Figure 7.

LTB₄ levels and cell death in genetically manipulated ARPE-19 cells. (A) Quantitation of LTB₄ levels in ARPE-19 cells untransfected and transfected with siScrambled (siSCR), siPNPLA2, PNPLA2 overexpression (PNPLA2-OE), or siPNPLA2 plus P1 and treated with indicated concentration of H₂O₂. The level of LTB₄ is normalized to total protein per well. Each condition was tested in triplicate wells per experiment ± SD (error bars). (B) Plot of relative cell death determined at the end point using intracellular protease activity as biomarker corresponding to conditions shown in (A). Relative cell death values are averages of triplicate wells ± SD (error bars). *P < 0.05, **P < 0.005, and ***P < 0.0005. (C) Total protein from cells harvested 72 hours post transfection with three different siRNAs. Lane 1, no silencing; lane 2, siScrambled; lane 3, siPNPLA2-1; lane 4, siPNPLA2-2; lane 5, siPNPLA2-3. Samples were resolved by SDS-PAGE followed by immunoblotting with anti-PEDF-R. The blot was stripped and immunoreacted with antibody to β-tubulin as loading control. (D) Quantitation of PEDF-R levels in siPNPLA2 transfected cells shown as percentage remaining compared to untransfected cells. Data are average from three independent experiments. (E) Total protein from cells harvested 72 hours post transfection with PNPLA2-OE. Lane 1, nontransfected; lane 2, PNPLA2-OE. Samples were resolved by SDS-PAGE followed by immunoblotting with anti-PEDF-R. The blot was stripped and immunoreacted with anti-V5 to probe V5-tag-PEDF-R-OE. The experiments were repeated three times.