Pre mir s

HeLa cells were transfected with 25 or 50 nM pre-miRs (Applied Biosystems, USA), 2.5 ng/cm²; pRL control vector, and 50 ng/cm²; pGL3_HSV TK_3′UTR hNCSTN WT or C460T (rs1043329), T623G (rs113810300) or delCA515–516 (rs141849450) mutant variant plasmids. Twenty-four hours post-transfection, cells were lyzed, and luciferase activity was measured according to the manufacturer’s instructions (Promega, USA). For western blots, cells were lyzed in RIPA buffer [50 mM Tris HCl, 1% NP40, 0.9% NaCl, 0.25% Na-deoxycholate, 1 mM EDTA, 1× proteinase inhibitors (Roche, Basel, Switzerland), 1 mM PMSF, 1 mM Na₃VO₄, and 1 mM NaF], mixed with LDS sample buffer (Invitrogen, Carlsbad, CA, USA) containing 5% β-mercapto-ethanol and boiled at 95°C for 8 min. Crude extracts (10 μg) were immunoblotted with the Aph-1a [clone B80.2, kind gift from B. De Strooper (Nyabi et al., 2003 (link))], Pen2 (Cell Signaling, clone D2G6), Nicastrin (Cell Signaling, clone D38F9), Presenilin 1 (Millipore, clone PS1-loop), and Gapdh (Millipore, clone 6c5) antibodies. Membranes were detected using the ECL detection kit (Millipore, Billerica, MA, USA). Quantifications were performed using the Multi Gauge software (FUJIFILM, Minato-ku, Tokyo, Japan).

Cells were plated and allowed to adhere and grow overnight at 37°C with 5% CO₂. The following day, cells were transfected with 50 nM of pre‐miRs (Ambion) or 30 nM of siRNAs (Dharmacon, Appendix Table S1) using the HiPerFect transfection reagent (Qiagen) according to the manufacturer's instructions. Twenty‐four hours later, cells were transfected with control (pCDNA 3.0) or HA‐MARK4 plasmid (kind gift from Dr. Drewes 6) using Attractene and according to the manufacturer instructions.

PremiRs (Ambion, Applied Biosystems) or antagomiR oligonucleotides (Eurogentec) were transfected (100 μM) using siIMPORTER (Millipore) according to the manufacturer’s procedures. Negative control premiR (Ambion) or scrambled antagomiR were transfected as controls. Chemically modified antisense oligonucleotides (antagomiR) have been used to inhibit miR expression in vitro. The sequences of antagomiR-145 used is as follows: 5′-AGGGATTCCTGGGAAAACTGGAC-3′. The scrambled antimiR sequence was 5′-CAGCTGAAGTAAATACCGACCAG-3′.

Pre-miRs (25 nM; Ambion) and anti-miRs (25 nM; Exiqon) were transfected into HUVECs and MDA-MB-231 cells using Dharmafect-4 (Dharmacon Research Inc.) according to the manufacturer's instructions. CCND2, CCND3 and control siRNAs (20 nM) were transfected based on the calcium phosphate transfection method. Transfected HUVECs and MDA-MB-231 cells were plated in EGM2 or complete DMEM, respectively. After a 24-hour transfection, the cells were washed and kept in their media for an additional 48 or 72 hours. Functional assays were performed as described above.

PremiRs (Life Technologies) for miR-9 were nucleofected into L2.3 cells using Rat Neuron 96-well Nucleofector Kit (VHPG-1003) in conjunction with an amaxa 96-well shuttle system (Lonza, Cologne, Germany). Observed transfection efficiencies using the 96-well shuttle system were consistently >95% for both the L2.2 and L2.3 NSCs. At four hours after transfection, L2.3 cells were differentiated by removing FGF2 from the growth medium and allowed to differentiate for 72 hours. At 3 days, cells were fixed with 2% paraformaldehyde and analyzed for anti-βIII-tubulin (TuJ1, 1∶500, Covance) expression by flow cytometry on the FACSCalibur System (BD Biosciences, San Jose CA). Data was analyzed using BD's CellQuest Pro v. software.