Journal: Cells
Article Title: C1orf106 ( INAVA ) Is a SMAD3-Dependent TGF-β Target Gene That Promotes Clonogenicity and Correlates with Poor Prognosis in Breast Cancer
doi: 10.3390/cells13181530
Figure Lengend Snippet: Increased C1orf106 in non-metastatic breast cancer cells promotes clonogenicity and enhances tumour initiation capacity in vivo. ( a ) Western blot analysis of C1orf106 protein levels in 67NR cells transfected with pCDNA4/TO empty vector, pcDNA4/TO C1orf106-GFP or pcDNA4/TO C1orf106-Flag following zeocin selection. α-tubulin was used as a loading control. ( b ) Mammosphere-forming efficiency of 67NR empty vector and C1orf106 overexpressing cells. Colonies > 50 μm were counted after 5 days in culture (n = 3 biological replicates). ( c ) 67NR empty vector and C1orf106 overexpressing cells were seeded at 100 cells/10cm dish to assess colony-forming capacity (n = 5 biological replicates). Cells were stained after 14 days in culture and colonies > 50 cells counted. Representative plates from each cell line are shown. ( d ) 67NR empty vector and C1orf106 overexpressing cells were seeded at 1 cell/well in 96-well plates and cells stained with sulphorhodamine B after 14 days (n = 4 biological replicates). Colonies > 50 cells were counted, and the surviving fraction was calculated. Representative plates from each cell line are shown. ( e ) 67NR cells were transfected with pcDNA4/TO empty vector, pcDNA4/TO C1orf106-GFP wildtype or pCDNA4/TO C1orf106-GFP ΔN(2–131) and protein expression was analysed by Western blotting. β-actin was used as a sample integrity control. ( f ) 67NR cells were assessed for clonogenicity as in ( d ) (n = 3 biological replicates). ( g ) 67NR empty vector and C1orf106-GFP cells were orthotopically transplanted into the mammary fat pad of female CD-1 mice. Tumours were monitored by calliper measurement thrice weekly, and time to measurable tumour initiation is shown (n = 10 mice). Statistical significance was determined by unpaired student’s t -test ( b – d , f ) and log-rank survival analysis ( g ). * = p < 0.1; ** = p < 0.01; **** = p < 0.0001; ns = non-significant.
Article Snippet: C1orf106 -tag cDNA was then subcloned into pcDNA4/TO mammalian expression vectors (Invitrogen, Renfrew, UK) using MluI and BamHI restriction sites.
Techniques: In Vivo, Western Blot, Transfection, Plasmid Preparation, Selection, Control, Staining, Expressing
New England Biolabs is a verified supplier 
New England Biolabs manufactures this product 
