Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration

Data Repository

Principal Investigator: Joshua Dunaief
Institution: University of Pennsylvania Perelman School of Medicine
Email: jdunaief@pennmedicine.upenn.edu

This data repository holds data used in the following paper:

Wojciechowski, A.M., Bell, B.A., Song, Y., Anderson, B.D., Conomikes, A., Petruconis, C., Dunaief, J.L., 2024. Inducible RPE-specific GPX4 knockout causes oxidative stress and retinal degeneration with features of age-related macular degeneration. Experimental Eye Research 247, 110028. https://doi.org/10.1016/j.exer.2024.110028

The authors encourage readers to refer to this paper for detailed scientific explanation and presentation of the included data. This repository primarily acts as a supportive document for any readers interested in looking closer at the details of our data or wish to replicate any of our findings.

Data was collected from 2022 to 2024 at the University of Pennsylvania, Philadelphia, PA.

This research was funded by the Research to Prevent Blindness, the Paul MacKall and Evanina Bell MacKall Trust, F.M. Kirby Foundation, and National Institutes of Health grants (RO1EY015240 and RO1EY028916 to J.L.D., T32GM008076 to A.W., T32EY007035–44 and T32GM008076 to B.D.A., S10OD026860 Shared Instrumentation Grant, and P30EY001583 Core Grant for Vision Research).

Protocols

Methods and reagents used can be found in the published paper’s “Methods” section. Antibodies and probes can be found at the end of this document. In addition to those methods, more detailed protocols can be found in this data repository in the “Protocols” folder, including:

•	Dunaief lab IHC sample preparation.rtf – used to generate the images seen in Fig. 8-10
•	Dunaief lab plastic section preparation.rtf – used to generate the images seen in Fig. 5c and 10c
•	Dunaief lab RPE cells isolation.rtf – used to prepare samples for the PCR analyses seen in Fig. 1, 3, and 7.
•	Dunaief lab RPE flat mount preparation.rtf – used to generate the images seen in Fig. 2b
•	Dunaief lab staining slides with toluidine blue.rtf – used after the “Dunaief lab plastic section preparation.rft” protocol to generate the images seen in Fig. 5c and 10c
•	Dunaief lab Western blot.rtf – used to generate images seen in Fig. 1b

Data organization

The data tables, located in the “Data” folder, are organized based on the paper’s figure numbers and the experiment type (e.g. PCR, immunohistochemistry). The columns in each data table are defined below.

•	Fig1,3,7 PCR data.csv
o	Column A: The figure the data is used for.
o	Column B: The plate the sample/probe was run on. All samples are normalized to the average WT values on each respective plate.
o	Column C: The sample identifier. Format: genotype (wild type or GPX4 KO), unique number.
o	Column D: Time when samples were collected, measured as weeks after the completion of the tamoxifen injection.
o	Column E: The tissue the sample was collected from (either retinal pigment epithelium (RPE) or neural retina).
o	Column F: The mRNA target was analyzed.
o	Column G: The average CT value (taken from technical triplicate), as reported by the PCR machine.
o	Column H: The average fold change that was derived from Column G, using the delta delta CT method. This number was graphed in their respective figures.
o	Column I: The probe ID used for that plate.
o	Column J: The lot number of the probe used for that plate.
•	Fig1b Western Blot Densitometry.csv
o	Column A: The tissue the sample was collected from (either RPE or neural retina).
o	Column B: The group (wild type or GPX4 KO) the sample belongs to.
o	Column C: The signal intensity of the GPX4 band.
o	Column D: The signal intensity of the B-actin band.
o	Column E: The ratio of the two intensities (Column C divided by Column D). This number is graphed in Fig. 1b. 
•	Fig2c RPE Cell Count.csv
o	Column A: The sample identifier. Format: genotype (wild type or GPX4 KO), unique number.
o	Column B: The location where the analysis was done (either central (near the optic nerve) or peripheral).
o	Column C: The amount of cells in the 550x550 pixel area. These numbers are graphed in Fig. 2c.
•	Fig5b OCT thickness.csv
o	Column A: The sample identifier. Format: genotype (wild type or GPX4 KO), unique number.
o	Column B: The eye analyzed. OD = right eye. OS = left eye.
o	Column C: The orientation of the OCT image. Either horizontal (sagittal plane) or vertical (transverse plane).
o	Column D: The distance (in µm) from the top of the outer nuclear layer (ONL) to Bruch’s membrane (BrM). These numbers are graphed in Fig. 5b. 
•	Fig5d ONL nuclei quantification - plastic sections.csv
o	Column A: Time when samples were collected, measured as weeks after the completion of the tamoxifen injection.
o	Column B: The sample identifier. Format: genotype (wild type or GPX4 KO), unique number.
o	Column C: Distance from the optic nerve the measurement was taken. See the images in the “Example plastic section distance locations” folder for a visual representation of this.
o	Column D: The thickness of the outer nuclear layer (ONL), reported as nuclei thick. The average of this data is reported in Fig. 5d. 
•	Fig6 ERG averages.csv
o	Column A: Time when samples were collected, measured as weeks after the completion of the tamoxifen injection.
o	Column B: The ERG wave being reported.
o	Column C: The flash illuminance (cd s m-2 for photopic waves or log cd s m-2 for scotopic waves) used to initiate a response. 
o	Column D: The response amplitude (µV) to the flash illuminance for GPX4 KO mice. Reported as the mean with the standard deviation in parentheses. The mean and standard deviation is graphed in Fig. 6. A response amplitude of 0 indicates a lack of the indicated wave form.
o	Column E: The median response amplitude (µV) to the flash illuminance for GPX4 KO mice, with quartile 1 and 3 in parentheses. A response amplitude of 0 indicates a lack of the indicated wave form.
o	Column F: The response amplitude (µV) to the flash illuminance for wild type mice. Reported as the mean with the standard deviation in parentheses. The mean and standard deviation is graphed in Fig. 6.
o	Column G: The median response amplitude (µV) to the flash illuminance for wild type mice, with quartile 1 and 3 in parentheses.
•	Fig8-10 IHC quantification.csv
o	Column A: The figure the data is used for.
o	Column B: The protein that was targeted by the antibody.
o	Column C: The group (wild type or GPX4 KO) the sample belongs to.
o	Column D: The amount of pixels that had a positive signal in the area of interest.
o	Column E: The total amount of pixels in the area of the image that was analyzed.
o	Column F: The ratio of positive pixels to total pixels (Column D divided by Column E). This number is reported in Fig. 8b, 9a, or 10c.

