Fig 2: Silencing Sox2 induces entry of cultured TRCs into dormancy. (A) B16 cells, transfected with Sox2 shRNA#1 or shRNA#2, were cultured as single individual cells in 90-Pa fibrin gels for 10 days. Left: bright field images were taken on days 5 and 10; right: B16 cells transfected with negative control, Sox2 overexpression plasmids, or Sox2 shRNA#1 (day 10) plus Sox2 overexpression plasmids. Colony sizes were quantified (mean ± SEM; n = 15 samples; three independent experiments. N.S.: not significantly different). (B) Cell cycles were analyzed by flow cytometry after seeding cells in 90-Pa fibrin gels for 5 days. (C) Quantification of cell cycle analysis (mean ± SEM; n = 3 independent experiments) **P < 0.01. (D) Representative images (left panels) and quantification (right panel) of double immunostaining of COUP-TF1 and Ki67 under various conditions. Scale bar: 50 µm. The values represent mean ± SEM from n = 10 randomly chosen fields of view from two different experiments. (E) Glucose consumption was assayed in B16 TRCs transfected with shRNA or treated with IFN-? (100 ng/ml) for 3 days. (F) SA-ß-gal staining was quantified. B16 TRCs were transfected with shRNA or treated with 50 µM Etoposide for 3 days. N.S.: not significantly different. (G) B16 cells transfected with negative control, Sox2 shRNA #1, Sox2 shRNA #2, or treated with IFN-? (100 ng/ml) were cultured in 90-Pa fibrin gels for 5 days and total mRNAs were extracted for quantitative analysis of IDO1 or Sox2 expression by real-time PCR (mean ± SEM; three independent experiments). *P < 0.05, **P < 0.01.