Figure 5. Effect of glucocorticoid receptor silencing on myocilin protein secreted by nontransformed human trabecular meshwork cells and perfused human organ culture treated with dexamethasone

Equivalent volumes of cultured HTM-55 cell supernatants and organ culture effluents from Individual number 5 transfected and/or perfused with scramble control or GR siRNA in the presence of DEX. Samples were concentrated, separated on 4-15% SDS-PAGE gels, and electro-transferred to PVDF membranes. MYOC protein was detected with a MYOC polyclonal antibody and visualized by chemiluminescence. Top: Representative gel shows the reduction of the secreted MYOC protein in HTM-55 cells (left) and organ cultures (right). Bottom: There is a percentage of reduction of MYOC protein on cells treated with GR siRNA and scramble controls, normalized to total protein concentration. Scramble negative control samples were normalized to a value of 0 (no reduction) and MYOC percent reduction values from scramble controls are expressed as the mean±SEM. HTM-55 cell supernatants (n=5) and perfused organ culture effluents of individual 5 (n=5). Transfection and/or perfusion of GR siRNA reduces protein levels of secreted MYOC in DEX-treated, cultured HTM cells and perfused organ cultures. An asterisk indicates that p=0.02 for HTM-55 cells and that p=0.0000002 for individual 5.