*2.8. Protein Analysis*

Retinal tissue was homogenized in lysis bu ffer (ThermoFisher, Waltham, MA, USA) containing 1% phosphatase and protease inhibitor cocktail (Sigma-Aldrich). Protein concentration was measured using the Bio-Rad protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's recommendation. Proteins from whole rat retinal tissue and HuREC lysates were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto polyvinylidene difluoride (PVDF) membrane. The membrane was blocked using 5% skim milk and incubated with the following primary antibodies: anti-HDAC6 (Abcam, Cambridge, MA, USA), anti-Trx-1, anti-sirtuin 1 (SIRT1) and anti-albumin (all from Cell Signaling Technology). After incubation with horseradish peroxidase–conjugated secondary antibody (GE Healthcare, Pittsburg, PA, USA), bands were detected using the enzymatic chemiluminescence reagent, ECL (GE Healthcare). Subsequently, the membranes were stripped using stripping bu ffer (Bio-Rad) and re-probed with anti-β-actin antibody (Sigma-Aldrich) to assess equal loading. Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

## *2.9. Dot BlotAanalysis*

Equivalent amount of proteins prepared from whole rat retinas and HuREC lysates were spotted on nitrocellulose membranes and dried for 5 min at room temperature. The membranes were blocked for 1 h by using 5 % non-fat dry milk in PBS and then probed for 1 h with either anti-3-nitrotyrosine (NT, Cayman) or anti 4-hydroxynonenal (4-HNE, Abcam) antibodies in PBS-tween bu ffer. The membranes were then washed three times in PBS-tween bu ffer and probed again with horseradish peroxidase-conjugate secondary antibody (Cell Signaling). After washing the membrane, the immuno-positive spots were visualized by using Clarity ECL- Blotting substrate (Bio-Rad). Scanned images of blots were used to quantify protein expression using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

#### *2.10. Quantitative PCR Analysis*

Gene expression at mRNA level was assessed in retinal and HuREC extracts by quantitative polymerase chain reaction (qPCR). Total RNA was isolated from the HuREC and rat retinas using RNeasy Kit (Qiagen, Germantown, MD, USA). cDNA was prepared using iScript ™ cDNA Synthesis Kit (Bio-Rad). Amplification of HDAC6, Trx-1, GCLC, GCLM, NQO1, and HO-1 mRNA was performed using power SYBR green PCR master mix (Applied Biosystems, Foster City, CA, USA). The conditions used for the PCR were as it follows: 95 ◦C for 3 min (1 cycle) and 94 ◦C for 20 s, 55 ◦C for 30 s, and 72 ◦C for 40 s (40 cycles). The relative mRNA abundance was determined by normalizing to mRNA for hypoxanthine phosphoribosyltransferase 1 (HPRT-1) for tissue or 18 s for cells, using the 2Ct method (Ct refers to the threshold value). A complete list of the di fferent primers used in this study is included in Supplementary Table S3.

#### *2.11. Reactive Oxygen Species Assays*

For detection of superoxide in retinal tissue, 10 μm-thick retinal cryosections, from di fferent experimental groups, were covered (at room temperature) with a 10 μM dihydroethidium (DHE) solution and incubated in a light-protected humidified incubator at 37 ◦C for 30 min. At the end of the incubation, sections were mounted with a coverslip and images were taken using Zeiss Axioplan-2 fluorescence microscope (Carl Zeiss). The fluorescence intensity was measured using NIH ImageJ software (http://rsb.info.nih.gov/ij/).

ROS detection from cellular sources was accomplished by CellROX green assay (ThermoFisher) performed according to the manufacturer's protocol. HuREC were loaded with 5 μM CellROX green in culture medium and stained in the dark for 30 min at 37 ◦C. Stained cells were washed in PBS twice, mounted using Fluoroshield mounting medium containing DAPI (Sigma-Aldrich) to visualize nuclei. Images were then immediately captured using a Zeiss Axioplan-2 fluorescence microscope (Carl Zeiss).

Mitochondrial superoxide production was measured using MitoSOX Red (ThermoFisher). HuREC were loaded with 5 μM MitoSOX red in Hank's balanced salt solution (HBSS) with calcium and magnesium for 30 min at 37 ◦C in the dark. Stained cells were then washed and suspended in HBSS, mounted using Fluoroshield mounting medium containing DAPI (Sigma-Aldrich) and immediately analyzed under a Zeiss Axioplan-2 fluorescence microscope (Carl Zeiss).

#### *2.12. Assessment of Senescence Markers*

To evidence senescent HuREC, we used senescence-associated β-Galactosidase (SA-β-Gal) reactivity-based assay using a commercially available kit (Cell Signaling) as previously shown [7]. Positive reactivity to SA-β-Gal, assessed at pH 6, is measured on images captured (10 frames per well) at 20× magnification by light microscopy using Zeiss Axioplan-2 microscope (Carl Zeiss). Percentage of SA-β-Gal positive cells/well was determined as number of cells positive for a blue color versus total number of cells counted in a blind fashion.