Additionally, for data tables created from image analysis, representative images are included to demonstrate what data is collected. These are organized in the following folders:

•	“Example OCT image analysis” contains two OCT images from a WT mouse. “WT6_OD_horizontal_original.bmp” is the original image, and “WT6_OD_horizontal_analyzed.tif” shows an overlay of the analysis done with the ImageJ macro referenced later in this document. The lengths of all vertical lines in the analyzed image are averaged and reported in “Fig5b OCT thickness.csv.” 
•	“Example plastic section distance locations” contains a cross section image from a WT mouse and a cross section image from a GPX4 KO mouse. These images demonstrate the location each measurement was taken, as reported in “Fig5d ONL nuclei quantification – plastic sections.csv.”
•	“Example RPE flatmount quantification” contains screenshots of analysis done in ImageJ from a WT mouse and a GPX4 KO mouse. The yellow square shows the 550x550 pixel area that was quantified for all samples. Blue dots within that box mark RPE cells that were identified by the program and the Cell Counter box displays the cell count. A summary of the cell count data from all samples can be found in “Fig2c RPE Cell Count.csv.” 
•	“Western blot images” contains the same images as seen in Fig. 1b, but uncropped. File names are organized as follows: figure, tissue, antibody, exposure time.

Code

An ImageJ macro was used to quantify OCT images. The quantification of these images can be found in Fig. 5b and an example of the quantification process can be seen in the “Example OCT image analysis” folder. This macro can be found at:

Anderson, B. (2023). Semiautomated OCT thickness analysis (Version 2.2) [Computer software]. https://doi.org/10.5281/zenodo.14947742

Probes and antibodies used

PCR probes (Fig1,3,7) (all probes are TaqMan probes from Applied Biosystems)
•	Gapdh (4352932E) for neural retina
•	18S rRNA (4352930E) for RPE
•	Gpx4 (Mm00515041_m1)
•	Rho (Mm00520345_m1)
•	Rpe65 (Mm00504133_m1)
•	C3 (Mm01232779_m1)
•	Hmox1 (Mm00516005_m1)
•	Slc7a11 (Mm00442530_m1)
•	Opn1sw (Mm00432058_m1)
•	Opn1mw (Mm00433560_m1)
•	Gfap (Mm01253033_m1)
•	Iba1 (Mm00479862_g1)
•	Cat (Mm00437992_m1)
•	Tfrc (Mm00441941_m1)
•	Ncoa4 (Mm00451095_m1)
•	Acsl4 (Mm00490331_m1)

Western blot (Fig1)
•	Rabbit anti-GPX4 (1:5000, abcam ab125066)
•	Mouse anti-beta-actin (1:1000, Cell Signaling 3700)

En face immunofluorescence (Fig2c)
•	Rabbit anti-ZO-1 (1:200, Invitrogen 40–2200)
•	Cy3 donkey anti-rabbit (1:200, Jackson ImunoResearch Laboratories 711-165-152)

Cross-sectional immunofluorescence (Fig8-10)
•	Mouse anti-GFAP (1:500, Cell Signaling 3670)
•	Mouse anti-CEP (1:300, a kind gift from John Crabb)
•	Rabbit anti-OPN1SW (1:500, Millipore Sigma AB5407)
•	Rabbit anti-OPN1MW (1:250, Millipore Sigma AB5405)
•	Cy3 donkey anti-mouse IgG (1:200, Jackson ImmunoResearch Laboratories 715-165-151) (paired with anti-CEP)
•	Cy3 donkey anti-rabbit IgG (1:200, Jackson ImunoResearch Laboratories 711-165-152) (paired with anti-OPN1SW and anti-OPN1MW)
•	488 donkey anti-mouse (1:200, Invitrogen A21202) (paired with anti-GFAP)