Gapdh

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.

Fetal bovine serum (FBS), Dulbecco’s modified Eagle’s medium (DMEM)/Ham’s F12 medium, L-glutamine, antibiotics and trypsin-ethylendiaminetetraacetic acid were purchased from Lonza (Switzerland). Human recombinant SERPINE2 was obtained from R&D System (MN, USA), while human recombinant IL-1α was purchased from Sigma (MO, USA). ERK 1/2 pharmacological inhibitor PD098059 was purchased from Sigma (MO, USA). For RT-PCR, a First Strand Kit, Master mix, primers for SERPINE2, MMP-13 and GAPDH were purchased from SABiosciences (MD, USA). Nucleospin kits for RNA isolation were from Macherey-Nagel (Germany).

RNA was extracted from cell culture lysates or liver tissue using a RNeasy Mini Kit (Qiagen, Germantown, MD) according to the manufacturer’s instructions. Synthesis of cDNA was accomplished using a Bio-Rad iScript™ cDNA Synthesis Kit (Hercules, CA). RT-PCR was performed as previously described [23 (link)] using commercially available primers designed against mouse TGFβ1, CCL2, CX3CL1, interleukin-1 beta (IL-1β), tumor necrosis factor alpha (TNFα), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (SABiosciences, Frederick, MD). A ΔΔCT analysis was performed using vehicle-treated tissue or untreated primary neurons as controls for subsequent experiments [24 (link), 25 (link)].

Total mRNA was extracted from cells with the RNeasy mini kit (Qiagen), and was reverse-transcribed into cDNA using the SuperScriptIII kit (Invitrogen) according to the manufacturer’s protocol. Using cDNA as the template, Cbx3/HP1γ transcript was amplified by qPCR and normalized to Gapdh (SA Biosciences).

The cochleae were dissected from the mouse and homogenized on ice. Because of limited tissue, we combined 10-15 mice cochleae for the study. Total RNA was isolated using the RNeasy Plus Mini Kit (Qiagen), and cDNA was generated using the RT2 First Strand Kit (Qiagen). cDNA was combined with RT2 SYBR Green Master Mix (Qiagen), specific qRT-PCR primers, and qRT-PCR analysis was run using the ViiaTM 7 Real-Time PCR System (ABI). Primer efficiencies were determined by standard dilution curve analysis. Three separate samples were used from 10 animals for each group. The experiments from each sample were performed in triplicate, and average cycle threshold (Ct) values were normalized to Gapdh expression. ΔΔCt values were determined relative to Cldn9^+/+ cochlear samples. Fold change was defined as 2(−ΔΔCt). Primers used include Gapdh (SA Biosciences) and Cldn9 (ThermoFisher).