Journal: Oncogene

Article Title: MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines

doi: 10.1038/onc.2013.131

Figure Lengend Snippet: A) 3D melanoma spheroids growing in a collagen matrix and treated with increasing doses of the MEK inhibitor UO126 for 72 h show invasive edges. Sample images are shown. Scale bar represents 200 microns for all panels except 1205Lu (600 microns). B) Quantitation of melanoma cell motility from spheroid edge in cell lines grown as spheroids as shown for (A). Values indicate the relative spheroid invasion area compared to the vehicle control-treated spheroids. The invasion observed in the treated spheroids was significantly higher than in the untreated spheroids (p=0.0095), controlling for cell line (p<0.0001). Experiments were conducted in triplicate. C) The early melanoma stage cell lines WM35 and WM793 are sensitive to UO126 and display reduced growth and invasion upon a 72 h treatment. Spheroid size differences are delineated in red, based on circumference of the untreated samples. Scale bar represents 600 microns. D) Transwell invasion assay showing DAPI staining of single cells invading through a collagen-coated semi-porous membrane in the presence of UO126 (10μM). Cells were allowed to invade for 72 h and representative images are shown. Scale bar represents 300 microns. E) Graph showing relative invasion for transwell invasion experiments as conducted in (D). Experiments were conducted in triplicate; (WM3918, p=0.010; WM983B, p=0.999; 1205Lu, p=0.010). F) Computer generated 3D model of a skin reconstruct section depicting invading 1205Lu melanoma cells (green). The model is based on 2-photon images and allows a 3D view of the invasion profile of GFP-tagged melanoma cells in a skin reconstruct, as well as the quantitation of the perpendicular distance traveled by individual 1205Lu cells from the basement membrane. Multiple skin reconstruct sections were imaged, recreated in 3D, and quantitated based on this model and results are summarized in (G). G) Histogram summarizing the average distance travelled by invasive 1205Lu cells (in microns) in skin reconstructs exposed to vehicle control or UO126 (10uM). The perpendicular distance was measured over three different sections per condition, tracking the 5 most invasive cells per section using the 3D modeling system shown in (F). This distance was significantly higher for MEK-inhibitor treated cells than for untreated cells (p<0.0001). Error bars represent standard errors.

Article Snippet: Invasion assay Transwell invasion chambers (Becton Dickinson), 24-well format, were used according to the manufacturer’s instructions and 50μL of collagen mix (as described above) were used to coat the membrane prior to use.

Techniques: Quantitation Assay, Transwell Invasion Assay, Staining, Generated

Journal: Oncogene

Article Title: MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines

doi: 10.1038/onc.2013.131

Figure Lengend Snippet: A) STAT3 knockdown cells were grown as spheroids, embedded in collagen and allowed to invade for 72 h in the presence of UO126 or vehicle control. Spheroid invasion differences are delineated in red and representative images are shown. Scale bar represents 300 microns. Experiments were conducted in triplicate. Right panel: STAT3 knockdown efficiency following melanoma cell infection with a lentiviral vector. B) UO126 (MEK inhibitor, 10μM), Dasatinib (SRC/RTK inhibitor, 1uM), and CPA-7 (STAT3 inhibitor, 10μM), were added to collagen-embedded spheroids for 72 h as single agents or combinations as indicated. Survival and invasion were monitored using a live/dead assay. Green fluorescence indicates metabolically active (live) cells, red fluorescence indicates membrane compromised (dead) cells. Experiments were conducted in triplicate and representative images are shown. Scale bar represents 600 microns. C) Graph depicting the invasion of melanoma cells using a transwell invasion assay following treatment with UO126 (MEK inhibitor, 10μM), Dasatinib (SRC/RTK inhibitor, 1uM), and CPA-7 (STAT3 inhibitor, 10μM), or combinations thereof. The ANOVA was significant for drug treatment but effects were different for the two cell lines. Specific comparisons show significant differences between UO126 and UO126+CPA-7 treated cells for both cell lines and a significant difference between UO126 and UO126+dasatinib for WM3918 only.

Article Snippet: Invasion assay Transwell invasion chambers (Becton Dickinson), 24-well format, were used according to the manufacturer’s instructions and 50μL of collagen mix (as described above) were used to coat the membrane prior to use.

Techniques: Infection, Plasmid Preparation, Live Dead Assay, Fluorescence, Metabolic Labelling, Transwell Invasion Assay

Journal: Oncogene

Article Title: MEK inhibition affects STAT3 signaling and invasion in human melanoma cell lines

doi: 10.1038/onc.2013.131

Figure Lengend Snippet: A) Transwell invasion assay showing DAPI staining of single melanoma cells invading through a collagen-coated semi-porous membrane in the presence of SB590885 (1μM). Cells were allowed to invade for 20 h. Representative photomicrographs are shown and scale bar represents 600 microns. A histogram shows the quantitation of melanoma cell invasion from transwell assays conducted in triplicate for two cell lines. Relative number of invasive cells in treated compared to the vehicle control-treated cells are shown. Relative invasion was significantly different for both cell lines (WM938B, p=0.016 and 1205Lu, p=0.004). Error bars depict standard errors. B) Western blot analysis of immunolysates from BRAFV600E mutant melanoma cell lines treated with SB590885 or vehicle control for 72 h. WT indicates the parental cell lines; BR indicates the BRAF inhibitor-resistant cell line counterpart. Phosphorylated and total levels of STAT3, MEK, and ERK are shown, as well as the HSP90 loading control. C) SB590885 (SB-885; 1μM) and CPA-7 (10μM) were added to collagen-embedded spheroids for 72 h. Spheroids were generated from BRAFV600E mutant melanoma cell lines (parental or BRAF inhibitor-resistant (BR)). Survival and invasion were monitored using a live/dead assay and representative images are shown. Green fluorescence indicates metabolically active (live) cells; red fluorescence indicates membrane compromised (dead) cells. Independent experiments were conducted in triplicate. Scale bar represents 600 microns.

Article Snippet: Invasion assay Transwell invasion chambers (Becton Dickinson), 24-well format, were used according to the manufacturer’s instructions and 50μL of collagen mix (as described above) were used to coat the membrane prior to use.

Techniques: Transwell Invasion Assay, Staining, Quantitation Assay, Western Blot, Mutagenesis, Generated, Live Dead Assay, Fluorescence, Metabolic Labelling

Journal: World Journal of Gastroenterology : WJG

Article Title: Fascin promotes the motility and invasiveness of pancreatic cancer cells

doi: 10.3748/wjg.v17.i40.4470

Figure Lengend Snippet: The effect of fascin overexpression on invasiveness of MIA PaCa-2 cells. A: Matrigel invasion assay showed that the overexpression of fascin dramatically increased cell invasive properties compared with control cells. (aP < 0.05); B: In vivo invasion assay. Tumors from fascin-overexpressing MIA PaCa-2 Fascin cells showed an increase in skin invasion when compared with control MIA PaCa-2 Vector cells; C: Histological examination of skin invasion of tumors from MIA PaCa-2 Fascin cells and control MIA PaCa-2 cells. (a) Tumor from MIA PaCa-2 Fascin cells had skin invasion and (b) tumor from MIA PaCa-2 Vector cells showed no skin invasion (HE, × 150). HE: Hematoxylin and eosin.

Article Snippet: Invasion assay For the invasion assay, the BioCoat Matrigel Invasion Chamber (Becton Dickinson Bioscience, United States) was used according to the manufacturer’s instructions.

Techniques: Over Expression, Invasion Assay, In Vivo, Plasmid Preparation