Data Availability StatementAll relevant data are within the paper and a minimal data set that has been provided and is available at doi:10. mRNA and protein analyses. Cellular ROS and mitochondrial superoxide were measured by CM-H2DCFDA and MitoSOX staining respectively. Mitochondrial viability was decided using MTT activity. qPCR-array system was used to investigate autophagic genes affected by p62. Nuclear and cytoplasmic levels of NFB p65 were evaluated after cellular fractionation by Western blotting. We statement that p62 is usually up-regulated in RPE cells under H2O2-induced oxidative stress and promotes autophagic activity. Depletion of endogenous p62 reduces autophagy by downregulation of ATG10 rendering RPE more susceptible to oxidative damage. NFB p65 phosphorylation at Ser-536 was found to be critical for p62 upregulation in response to oxidative stress. Proteasome inhibition by H2O2 causes p62-NFB signaling as antioxidant pre-treatment reversed p62 expression and p65 phosphorylation when RPE was challenged by H2O2 but not when by Lactacystin. p62 protein but not RNA levels are elevated in mice, which at the age of 16 months were switched from a normal rodent diet (ND) (Isopurina 5001; Prolab, Dewitt, NY) to a high excess fat, cholesterol (HFC) enriched diet (TD 88051; Harlan Teklad, Madison, WI) for 2 months as explained in Malek mice constantly maintained on a ND (mouse samples, RNA was extracted from your RPE/choroid lysates using a combination of TRIzol reagent and Qiagen RNeasy Mini kit. 500ng of total RNA was reverse Gata6 transcribed to cDNA using the iScriptTM Reverse Transcriptase kit (Bio-rad, Berkley, CA) (20l reactions). Further downstream actions for Ginsenoside Rg2 qRT-PCR were the same as explained above. Proteasomal peptidase assay Proteasomal peptidase assay was performed as explained previously (39). Briefly, after treatment RPE cells were harvested and lysed in hypotonic buffer (10mM HEPES, 5mM MgCl2, 10mM KCl, 1% sucrose, and 0.1% CHAPS). Lysates were then incubated with fluorogenic substrate Suc-LLVY-AMC (75M) in the assay buffer (50mM Tris-HCl, 20mM KCl, 5mM MgOAc and 10mM Ginsenoside Rg2 dithiothreitol, pH 7.6) for 30 min at 37C. Cleaved fluorescent products were then examined at the excitation wavelength of 380 nm and emission wavelength of 460 nm by a fluorescence plate reader (Biotek, Winooski, VT). Enzymatic activities were normalized by protein concentration, which was measured using the Bradford method. Transfection experiments ARPE-19 cells were transfected by Lipofectamine? RNAiMAX with duplex oligoribonucleotides against p62/SQSTM1 or with scrambled siRNA (Silencer? Select siRNA, Life Technologies, Carlsbad, CA) for 24 h. Cells were then treated with 400M H2O2 as explained above. For ATG10 knockdown experiments ARPE-19 cells were transfected with either scrambled or Flexitube ? siRNA oligos from Qiagen. For overexpression experiments, ARPE-19 cells were transfected with a p62 construct (pTR-SC-smCBA-p62/SQSTM1) or vacant vector (pTR-SC-smCBA) using Lipofectamine? LTX reagent. Similarly, p65 wild type (p65-WT), p65 dominant positive mutant serine to aspartic acid (p65-S536D) and p65 phosphorylation unfavorable mutant serine to alanine (p65-S536A) on vector pcDNA3.1 were transfected in ARPE-19 cells for the NFB p65 overexpression experiments. Cellular reactive oxygen species (ROS) and superoxide detection CM-H2DCFDA (Thermo Scientific, Cat# C6827) and MitoSOX? Red (Invitrogen, Cat # Ginsenoside Rg2 “type”:”entrez-nucleotide”,”attrs”:”text”:”M36008″,”term_id”:”214108″,”term_text”:”M36008″M36008) were used to detect cellular ROS and mitochondrial superoxide formation respectively in ARPE-19 cells following stress procedures. After incubating cells with or without H2O2 (6 Hrs for CM-H2DCFDA and 1 Hrs for MitoSOX?) or Lactacystin (12 Hrs) on black, clear-bottom cell culture treated 96-well tissue culture plate (CorningTM, Cat # 3603), cells were Ginsenoside Rg2 loaded with CM-H2DCFDA (10 M) or Mitosox (2M) dye for 10 minutes at 37C. Following the incubation, the plate was first centrifuged at 200g at.