qRT-PCR:
RPGSMCs were grown in 6-well plates until approximately 90% confluent
before being washed and switched to serum and growth factor starved
media. Cells were then subjected to 48-hour drug and/or peptide
treatment before lysis in TRIzol reagent (ThermoFisher, 15596026). The
Direct-zol RNA miniprep plus (Zymo, R2051) manufacturer’s protocol was
used to isolate RNA from cells. For cDNA synthesis, the SuperScript IV
First Strand Synthesis (ThermoFisher, 18091050) kit manufacturer’s
protocol was used. For quantitative real time PCR analysis, the PowerUp
SyBr Green (ThermoFisher, A25742) and 1 μM target primer (Table 2) were
mixed according to manufacturer’s protocol with settings for 40 PCR
cycles, 95°C melting temperature, 58°C annealing temperature, and 72°C
extension temperature set on a QuantStudio 5 Real-Time 384-well PCR
System (ThermoFisher A28140) for amplification. The Δ-Δ-ct value fold
change in expression was used in order to control for cell number and
RNA quality with values normalized to an 18S housekeeping gene
transcript.