Lipofectamine ltx reagent

ERK5-overexpressing cells were obtained by transient transfection with pCMV-ERK5 (carrying the human ERK5 cDNA, kindly provided by J.E. Dixon) and with pCMV-MEK5DD (carrying a phosphomimetic mutant sequence of human MEK5 cDNA, kindly provided by C.J. Marshall). Control cell lines were obtained by transfection with the empty vector. Cells were transfected with Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol, and collected 48 h after transfection. YAP-overexpressing cells were obtained by transient transfection with pQCXIH-Myc-YAP or pQCXIH-Myc-YAP5SA (gift from Kunliang Guan, Addgene plasmids # 33091 and # 33093) [50 (link)], respectively, using Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific) according to the manufacturer’s protocol. Cells were collected 48 h after transfection or utilized for treatments. Notably, YAP5SA protein, carrying mutations of LATS1/2-dependent phosphorylation sites (S61A, S109A, S127A, S164A, S381A), results constitutively active [50 (link)].

Protospacer sequences of CRISPR/Cas9 against p53 and Yap were designed by CRISPR DESIGN (http://crispr.mit.edu/). All specific target sequences were cloned into pX459 or pX330 vectors (Addgene plasmid #62988 and #42230 respectively). and verified by DNA sequencing. After the transient transfection of CRISPR/Cas9 into cells using Lipofectamine LTX reagent (Life Technologies), transfected cells were selected by DMEM culture medium supplemented with puromycin (2 μg/mL) (Sigma) for 48 hours and then expanded in regular culture medium. sgRNA oligo sequences targeting p53 and Yap are listed in (Table S2, supplementary methods).

LTR basal activity from strains A4 [21 (link)], KV1772 [43 (link)], Ov496 [19 (link)] and CAEV-Co [42 (link)] was assayed using a luciferase reporter system. Briefly, DSF, FSF and GSM cells (10⁵ in 24-well plates) were transfected with 200 ng of each LTR construction using 4 µL of Lipofectamine-LTX Reagent and 0.2 µL of PLUS-Reagent (Life Technologies). Plasmids pGL4.13 [luc2/SV40] and “empty” p-GL4.10 [luc2] were used as positive and negative controls, respectively. At the same time, 20 ng of pGL4.73 [hRluc/SV40] Vector (Promega) were cotransfected, so that the firefly activity was standardized according to the Renilla luciferase activity. After 24 h, cells were harvested with Passive Lysis 5X Buffer (Promega), and firefly and renilla luciferase activity were measured following manufacturer’s instructions. Results were expressed as: Relative Luminiscence Units (RLU) firefly luminescence/RLU Renilla luminescence.

Hoxa5 3′ UTRs were individually cloned into the psiCHECK-2 vector (Promega). iMir-27OE or iMir-27SP cells were plated at a density of 8 × 10³ per well (96-well plate), expanded for 20 h and transfected with 150 ng of reporter plasmids using 0.8 μl PLUS Reagent and 0.4 μl Lipofectamine LTX Reagent (Life Technology). Cells were lysed 24 h later and processed for luciferase assay using the Dual-Luciferase Reporter Assay System (Promega). Luciferase activity was measured using the Enspire Multimode Plate Reader (Perkin Elmer).

The human colon cancer cell line HT29 was obtained from American Type Culture Collection (ATCC, USA) and was routinely maintained in Dulbecco’s Modified Eagle Medium (DMEM) containing 10% FBS, 1.8 mM Ca²⁺, 2 mM L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin (all from Life Technologies) in a 5% CO₂/humidified air incubator maintained at 37°C. Cells were periodically tested for mycoplasma contamination and authenticated by STR DNA profiling (DNA Diagnostic Center, UK).
HT29 cells were transfected with pcDNA3.1/Zeo⁽⁺⁾ (EMP) or an expression vector encoding the full length CaSR cDNA (constructs kindly provided by Prof. Romuald Mentaverri, University of Picardie Jules Verne, France) using Lipofectamine LTX reagent (Life Technologies) as previously described [18 ]. Stable transfectants were selected by culturing the cells in the presence of Zeocin (150 μg/ml) for over 6 months.

For live cell imaging of MG53-mediated cell membrane repair in hCEC, transfection of GFP-MG53 into hCECs was performed using the Lipofectamine LTX reagent (Life Technologies), per manufacturer’s instructions. hCECs expressing GFP-MG53 were subsequently subjected to microelectrode penetration-induced injury to the plasma membrane as previously described^{12 (link)}. Cells were imaged using confocal microscopy (Zeiss LSM780). For visualizing the dynamic process of rhMG53 entry into the corneal fibroblasts, rhMG53 and bovine serum albumin (BSA) were labeled with Alexa Fluor^TM 647 by Alexa Fluor™ 647 Protein Labeling Kit (Life Technologies, Cat. No. A20173). Labeled rhMG53 or BSA was added to the culture medium of primary corneal fibroblasts and intracellular signal of Alexa 647 was imaged at indicated time points by a confocal microscope. Intracellular fluorescent intensity of Alexa 647 at each time point was quantified by ImageJ software. To visualize cell morphology for fluorescence quantification, the cells were counterstained with MitoTracker Green (Life Technologies, Cat. No. M7514).

Human neuroblastoma SH-SY5Y cells (ATCC number CRL-2266) were grown in DMEM–F12, 10% Fetal Bovine Serum (FBS) at 37°C. The plasmid pcDNA3.1 containing cDNAs coding for DRD1-Flag or DRD2-Flag were transfected using Lipofectamine^® LTX Reagent (Life Technologies) according to the manufacturer’s protocol. The different SH-SY5Y clones were maintained under selection by 400 μg/mL of G418. Individual clones were picked after 14 days of selection, moved in a 96 well plate, and maintained under selective medium until confluence growth. Different individual clones were analyzed for DRD1 or DRD2 expression by western blot and immunofluorescence.