X tremegene hp dna transfection reagent

For differential display (Figure 4), HEK293 cells were transfected with control siRNAs (Sense; 5′-CAGCGACUAAACACAUCA-3′ Antisense; 5′-UUGAUGUGUUUAGUCGCUGTT-3′) or TBX3 specific siRNA A (Sense: GACCAUGGAGCCCGAAGAA, Antisense: UUCUUCGGGCUCCAUGGU) or CAPERα-specific siRNA (Sense: GACAGAAAUUCAAGACGUU, Antisense: AACGUCUUGAAUUUCUGUC) using lipofectamine 2000 (Invitrogen) or X-treme GENE HP DNA transfection reagent as per manufacturer's instructions.
HNRNP A1 siRNA for knockdown in HFFs (Figure 6) was obtained from Cell Signaling (cat. #7668).

Oligonucleotides of siRNAs targeting ACYP2, PMCA4 and PTP1B were obtained from Gene Pharma (Shanghai, China) and Ribobio (Guangzhou, China), respectively. The sequences were presented in Additional file 1: Table S3. Cells were transfected at 50% confluence using Lipofectamine 2000 (Invitrogen, Grand Island, NY) according to the instructions of the manufacturer, with a final siRNA concentration of 50 nM. All silencing experiments were carried out in triplicate. Two oligonucleotides with maximal knockdown efficiency were selected among three different sequences.
Open reading frame (ORF) of ACYP2 with stop codon was amplified and then cloned into pcDNA3.1(−) mammalian expression vector, termed pcDNA3.1(−)-ACYP2. The primer sequences were shown in Additional file 1: Table S4. Cells were transfected with the indicated constructs at 70% confluence using X-treme GENE HP DNA Transfection Reagent (Invitrogen, Grand Island, NY) according to the instructions of the manufacturer. Lentivirus encoding shRNA targeting ACYP2 and control lentivirus were obtained from HanBio Biotechnology Co., Ltd. (Shanghai, China). Cells were transfected at 50% confluence with a final lentivirus multiplicity of infection (MOI) of 50–100 according to the instructions of the manufacturer. Cells stably knocking down ACYP2 were selected by puromycin.

The plasmid expressing IGF1R (pLVX-AcGFP1-IGF1R) and empty vector were obtained from MiaoLingBio (Wuhan, P.R. China). Cells were plated at approximately 60%-80% confluence in a 6-well plate and transfected with 2 μg of the above plasmids with X-tremeGENE HP DNA Transfection Reagent (Invitrogen). Functional experiments were carried out 48 h after transfection.
Oligonucleotides of siRNA targeting IGF1R (si-IGF1R) and control siRNA (si-NC) were purchased from RiboBio Co., Ltd. (Guangzhou, P. R. China). Cells were transfected with a final siRNA concentration of 80 nmol/L with X-tremeGENE siRNA Transfection Reagent (Roche Diagnostics GmbH, Mannheim, Germany). The sequences of siRNA were presented in Supplementary Table 3.

Baby hamster kidney cells (BHK-21) were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum (FCS) in a six-well tissue culture plate under 5% CO₂ at 37°C. BHK-21 cells were transfected using the X-tremeGENE HP DNA transfection reagent (Roche) per the manufacturer's instructions. In brief, 2 μg plasmid pRc/CMV-VP1 in 100 μl OPTI (Invitrogen) was mixed with 6 μl X-tremeGENE HP DNA transfection reagent at room temperature and incubated for 20 min. The DNA/transfection reagent complexes were slowly added to cells and incubated for 48 h. The cells in each well were fixed with an ice-cold acetone and methanol mixture (1∶1) for 30 min at -20°C. After being washed three times with 0.01 M pH 7.2 Phosphate Buffered Saline with Tween 20 (PBST) (containing 0.05% Tween-20), primary rabbit anti-FMDV type O-IgG antibody (LVRI) (1∶50 Phosphate Buffered Saline, PBS) was applied to the wells for 1 h at 37°C. The cells were then washed three times and incubated with FITC-conjugated goat anti-rabbit-IgG (Sigma) (1∶200 in PBS) for 30 min at 37°C. The cells were then washed five more times. The cells were examined under an Olympus fluorescence microscope.

Cells after 3 days in ED-medium were transfected overnight with oestrogen responsive element (ERE)–luciferase and β-galactosidase constructs using X-treme GENE HP-DNA transfection reagent (Invitrogen) in phenol red-free Opti-MEM reduced-serum medium (HyClone, Logan, UT) as previously described^{18 (link)}. Cells were then treated for additional 24 h with ED, E2 (10^–9 M), or 10% FBS, plus tamoxifen (10^–7 M), fulvestrant (10^–7 M), or AZD9496 (10^–7 M). Relative luciferase activity was determined and analysed as previously described^{18 (link)}.