RNA Extraction and Real-Time PCR Analysis

Lung tissues were dissociated using TRIzol (Life Technologies) using beads and a high-speed homogenizer (Thermo Fisher). RNA was isolated from lung tissues, primary mouse ASMs, or primary asthmatic human bronchial ASMC (Lonza, Walkerville,MD, USA) using an RNAeasy mini kit (QIAGEN) following the manufacturer’s instructions and as previously described in our previous studies^{28 (link),72 (link)}. cDNA was synthesized using Superscript III (ThermoFisher), and real-time PCR was performed using SYBR Select Master Mix (Bio-Rad) and a CFX384 Touch Real-Time PCR instrument (Bio-Rad). Data were analyzed using CFX Maestro software version 4.0. Target gene transcripts were normalized using hypoxanthine-guanine phosphor ribosyl transferase (HPRT, for murine cells and tissues) or β-actin (for human cells) as housekeeping genes. The list of primers used to measure the transcript levels of the genes of interest can be found in the supplementary material (Table S1).