Fig. 3
From: A new mouse model to study the role of ectopic Nanos3 expression in cancer
Nanos3 and eGFP expression in the lungs of transgenic mice. a. Scheme of inducible Nanos3 expression in the lungs. The club cell secretory protein (CCSP) promoter, active in club cells and type-II alveolar cells, leads to transcription of the reverse tetracycline transactivator gene (rtTA). In the presence of doxycycline (Dox), rtTA binds the tetracycline operator (tetO) in the tetO-CMV promoter, leading to Cre recombinase expression. Cre recombinase mediates recombination between loxP sites and thereby deletes the floxed STOP cassette (LSL) and allows transcription of Nanos3 and enhanced green fluorescent protein (eGFP). IRES: internal ribosomal entry site. To induce transgene expression both control and Nanos3 NSCLC mice were fed doxycycline-containing food and killed about 38 days after Dox induction. b. Total lung lysates of two Cre-negative control mice, three Cre-positive control NSCLC mice (LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/−;TetO-Cre+/−) and three Nanos3 overexpressing NSCLC mice (Nanos3LSL/−;LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/−;TetO-Cre+/−) were tested for Nanos3 and the associated eGFP expression by western blotting. Actin was used as a loading control. c. RNA prepared from total lung lysates of control and Nanos3-overexpressing mice was used to detect eGFP and Nanos3 transcripts by RT-qPCR. CNRQ, calibrated normalized relative quantity; error bars, SEM; n = 3, **: P ≤ 0.01 and ****: P ≤ 0.0001. Gene expression was normalized to reference genes (rpl13A and hprt1) using qbase+ (Biogazelle) [35]. d. Lung tumor sections of control NSCLC mice and Nanos3-overexpressing NSCLC mice (Nanos3LSL/−;LSL-KRasG12D;p53fl/fl;CCSP-rtTA+/−;TetO-Cre+/−) were stained with a GFP- and a Nanos3-specific antibody. Bars: 5 mm for the total lungs, 500 μm for the magnifications of the eGFP staining and 200 μm for the magnifications of the Nanos3 staining