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. 2014 May 19;9(5):e97764. doi: 10.1371/journal.pone.0097764

Figure 2. Conditional silencing using TetR-KRAB or TetR repressor proteins.

Figure 2

U2OS cells constitutively expressing TetR-KRAB (A,B,C) or TetR (D,E,F) were infected with pGLTR-FP-GFP-CDC27 and cultured in the absence or presence of 1 µg/ml doxycycline (Dox) for 3 days (A,D). In the presence of TetR-KRAB, GFP expression was Dox dependent (A). For time-course experiments (B,E) cells were treated with 1 µg/ml Dox and samples taken at the indicated time points for immunoblotting for CDC27 levels as well as GAPDH as loading control. For dose-dependent experiments (C), cells were treated with increasing doses of Dox for 3 days and total cell extracts analysed for CDC27 and GAPDH expression. F) Inducibility and leakiness of TetR-regulated shRNA expression was tested in pGLTR-FP-GFP-CDC27 infected U2OS-TetR cells analysed for CDC27 and GAPDH expression. Shown are representative results from 3 independent experiments.