Lipofectaminetm ltx

Fibroblasts at early confluence were transiently transfected with [SBE]₄-TK-luc reporter constructs or appropriate empty vectors [29 (link)] using Lipofectamine^TM LTX (Invitrogen, Carlsberg, CA). Following incubation with roscovitine and TGF-β for 24 h, cultures were harvested and whole cell lysates were assayed for their luciferase activities using the dual-luc reporter assay system (Promega, Madison, WI) [30 (link)]. pRL-TK Renilla luciferase (pRL-TK-Luc) was used in each experiment as an internal control and experiments were performed in triplicate.

HERS01a, a mouse HERS cell line, was cultured as described previously (Akimoto et al., 2011). MDPC‐23 cells (Hanks et al., 1998), a murine dental papilla cell line, were maintained in Dulbecco's modified Eagle medium (Invitrogen) with 10% fetal bovine serum (Invitrogen), and 100 IU/ml penicillin‐100 μg/ml streptomycin (Invitrogen). To generate retroviral particles, small hairpin RNA (shRNA) against mouse Ctnnb1 (TG500280) and control shRNA (TR30013) were purchased from OriGene Technologies (Rockville). The establishment of stable cell lines by viral transductions were conducted as previously described (Choi et al., 2017). After establishment of stable cell lines with shRNA, conditional media were harvested from HERS cells, mixed 50:50 with the growth medium for MDPC‐23 cells, and then administered to MDPC‐23 cells for 24 h. The plasmid driving the expression of mouse β‐catenin S33Y was a gift from Shinya Yamanaka (Addgene plasmids #13371). DNA constructs were transfected by using Lipofectamine^TM LTX and PLUS reagent (Invitrogen) according to the manufacturer's instructions.

The full length open reading frame of mouse Osx (Accession No. NM_130458), cloned into the pCMV6 vector, was purchased from OriGene Technologies. A GFP construct was also transfected as a control. The luciferase Osx promoter plasmid + 1269/-91 was kindly provided by Dr. Mark Nanes (Emory University, Atlanta, GA, USA). Transfection experiments were performed with Lipofectamine^TM 3000 (Invitrogen) according to the manufacturer’s instructions. After 24 h, transfected cells were harvested for whole cell lysate preparation or cultured with OM for further differentiation. Viral particles were generated by transfecting a 293T-based amphotropic retroviral packaging cell line, Phoenix with a plasmid expressing shRNA for mouse Tgfbr2 (OriGene Technologies, Rockville, MD, USA) using Lipofectamine^TM LTX and PLUS reagent (Invitrogen). Supernatants containing viral particles were collected between 48 and 72 h after transfection, filtered through a 0.45-μm filter, and used immediately. Subconfluent OCCM-30 cells were infected overnight with the retroviral particles expressing shRNA for mouse Tgfbr2 in the presence of Polybrene. Forty-eight hours later, transduced OCCM-30 cells were selected from growth medium containing 10 μg/ml puromycin.

Cells were transfected with 12 μg of plasmid DNA (pMXs-gw and pMXs-STAT3C) by Lipofectamine^TM LTX (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instruction and then incubated for 24 h prior to the treatment of TMS-TMF-4f.

Human Embryonic Kidney (HEK‐293) cells stably expressing WT hERG were kindly donated by Prof Craig January. HEK 293 cells used for transient transfection were obtained from ECACC (catalog number 85120602). Cells were passaged using a nonenzymatic agent (Enzyme Free, Chemicon International^®) and maintained as previously described (McPate, Duncan, Milnes, Witchel, & Hancox, 2005; Milnes, Crociani, Arcangeli, Hancox, & Witchel, 2003; Ridley, Dooley, Milnes, Witchel, & Hancox, 2004). For transient transfection experiments, 24 hr after plating cells out, cells were transiently transfected with 0.5 µg of the F656V hERG construct using Lipofectamine^TM LTX (Invitrogen) according to the manufacturer's instructions. Expression plasmid encoding CD8 was also added as a transfection marker (El Harchi, Zhang, Hussein, Dempsey, & Hancox, 2012). Cells were plated onto small sterilized glass coverslips 6 hr after transfection and recordings were made after at least 24 hr incubation at 37°C. Successfully transfected cells were identified using Dynabeads^® (Invitrogen). All experimental data for the F656V hERG mutant channel were obtained from cells from a minimum of two transfections.

Transient transfection of reporter plasmids and luciferase assays was performed as described previously^{4 (link), 80 (link)}. 1 × 10⁵ cells were seeded per well in 24-well plates 24 h before transfection. Lipofectamine^TM LTX and Plus^TM reagents (Invitrogen) were used for DNA transfection, according to the manufacturer’s instructions. 400 ng of pGL3p-N3Int2 was transfected along with or without 400 ng of pBABE-bla-ZEB1, pBABE-bla-ZEB2 of pBABE-bla (empty vector control). 5 ng of phRL-SV40-renilla luciferase vector (Promega) was co-transfected to calibrate the variation of transfection efficiencies among wells. Cells were incubated in the presence or absence of 1 µg/ml DOX to induce ICN1 in cells expressing ICN1^TetOn for 48 h before cell lysis. Alternatively, 5 ng/ml TGFβ1 was added at 24 h after transfection and incubated for an additional 48 h before cell lysis. Luciferase activities were determined using Dual-Luciferase^TM Reporter Assay system (Promega) and ORION Microplate Luminometer (Berthold Detection Systems, USA, Oak Ridge, TN). The mean of firefly luciferase activity was normalized with the co-transfected renilla luciferase activity. Transfection was carried out at least three times, and variation between experiments was not greater than 15%.

For transfection of cortical neurons, cells were maintained in culture for 5 days, and 2–3 μg of plasmid was added together with LipofectamineTM 2000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. After 1 h, the cells were washed 2 times with HEPES buffer (10 mM HEPES, pH 7.5, 135 mM NaCl, 5 mM KCl, 15 mM glucose, 2 mM MgCl₂, and 2 mM CaCl₂) and maintained in culture medium. Finally, cells were kept in culture for 2 days.
For transfection of CHO cells, cells were kept for 1 day in culture in a 6-well plate before transfection, and 3 μg of plasmid was added to the cells together with 3 μL of PlusTM Reagent and 9 μL of LipofectamineTM LTX (Thermo Fisher Scientific) following the manufacturer’s protocol. After 6 h, cells were washed 2 times with HEPES buffer, and maintenance medium was again added. Finally, cells were kept in culture for 1 day.