Tet on advanced inducible gene expression system

To generate a stably transfected Lhx5-expressing Neuro2a cell line, a construct was generated that added a FLAG-tag to the C-terminus of LHX5 and that controls the expression of Lhx5 under the Ptight-promotor of the Tet-On Advanced Inducible Gene Expression System (Clontech). The cells were cotransfected with this construct and the pTet-ON-Advanced vector (3:1) by using FUGENE HD Transfection Reagent (Promega). The cells were selected in medium containing 1 mg/ml G418 and clones that showed a doxycycline-dependent, homogenous expression of Lhx5 were frozen for further experiments.

A pool of positive cell clones with stable forced expression of TFF3 in immortalized-HMECs was generated as previously described^{8 (link),20 (link)}. Briefly, positive transfectants were selected in 200–400 μg/ml G418 (Calbiochem) in the appropriate culture medium for the respective cell lines. Individual colonies were selected to determine TFF3 expression level by western blot analysis. Cell lines were established as HMEC-hTERT-TFF3, MCF10A-TFF3, and MCF12A, respectively, by pooling more than 15 individual colonies with high TFF3 expression. Tet-On® Advanced inducible gene expression system (Clontech Laboratories Inc, CA) obtained from Prof. Daniel G. Tenen at The Cancer Science Institute of Singapore (CSI), National University of Singapore (NUS), Singapore. For the inducible TFF3 expression system, forward and reverse oligonucleotides were annealed to produce the dsDNA, digested with BamHI and EcoRV and cloned into a pTRE-Dual2 plasmid (Clontech Laboratories Inc, CA) and correct insertion and insert sequence checked by sequencing. Stable transfection of HMEC-hTERT cells with a plasmid containing Tet-On advanced and pTRE-Dual2-TFF3 was carried out using X-tremeGENE HP DNA transfection reagent, according to the manufacturer’s instructions (Clontech Laboratories Inc, CA). Stable pooled clones are designated as HMEC-hTERT-TetON-Dual2 and HMEC-hTERT-TetON-Dual2-TFF3 cells.

The circ_0000199 vector was synthesized by Invitrogen Co., Ltd. circ_0000199 sequence was inserted into pcDNA3.1. Circ_0000199 without the downstream reverse sequence was used as a negative control. Circ_0000199 vector was finally cloned into the Tet-On Advanced Inducible Gene Expression System (Clontech Laboratories, Inc. Mountain View, CA, USA) according to the manufacturer's protocol. The target sequence of circ_0000199 small interfering RNA (siRNA) was 5′-TACTATTTTTCGACAAAAAGGTAAACAGC-3′. These adenoviruses were constructed using the AAVPrime AAV System (GeneCopoeia, Inc.) according to the manufacturer's protocol. SCC9 and HN12 were infected with viral at multiplicity of infection = 50 for 48 h.