Il 17a

Total RNA was isolated from lumbar spinal cords using RNeasy Mini kit (Qiagen) according to the manufacturer’s instructions and stored at −80 °C. The concentration and purity of RNA were determined by measuring absorbance at 260/280 nm using a nanodrop spectrophotometer (Thermo Fisher Scientific). First strand cDNA synthesis was performed with 1 μg total RNA using miScript II RT Kit (Qiagen) for mRNA expression analyses according to the manufacturer’s instructions. Real time PCR was performed using QuantiFast SYBR Green master mix (Qiagen) and Quantitect Primers: Il10 (Qiagen,QT00106169); Il17a (Qiagen, QT00103278); Il1a (Qiagen, QT00113505); Arg1 (Qiagen, QT00134288); NOS2 (Qiagen, QT01547980); Il2 (SA Biosciences, PPM03020E); Actb (Qiagen, QT00095242) and Gapdh (Qiagen, QT01658962) with the following cycling conditions: 95 °C for 5 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s and extension at 72 °C for 30 s. βactin and Gapdh housekeeping genes were used to normalize mRNA expression. The relative expression levels were analyzed by 2^-ΔΔct method.

Embryonic day 16 fetal mouse lungs were minced into 0.5–1.0 mm³ pieces. Explants were placed onto polyethylene hanging insert filters (0.4 μm, EMD Millipore Corp, Billerica, MA) in 12-well plates and were cultured in the presence or absence of recombinant mouse TNF-α (50 ng/ml) and IL-17a (100 ng/ml), both from Peprotech, (Rocky Hill, NJ), for 72 h. Total RNA was extracted using RNeasy Plus Micro kit (Qiagen, Valencia, CA). Gene expression was analyzed by RT-qPCR. Selected explants were fixed in 4% paraformaldehyde overnight and stained with fluorescently labeled, monoclonal antibody specific for α-smooth muscle actin (Sigma-Aldrich).

Total RNA was extracted from RSCs by using an RNeasy Mini Kit (Qiagen, Hilden, Germany), and equal amounts of RNA were synthesized to cDNA with Superscript II RNase H Reverse Transcriptase (Invitrogen) according to the manufacturer's instructions. Quantitative RT-PCR (qRT-PCR) was performed using RT² SYBR Green ROX qPCR Mastermix (Qiagen). cDNA was amplified with primers β-actin, Il-6, Il-17rc, or Il-17a (Qiagen) separately for 50 cycles. All data were normalized to the β-actin mRNA level. Expression fold-changes were calculated by 2^−ΔΔCT.