Tet on 3g inducible expression system with zsgreen1

The Tet-On 3G Inducible Expression System with ZsGreen1 (Clontech) was used to establish the tet-on cell lines, according to the manufacturer's instructions, to avoid difficulty in obtaining stable cell lines because of unexpected growthinhibitory effects caused by the transfected genes. In brief, clones expressing tet-on 3G transactivator protein were obtained by the transfection of H69, H889, H358, and H1975 with the pCMV-Tet3G vector (Clontech). Selected positive clones were cultured in each medium containing G418 (500 mL/mL; Clontech). Selected clones were then cotransfected with pTRE3G-Zsgreen1-INSM1 (for H358 and H1975), pTRE3G-Zsgreen1-Notch1 (for H69 and H889), pTRE3G-Zsgreen1-Hes1 (for H69 and H889), and Linear puromycin Marker (Clontech). Transfected cells were selected with 5 mg/mL puromycin (Clontech). The resistant clones were expanded and examined for the induction of each protein and ZsGreen1 on the addition of 0.5 mg/mL doxycycline (Dox) by WB analysis, quantitative RT-PCR, and IFA. Cells were transfected by using a NEPA21 pulse generator (Nepa Gene), as described in the manufacturer's instructions.

The tet-on H358 and H1975, which express INSM1 in the presence of Dox, were established by using a Tet-On 3G Inducible Expression System with ZsGreen1 (Clontech). A total of 2 Â 10 6 cells were injected s.c. into the back of 10 mice [Rag À/À :Jak3 À/À mice; a generous gift from Prof. Seiji Okada (Kumamoto University)]. After tumor formation was confirmed after 3 weeks, the mice were divided into two groups, in which the average tumor sizes were similar (approximately 1000 mm 3 ). One group was given drinking water supplemented with 4 mg/L Dox, and the other group was given normal water. Five weeks after the first injection, the tumors were removed and measured. The samples were fixed with 10% formalin and embedded in paraffin. Tissue sections were stained with hematoxylin and eosin, and additional sections were IHC stained, as described above. All animal experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Kumamoto University.