In Vitro Luciferase Assay Protocol

Total RNA was isolated using a RNeasy Plus Mini Kit and quantitative polymerase chain reaction (qPCR) analysis was performed, as previously described [38 (link),43 (link),50 (link),51 (link)]. Total RNA was quantified using a NanoDrop 2000 apparatus (Thermo Fisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti^® Thermal Cycler using the High Capacity RNA-to-cDNA Kit (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). qPCR was performed using individual TaqMan^® Probe-Based Gene Expression Assays (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Individual TaqMan gene expression assays were selected for nuclear factor (erythroid-derived 2)-like 2 (Nrf2) regulated genes, NADPH: quinone oxidoreductase-1 (NQO1), glutathione S-transferase mu 1 (GSTM1), and heme oxygenase-1 (HO-1). qPCR was performed using 50 ng of cDNA per reaction well on an Applied Biosystems QuantStudio 6 Flex Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA). Gene expression data are presented normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Gene expression analysis was also performed using two additional housekeeping genes, 18S ribosomal RNA (18S rRNA) and β-actin for validation. All data were calibrated to the control, untreated samples (RPE Ctrl) according to the ΔΔCT method as previously described [52 (link)].

Total RNA was isolated from huPCLS and qPCR analysis was performed as previously described [15 (link),25 (link),36 (link)] using individual TaqMan^® Probe-Based Gene Expression Assays (Applied Biosystems, Life Technologies, Carlsbad, CA, USA) selected for proinflammatory cytokines (IL-1β, IL-6, TNFα, and IL-1α), inflammation (COX-2), relevant cytoprotective and antioxidant enzymes (heme oxygenase-1 (HMOX1) and NADPH:quinone oxidoreductase-1 (NQO1), and cell cycle markers (TP53, CDK2, CDKN1A, CDKN2A, CDK4, CDK6, RB, and E2F). Transcript levels of tested genes were normalized to β-Actin.

Quantitative Polymerase Chain Reaction (qPCR) was performed using TaqMan^® Probe-Based Gene Expression Assays supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Individual TaqMan gene expression assays were selected for GADD45α, Survivin (Birc5: baculoviral inhibitor of apoptosis repeat-containing 5), and Bcl-2-associated X protein (BAX). Briefly, cells were lysed and RNA was isolated using a commercially available kit, QIAprep Spin Miniprep Kit, supplied by Qiagen (Valencia, CA, USA). Total RNA was quantified using a NanoDrop 2000 (ThermoFisher Scientific, Waltham, MA, USA). Reverse transcription of RNA to cDNA was then performed on a Veriti^® Thermal Cycler using the High Capacity RNA to cDNA kit supplied by Applied Biosystems, Life Technologies (Carlsbad, CA, USA). Quantitative real-time PCR was performed using 50 ng of cDNA per reaction well on a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Life Technologies, Carlsbad, CA, USA). Gene expression data was normalized to 18S ribosomal RNA housekeeping gene and calibrated to the control samples according to the 2^−ΔΔCt method as previously described [40 (link),49 (link),50 (link)].