Wwp1 sirna

Wwp1 siRNA and control siRNA were purchased from Ambion^® (ThermoFisher Scientific). For gene silencing experiments, mMSCs were seeded in 24-well plates at 8000 cells/cm² one day prior to siRNA/NP treatment. Nanoparticles with complexed siRNA (40 nM) were used to treat cells at charge ratios of 1:1–8:1. Cells were also treated with siRNA complexed with Lipofectamine 2000 as a positive control. After 48 h, the cells were washed with PBS (0.5 ml) twice and lysed with TRK Lysis Buffer (OMEGA Bio-Tek). RNA was extracted using Homogenizer mini columns, Hibind mini columns and RNA wash buffers with on-column DNase digestion (Omega Bio-Tek). Extracted RNA was quantified using a NanoVue (GE Healthcare) and RNA concentrations were normalized amongst samples to 50 ng/µl. Then, RNA (2 µL) sample was reverse transcribed to cDNA using an iSCRIPT reverse transcription kit (Bio-Rad) and 2 µl of cDNA was used in RT-PCR reactions using SsoFast Eva-Green Supermix (Bio-Rad) on a CFX96 Real-time PCR Detection System (Bio-Rad). β-actin was used as the housekeeping gene RT-PCR. Primer sequences for Wwp1 and β-actin are listed in Table S1. Wwp1 expression was quantified based on the reaction efficiency [29 (link)] and normalized to β-actin expression and comparing to untreated cell populations [30 ].

Wwp1 siRNA and control siRNA were purchased from Ambion^® (ThermoFisher Scientific). Mouse MSCs (mMSCs) derived from C57BL/6 mice were cultured at 37 °C and 5% CO2 in growth media consisting of low glucose Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS), 100 units/ml Penicillin-Streptomycin (Gibco). For gene silencing experiments, mMSCs were seeded in 24-well plates at 8000 cells/ cm² one day before siRNA/NP treatment. For Wwp1 siRNA/NP treated groups, nanoparticles complexed with siRNA were used to treat cells at 40 nM and charge ratios of 4:1. For Wwp1 siRNA/NP hydrogel treated groups, Wwp1 siRNA (0.05 nmole) was complexed with NPs, and entrapped in hydrogels (40 µl). Hydrogels were placed in transwell inserts with 0.5 ml cell culture media, to approximate 40 nM siRNA in hydrogel-released complexes over the first 2 days of treatment to emulate the free siRNA/NP treatments. Media was replaced every 3 days. At each time point, cells were washed with 0.5 ml PBS twice and lysed with TRK Lysis Buffer (OMEGA Bio-Tek). RNA extraction, cDNA synthesis, and RT-PCR were performed as described previously. Wwp1 expression was quantified based on the reaction efficiency and normalized to β-actin expression and comparing to untreated cell populations [29 (link)].