Tet on 3g expression system

All transfections were performed with the Xfect TM transfection kit (Clontech) according to the manufacturer's protocol. First, the wild-type BLM cells were transfected with the pEF1a-Tet3G plasmid (Tet-On 3G Expression System, Clontech) and grown under geneticin (1500 μg/ml) selection for 2 weeks. Single cells were sorted with a FACS Aria III flow cytometer (Becton Dickinson, NJ, USA), expanded in geneticin selection medium and resistant clones individually probed for Tet-ON 3G transactivator expression levels by a luciferase assay with the inducible plasmid pTRE3g-Luc (Tet-On 3G Expression System, Clontech) according to the manufacturer's protocol. The highest expressing clone (Tet-on 3G BLM) was subsequently co-transfected with the pTRE3G-ChR2(D156A)-YFP plasmid and a linear puromycin selection marker and grown under puromycin (0.75 μg/ml) selection for 2 weeks. Twenty-four hours before single cell sorting, 1 μg/ml doxycycline was added to the culture medium in order to induce ChR2(D156A)-YFP expression. Single cells with high YFP signal were sorted in 96-well plates using the FACS Aria III (Becton Dickinson) and the clone with highest ChR2(D156A)-YFP expression levels qualitatively selected for further experiments (in this manuscript referred to as ChR2(D156A)-YFP BLM cell line).

To stably express RepA-WH1(WT)-TagGFP2 and TagGFP2 in the SH-SY5Y cell line, a Tet-ON 3G expression system (Clontech) was used. It was composed of pCMV-Tet3G (regulatory) and pTRE3G (response) plasmids. To generate SH-SY5Y cells expressing the transactivator protein (rtTA) upon genomic integration of pCMV-Tet3G, 1 μg of this plasmid was transfected into 5 × 10⁵ SH-SY5Y cells with Lipofectamine LTX Plus (1:1 ratio; Invitrogen) and selected for colonies with G418 (0.5 mg/ml). Individual clones, isolated using a cloning cylinder (Sigma), were then screened for luciferase activity (see below). Suitable THN-rtTA/SH-SY5Y clones (i.e., those with high levels of luciferase induction and low levels of basal expression) were subsequently cotransfected with a mixture (10:1) of pTRE3G-WH1(WT)-TagGFP2 (or pTRE3G-TagGFP2) and a hygromycin linear selection marker. Doubly stable SH-SY5Y transfectants were then selected by screening with hygromycin (0.4 mg/ml) and G418 (0.5 mg/ml) for 2 weeks followed by testing for the best expression of repA-WH1(WT)-TagGFP2 and TagGFP2 genes in the presence/absence of doxycycline (0.5 μg/ml) after 48 h by Western blotting (Fig. S3B; also see above), flow cytometry (Fig. S3C), and confocal microscopy (Fig. S3D) (see below).