Lenti x tet on advanced inducible expression system

Inducible overexpression of RBM10-EGFP wild type (WT) or mutants (MUTs) in A549 cells was achieved using the Lenti-X Tet-On Advanced Inducible Expression System (Clontech) according to the manufacturer’s manual. Briefly, lentiviral plasmids (pLVX-tight-puro) carrying RBM10-EGFP WT or MUTs or TetR (pLVX-Tet-On) were transfected into HEK293-FT cells together with three packaging plasmids (pLP1, pLP2, and pLP/VSVG, Invitrogen). Medium containing the lentiviral particles was harvested 48 or 72 hrs after transfection.
Lentiviral particles carrying TetR was added into the A549 cell cultures and selected by 200 μg/ml G418 (Invitrogen). The G418 selected cells were transfected with lentiviruses carrying RBM10 WT or MUTs followed by selection in medium containing 2 μg/ml puromycin (Sigma). Stably transfected A549 cells were maintained under standard conditions and overexpression of RBM10-EGFP proteins were induced by 1 μg/ml doxycycline treatment (Sigma) for 2 days unless otherwise stated.

Tet-inducible TUSC2-expressing H1299 and H157 cells were produced using the Lenti-X Tet-On advanced inducible expression system (Clontech, Mountain View, CA) as described previously. TUSC2-induced H1299 and H157 cells were selected after exposure to 2 μg/mL doxycycline for 48 hours and assessed for TUSC2 expression using Western blot analysis.

Stable, inducible cell lines were generated using the Lenti-X Tet-On-Advanced Inducible Expression System (Clontech Laboratories, Inc) according to the manufacturer’s instructions. Briefly, JCaM1.6 were transduced with lentiviral particles (as described above) containing the PLVX-Tet-On-Advanced vector, which constitutively expresses the tetracycline-controlled transactivator rtTA-Advanced. Forty eight hours after transduction, the cells were subjected to selection by Geneticin (1 mg/ml) to generate a stable JCaM1.6-Tet-ON cell line. This parental cell line was then transduced with lentiviral particles of pLVX-Tight-Puro containing the Lck constructs and, 48 h after transduction, subjected to selection by Puromycin (10 μg/ml) and Geneticin (1 mg/ml) to generate the respective stable cell line. Expression of the Lck constructs was induced by 1 μg/ml doxycycline (dox, Sigma-Aldrich) added to the cell culture medium, routinely 14 to 18 h prior to each experiment. Potential phenotypic drift of cell cultures was reduced by conditionally expressing Lck or chimeras in JCaM1.6 by doxycycline induction for 14 to 16 h.

HUVEC plated at 3 × 10⁶ cell density were kept as above and serum-deprived for 4 h, 24 h after plating. Culture medium was replaced by fresh DMEM high glucose medium without serum and antibiotics. Plasmids (20–30 μg) or siRNAs (500–1000 nM) were diluted in transfection medium and lipofectamine according to manufacturer’s instructions (Invitrogen) and incubated with cells for 8 h, followed by change to RPMI plus 10% FBS. Cells were used 24, 48 and 72 h after transfection. b) Lentiviral-carried Tet-on system: Inducible PDI overexpression was achieved in rabbit VSMC coinfected with 2 different lentiviruses, one carrying rat PDIA1 gene (with a myc tag inserted at the C-terminus before the C-terminal KDEL sequence, a kind gift of Drs. Tomohiro Nakamura and Stuart Lipton (Burnham Institute for Medical Research, La Jolla, USA) under control of the Tet-inducible promoter/modified Tet-Responsive Element (TREmod), and the regulatory plasmid, which carries the reverse tetracycline trans-activator (rtTA), (Lenti-X Tet-On Advanced Inducible Expression System, Clontech).

Lentiviruses were produced according to the Lenti‐X Tet‐ON Advanced Inducible Expression System (Clontech). HEK293T helper cells were transfected with packaging plasmids pMd2.G and psPAX2 using Lipofectamine 2000 (Life Technologies) to generate responsive lentiviruses carrying pLVX‐Tight‐Puro, pLVX‐Tight‐Puro‐H2B‐GFP/α‐Tubulin mCherry 24, pLVX‐Tight‐Puro‐GFP‐MCAK, pLVX‐Tight‐Puro‐GFP‐Kif2b, or pLVX‐Tight‐Puro‐mEOS‐α‐Tubulin, as well as transactivator lentiviruses carrying the rtTA expressing construct (pLVX‐Tet‐On Advanced). Human fibroblasts were then co‐infected for 6 h with both the responsive and the transactivator lentiviruses (2:1 ratio) in the presence of 8 μg/ml polybrene (AL‐118, Sigma‐Aldrich). Co‐transduction was induced with 500 ng/ml doxycycline (D9891, Sigma‐Aldrich). Transfection efficiencies of all experiments were determined by scoring the number of fluorescent cells, or protein levels by Western blot analysis.