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. 2010 Nov 17;5(11):e14026. doi: 10.1371/journal.pone.0014026

Figure 2. Dual-luciferase reporter constructs to measure TF activity.

Figure 2

(A–D) MCF-7/WS8 cells within hydrogels were transfected in parallel with pFLuc reporter constructs containing enhancer elements for specific TFs (AP1, ER, and p53), vector control (TA), or not transfected (NC). For wells with reporter constructs, a constituently active pGLuc construct was delivered to normalize for transfection efficiency. (A) Cells were extracted and lysed after 48 h to determine FLuc production, which was normalized to transfection efficiency. (B–D) Bioluminescence imaging was used to non-invasively quantify FLuc production after 48 h from cells growing within Matrigel. (B) Pseudo-color mapping demonstrates localized luminescence output from cells in hydrogels seeded in alternating wells of a 96-well plate. (C) Raw FLuc signals were significantly greater than bioluminescence noise from NC. (D) FLuc signals from bioluminescence imaging were normalized to transfection efficiency and normalized TF activities for all three factors were significantly greater than TA. Values are means ± s.d. from at least three independent experiments carried out in triplicate.