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. 2009 Apr 1;20(7):2041–2048. doi: 10.1091/mbc.E08-07-0699

Figure 3.

Figure 3.

Activation of the conditional FKHRL1(A3)ERtm fusion protein by 4OHT causes repression of Survivin on mRNA and protein level. (A) For analysis of Survivin expression FKHRL1(A3)ERtm-transgenic and mock-transfected cells were treated with 75 nM 4OHT for the times indicated, and protein extracts were analyzed by immunoblotting by using antibodies directed against Survivin and α-Tubulin. Densitometry of 16-bit digital images was done using LabWorks 4.6 software. (B) Activation of FKHRL1(A3)ERtm by 4OHT (75 nM) represses Survivin on mRNA level as analyzed by means of quantitative RT-PCR in SH-EP/FKHRL1 and NB15/FKHRL1 cells. Shown is the mean of three independent experiments each performed in triplicate. (C) SH-EP/Ctr and SH-EP/FKHRL1 cells were transfected with a survivin promoter firefly luciferase vector, and cultured in the presence (black bars) or absence of 75 nM 4OHT (white bars) for 24 h to analyze changes of survivin promoter steady-state activity during FKHRL1 activation. Relative luciferase activity was calculated as percentage of untreated controls. Shown is the mean of three independent experiments each performed in triplicate. (D) SH-EP/FKHRL1 cells were treated with 75 nM 4OHT and 10 μg/ml cycloheximide (CHX) for 6 h alone or in combination, respectively. Survivin mRNA expression was quantified by quantitative RT-PCR.