with ZEB1 knockdown siRNA or control oligos. (g) WST1 and liquid colony formation assays of MeT-5A cells transfected with ZEB1 knockdown siRNA or control oligos. **P < 0.01, ***P < 0.001 (Mann–Whitney U-test)

FIG. 2 — Transient ZEB1 knockdown suppresses cellular proliferation and liquid and soft agar colony formation in MPM cell lines. (a) Western blots of ZEB1, E-cadherin, and vimentin in ACC-MESO-1 and H2052 transfected with ZEB1 knockdown siRNA or control oligos. Actin was used as a loading control. (b) Cell proliferation (WST-1) assay for ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were transfected with 3 different siRNA oligos targeting ZEB1 or control oligos. Cells were counted 48 h after transfection, and 1000 cells were plated in 96-well plates. Absorbance values were determined 96 h after transfection. Results are from 3 independent experiments with 8 replicates each and shown as mean ± SD. Values of cells transfected with control oligos are set as 100%. (c) Liquid colony formation assay of ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were counted 48 h after transfection, and 500 to 1000 cells were plated in 6-well plates in triplicate, cultured for 2 weeks, and stained with methylene blue. Colonies >2 mm were counted. Results are from 3 independent experiments and shown as mean ± SD. Colony numbers of cells transfected with control oligos are set as 100%. (d) Soft agar colony formation assay for ACC-MESO-1 and H2052 cells transfected with ZEB1 knockdown siRNA or control oligos. Cells were counted 48 h after transfection, and 1000 cells were suspended in 0.37% SeaKem GTG Agarose (Lonza, Rockland, ME) in triplicate 12-well plates. About 2 weeks later, colonies (>50 cells) were counted. Results from 3 independent experiments are shown as mean ± SD. Colony numbers of cells transfected with control oligos are set as 100%. (e) qRT-PCR analysis of ZEB1, E-cadherin, and vimentin in Y-MESO-8A and Y-MESO-29 transfected with ZEB1 knockdown siRNA or control oligos. (f) Liquid colony formation assay of Y-MESO-8A and Y-MESO-29 cells transfected with ZEB1 knockdown siRNA or control oligos. (g) WST1 and liquid colony formation assays of MeT-5A cells transfected with ZEB1 knockdown siRNA or control oligos. **P < 0.01, ***P < 0.001 (Mann–Whitney U-test)