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. 2016 Apr 18;5(4):e220. doi: 10.1038/oncsis.2016.29

Figure 1.

Figure 1

Nupr1 is low in soft matrix and independent of Sox2. (a) Nupr1 mRNA expression is lower in 3D fibrin gels than on rigid plastic. 2D: B16-F1 cells were cultured on rigid plastic. 3D 90-Pa, 3D 1050-Pa: cells were cultured in 90 or 1050-Pa fibrin gels for 5 days. After 5 days, the mRNA was extracted for quantitative analysis of Nupr1 mRNA expression by real-time PCR. Cells that are grown in 3D 90 Pa gels are defined as TRCs. (b) Nupr1 mRNA expression increases with substrate stiffness. Cells were cultured on the surface of collagen-1-coated polyacrylamide gels of various stiffnesses or on glass-bottomed dishes for 24 h. All were compared with ‘0.15 kPa'. (c) Silencing Nupr1 does not affect Sox2 expression. Cells were transfected with negative control siRNA, or Nupr1 siRNA #1, #2, for 24 h respectively, and then analyzed for Sox2 mRNA expression by real-time PCR. (d) Silencing Sox2 do not affect Nupr1 expression. TRCs were extracted from 3D fibrin gels and re-plated onto 2D 90-Pa fibrin gel surface for 3 h, and then transfected with Scramble control, or Sox2 shRNA#1, #2, for 12 h (or for 24 h, Supplementary Figure 10c) and then analyzed for Nupr1 mRNA expression by real-time PCR. Mean±s.e.m.; n=3 independent experiments for all subfigures; *P<0.05; **P<0.01; NS, not statistically significant.