Journal: bioRxiv

Article Title: BCL-3 enhances β-catenin signalling in colorectal tumour cells promoting a cancer stem cell phenotype

doi: 10.1101/178004

Figure Lengend Snippet: ( A ) Western analysis confirming BCL-3 suppression and LGR5 downregulation in SW1463 cells seeded into Matrigel. α-Tubulin serves as a loading control. ( B ) Widefield microscopy image of a spheroid grown from a single SW1463 cell following 10 days of culture. 20x objective. Scale bar = 50μm. ( C ) Widefield microscopy images of wells containing BCL-3 knockdown and control SW1463 spheroids. 5x objective. Scale bars = 250μm. ( D ) and ( E ) Spheroid forming assay. ( D ) SW1463 cells transfected with BCL-3 siRNA or negative control siRNA were re-suspended in Matrigel and seeded into 48 well plates, 24 hours post-transfection. Spheroids were cultured for 10 days as previously described . Wells were imaged and analysed using Matlab R2015a software, with subjective gating applied to exclude any cells/debris less than 3000μm 2 in cross-sectional area. ( E ) SW620 cells stably-transfected with pcDNA-BCL-3 WT vector (SW620 BCL-3 ) or empty pcDNA vector (SW620 pcDNA ) as a negative control were seeded into Matrigel, cultured and analysed as in ( D ). One sample t-test. N =3, ± SEM * P <0.05. ( F ) and ( G ) Tumoursphere forming assay. SW1463 cells transfected with BCL-3 siRNA or negative control siRNA were re-suspended in tumoursphere medium and cultured as described previously . Tumourspheres were dissociated and cells counted after 6 days of culture. ( G ) SW620 cells stably-transfected with pcDNA-BCL-3 WT vector or empty pcDNA vector as a negative control were seeded, cultured and analysed as in ( F ). One sample t-test. N =4, ± SEM * P <0.05.

Article Snippet: Images were analysed using Matlab R2015a software (Mathworks, MA, USA).

Techniques: Western Blot, Control, Microscopy, Knockdown, Transfection, Negative Control, Cell Culture, Software, Stable Transfection, Plasmid Preparation