Trk lysis buffer

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)].

After treatment for four hours, cells were lysed in TRK lysis buffer (Omega Biotek) containing β-mercaptoethanol (Sigma) and RNA isolated (E.Z.N.A.® RNA Isolation Kit, Omega Biotek). Equal amounts of RNA were reverse transcribed into cDNA (High-Capacity cDNA Reverse Transcription Kit, Thermo Scientific). Quantitative PCR was performed on a CFX96™ Real-Time PCR Detection System (Biorad) with Fast SYBR Green Master Mix (Roche). Custom primers were validated and efficiencies [47 (link)] listed in Table 1. Data are presented as expression relative to the housekeeping gene Rpl32 calculated by $E_{HKG}^{C{T}_{HKG}}/E_{GOI}^{C{T}_{GOI}}$ [47 (link)]. Fold-change calculated by (RE_hydrogel – RE_ref)/RE_ref; ref is the reference substrate. CT values for Rpl32 were consistent across all samples in macrophages from WT and MyD88^−/− mice. After treatment for 24 hours, media were collected and assessed for the cytokines tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) by standard enzyme-linked immunosorbent assay kits (ELISA, R&D Systems).

Primary SMG isolates (Day 0) or SMG aggregates (Day 2) were pelleted by centrifugation and lysed in 350 μL TRK lysis buffer (Omega Biotek) with β-mercaptoethanol (β-ME, 20 μL per 1 mL lysis buffer). Samples were transferred to a homogenizer column (Omega Biotek) and centrifuged at 10,000 RCF for 2 minutes. Sample flow through was precipitated with an equivalent volume of 70% ethanol (~350 μL) and transferred to an E.Z.N.A. RNA purification column (Omega Biotek). RNA was purified and isolated according to manufacturer’s instructions. RNA concentration and quality were determined using a NanoVue UV-vis spectrophotometer. Samples were reverse transcribed into cDNA using the iScript™ cDNA synthesis kit (BioRad). QPCR was performed on a CFX96 Real-Time System (BioRad) using SsoFast Evogreen Supermix (BioRad) according to manufacturer’s instructions, with 10 μM forward and reverse primers (Supplemental Table 1) and cDNA. Gene expression was analyzed using the Pfaffl method with LE32 as the housekeeping gene (Pfaffl, 2001 (link)).