Transfection. Lipofectamine 2000 (Invitrogen) was used to transfect DNA plasmids and LNA- RNA into Neuro2A cells. Magnetofection using Neuromag (OZBioscience) was used to transfect xxXXX inhibitors into primary cultured neurons according to according to manufacturer's instructions. Fluorescence in situ hybridization for miRNAs on brain sections and cells. Mature hippocampal neurons or brains from p21 mice were used for in situ hybridization as described previously (Xxxxxxxxxxx et al., 2011) Western blot analysis Western blot analysis was performed as described before (Xxxxx et al., 2010). Rabbit anti-Kv4.2 antibodies were used at 1:1000 (Millipore); mouse anti-GluN1 antibodies were used at 1:1000 (BD Pharmingen); and mouse anti α-tubulin (Sigma) antibodies wer used at 1:1,000, 000. Specific signals were detected with horseradish- peroxidase-coupled secondary antibodies (GE Healthcare) and Enhanced ChemiLuminescence (Thermo Scientific). Biotinylation assay High density cortical neurons were used at 14-18 DIV to perform biotinylation assay. Cells were incubated with 1.0 mg/ml EZ-Link sulfo-NHS-Biotin (Thermo Scientific) in 1X PBS (Hyclone) for 30 min at 4°C. Cell lysates were prepared and biotinylated proteins were pulled down as decribed previously (Xxxxxx, 2000). Western blot analysis was used to assess the specific protein levels (Xxxxx et al., 2011). Dual-luciferase assay Neuro2A cells were used to perform the luciferase assays and luciferase activity was measured by dual luciferase assay using Renilla and Firefly luciferase according to the manufacturer’s protocol (Promega). Statistics. All statistical analyses were performed in SPSS 17.0 and PASW Statistics 18. Data were tested for normality and homogeneity of variances, and appropriate tests were used as indicated for each figure Results Kv4.2 mRNA is a putative target of miR-324-5p
Transfection. HepG2 cells were seeded into six p100 dishes. XGtremeGENE 9 DNA Transfection Reagent (Roche) was mixed with serum free media, and same amount of Vector and MTSGCore plasmid DNAs were each added to two sets of transfection reagent, at two ratios of transfection reagent to plasmid DNA (3:1 and 6:1). Incubated transfection complexes were added to the cells in a dropwise manner, and after incubation and switching to media containing G418, the cells were split into new p100 dishes at a dilution of 1:50. Cell Culture and Plasmid DNA Verification. Cells were trypsinized once a week and media were changed every other day. At the time cell colonies were visible, big colonies were selected using cloning cylinders, trypsinized, and added to G418 media in 6Gwell plates for better growth. After 2 weeks of growth, DNA was isolated from samples of the transfected cells using DNA lysis buffer and ethanol precipitation, and the plasmid taken up by the cells was verified by PCR using primers specifically designed for each insert. SDSHPAGE, Western Blotting, and Protein Detection. Cells with pCMV vector and cells with MTSGCore clones were each lysed using RIPA buffer (SigmaGAldrich) to obtain the nuclear and mitochondrial fractions. Protein concentration of each sample was determined by Bradford Protein Assay. Following a standard western blot protocol, 20 μg of each sample were run on a preGmade polyacrylamide gel, electrophoretically transferred to a membrane, and incubated overnight in a blocking solution of 5% nonGfat dried milk and 0.1% Tween 20. Primary and secondary antiGSHDB (mitochondrial marker, Novus Biologicals, dilution of 1:5000) and antiGHA antibodies (MTSGCore marker, Thermo Scientific, dilution of 1:500) were added, and West Pico Chemiluminescent Substrate (Thermo Scientific) was used for protein detection. The membrane was exposed for 15 minutes on a film.
Transfection. For all transfections, cells were plated in 6-wells plates and grown over-night to a 30% confluency with mostly single cells. Cells were incubated with 15 (g DNA con- struct, for 2 days using Fugene6TM transfection reagent (Boehringer) according to the manufacturer’s recommendations. The pInd/pVgRXR transfected clones were selected by neomycin (G418, Gibco BRL), the pTracer transfected cells were not selected, but traced with GFP. To enhance transcription of the construct in the pInd/pVgRXR system, 25 μM MuristeroneA (MuA) was added 72 hours prior to experiments. Detergent extractions, cell lysis, and Western blot analysis Cells were grown to 75% density, left on ice for 10 minutes and rinsed with ice- cold PBS/1mM CaCl2 /1mM MgCl2. For extraction, cells were incubated with 0.5% Triton-X-100 in extraction buffer (50 mM TrisHCl, pH7.4, 100 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 300 mM sucrose, 1 tablet complete protease inhibitor (Boehringer Mannheim), gently scraped from the dish, collected and rotated for 30 minutes at 4˚C. The samples were collected and spun down (15 minutes, 15000 rpm). The detergent insoluble fraction (pellet) was separated from the supernatant and further lysed in 100°C SDS buffer (1% SDS, 10 mM EDTA, 10 mM TrisHCl pH7.4, 1 tablet complete protease inhibitor). For total protein lysate, cells were lysed in hot-SDS buffer immedia- tely after rinzing. Protein concentrations in lysates were measured using Bio-Rad DC Protein Assay (Bio-Rad Lab., Hercules, CA), for equalized loading of the individual samples. Lysates were size fractionated by SDS/PAGE and semi-dry blotted onto polyv- inyl difluoride membrane (Millipore, Bedford, MA). Filters were blocked for 1 hour in 5% skim milk, incubated with a primary antibody for two hours, washed in PBS/ 0.05% Tween-20, and incubated for 1 hour with an anti-mouse IgG HRP-conjugate (Transduction Laboratories, Lexington, KY). The filter was developed with ECL detecti- on substrate (Amersham Pharmacia Biotech, Buckinghamshire, UK). Immunofluorescence Microscopy Cell cultures transfected with the pIND vector were incubated 72 hours with 25 μM MuA to induce transcription. Then they were rinsed with ice-cold PBS / 1mM CaCl2/1mM MgCl2 and fixed with either cold methanol (15 minutes, -20°C) or 4% paraformaldehyd (as previously described; Balzar et al., 1999b) and air-dried. Fixed cells were re-hydrated with PBS, blocked with 5% skim milk in PBS, incubated with a primary antibody for 2 hours, followed by 1 hour inc...
Transfection. To verify the effects of xxXXX-302 family on MSC fate decisions, xxXXX inhibitors were introduced into the cells to decrease xxXXX expression level (Xxxxxxxx, Xxxxx et al. 2012). Subsequently specific xxXXX mimics were also used to simulate over expression of xxXXX-302 family members. The phenotypical consequences of transfection of either of inhibitor or mimic were investigated after treatment with differentiation media. Having opted for non-virally induced transfection, lipofection and electroporation were performed as two compared methods of xxXXX transfection into MSCs and then the experiment followed by transfecting xxXXX inhibitor or mimic via lipofection.
