Gene expression profile of human trabecular meshwork cells with silenced expression of autophagy genes Atg5 and Atg7 subjected to cyclic mechanical stretch. Gene expression profile of human trabecular meshwork cells with silenced expression of autophagy genes Atg5 and Atg7 subjected to cyclic mechanical stretch

In order to investigate the effects of downregulated autophagy in TM cells response to mechanical stretch, we conducted gene expression analysis in human TM cells deficient in autophagy and subjected to cyclic mechanical stretch. For this, three independent strains of primary human TM cells were transfected with a cocktail of siRNAs to specifically silence the expression of the autophagy genes Atg5 and Atg7 (siAtg5/7), and subjected to cyclic mechanical stress (15% elongation, 1 cycle/sec, 24h). Overall design: siRNA transfection was performed as described previously with minor modifications (Kristine M Porter, Jeyabalan, & Liton, 2014). In brief, primary human TM cells were plated on 24-well plate, with 60-80% confluence, in 0.5 mL of the growth media. After 24 h, the cells were transfected with either 5 pmol of siRNA against ATG5 (siAtg5, sc-41445) and ATG7 (siAtg7, sc-41447) or 5 pmol of non-targeting siRNA (siNC, sc-37007) using Lipofectamin® RNAiMAX Reagent, according to the manufacturer’s instructions. At 72 h post-transfection, cells were subjected to cyclic mechanical stress for 24 h using the Flexcell System. A regime of 15% cyclic stretch, 1 cycle/sec was applied. Non-stretched control cells were maintained under same culture conditions, but no mechanical forces were applied. Cells were washed with cold PBS and fixed in RNAlater (Qiagen). Total RNA was isolated using RNeasy kit (Qiagen, Valencia, CA), following the manufacturer's protocol, and treated in-column with DNase I. RNA concentration and quality were determined using the Agilent 2100 Bioanalyzer. Total RNA (5 µg) from human TM primary cultures with transient silenced autophagy and controls were independently hybridized to human Clariom D microarrays (ThermoFisher) following the manufacturer's instructions at the Duke Microarray Core Facility. Data analysis was performed using the Partek Flow and Partek Genome Suite statistical analysis software (Partek Incorporated).