DOI: 10.5061/dryad.3j9kd51vx
title: Soluble RAGE enhances muscle regeneration after cryoinjury in aged and diseased mice

description: The Receptor for Advanced Glycation End Products (RAGE), classically
 considered a mediator of acute and chronic inflammatory responses, has
 recently been implicated by genetic knockout studies as a regulator of
 skeletal muscle physiology during development and following acute injury.
 Yet, the role of its soluble isoform, soluble RAGE (sRAGE), in muscle
 regeneration remains relatively unexplored. To address this knowledge gap,
 Adeno-Associated Virus (AAV) mediated and genetic knockin supplementation
 strategies were developed to specifically assess the effects of changing
 levels of sRAGE on muscle regeneration. We evaluated general muscle
 physiology and histology, including central nucleation, and myofiber size.
 We found that acute induction of sRAGE in aged and atherosclerotic animals
 accelerates muscle repair after cryoinjury. Similarly, genetic
 modification of the endogenous Ager gene locus to favor production of
 sRAGE over transmembrane RAGE accelerates repair of cryo-damaged skeletal
 muscle. However, increasing sRAGE via AAV delivery or using our transgenic
 mouse lines had no impact on muscle repair in aged or diseased mice after
 barium chloride (BaCl2) injury. Together, these studies identify a unique
 muscle regulatory activity of sRAGE that is variable across injury models
 and may be targeted in a context-specific manner to alter the skeletal
 muscle microenvironment and boost muscle regenerative output.
description: # Soluble RAGE enhances muscle regeneration after cryoinjury in aged and
 diseased mice
 [https://doi.org/10.5061/dryad.3j9kd51vx](https://doi.org/10.5061/dryad.3j9kd51vx) ## Description of the data and file structure Adeno-Associated Virus (AAV) mediated and genetic knockin supplementation strategies were developed to specifically assess the effects of changing levels of sRAGE on muscle regeneration. We evaluated general muscle physiology and histology, including central nucleation, and myofiber size. We found that acute induction of sRAGE in aged and atherosclerotic animals accelerates muscle repair after cryoinjury. Similarly, genetic modification of the endogenous *Ager*gene locus to favor production of sRAGE over transmembrane RAGE accelerates repair of cryo-damaged skeletal muscle. However, increasing sRAGE via AAV delivery or using our transgenic mouse lines had no impact on muscle repair in aged or diseased mice after barium chloride (BaCl2) injury. Together, these studies identify a unique muscle regulatory activity of sRAGE that is variable across injury models and may be targeted in a context-specific manner to alter the skeletal muscle microenvironment and boost muscle regenerative output. ### Files and variables #### File: Fig1A.zip **Description:** ELISA measurement of serum sRAGE concentration in 4, 7, 12 and 18 month old C57BL/6J mice along with corresponding measurements of total RAGE levels from muscle. xlsx and xpt files correspond to ELISA absorbance measurements of mouse RAGE (R&D #MRG00) captured via microplate reader of either serum or muscle tissue. Readings at 540nm are subtracted from readings at 450nm. xlsx files have been analyzed based on standard curve measurements. Data was further compiled and analyzed for statistical significance using prism. Prism pzfx file is included as well. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. Raw data .xtp were created using a Cytation 3 Plate Reader and can be opened and viewed using BioTek Gen5 Software for imaging & microscopy. All relevant measurements were exported to corresponding .xlsx files from the Gen5 Software and further annotated/analyzed on Microsoft excel. Briefly, absorbance values at 450nm (exported directly in blue highlight) were normalized by subtracting the zero standard, further corrected by subtracting the corresponding readings at 540nm, then estimated concentration values were derived based on the standard curve, and dilution factors were accounted for at the end to arrive at correct concentrations of RAGE protein. All analyses were performed and can be found below the exported absorbance values (in blue). pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** 6 xlsx and xpt files captured on 7/6/22 contain absorbance readings (either at 450nm or 540nm) corresponding to serum levels of sRAGE. 4 xlsx and xpt files captured on 7/12/22 contain absorbance readings (either at 450nm or 540nm) corresponding to total RAGE protein in injured muscle. 1 pzfx file contains the corresponding plot and statistical analysis for these measurements.  #### File: Fig1D\_G.zip **Description:** In vivo kinetics of serum sRAGE levels in vehicle (FBB) and AAV9-sRAGE injected adult and aged mice at 5, 20, and 31 days post injection. xlsx and xpt files correspond to ELISA absorbance measurements of mouse RAGE (R&D #MRG00) captured via microplate reader of serum following AAV and vehicle injections. Readings at 540nm are subtracted from readings at 450nm. xlsx files have been analyzed based on standard curve measurements. Data was further compiled and analyzed for statistical significance using prism. Prism pzfx file is included as well. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. Raw data .xtp files were created using a Cytation 3 Plate Reader and can be opened and viewed using BioTek Gen5 Software for imaging & microscopy. All relevant measurements were exported to corresponding .xlsx files from the Gen5 Software and further annotated/analyzed on Microsoft excel. Briefly, absorbance values at 450nm (exported directly in blue highlight) were normalized by subtracting the zero standard, further corrected by subtracting the corresponding readings at 540nm, then estimated concentration values were derived based on the standard curve, and dilution factors were accounted for at the end to arrive at correct concentrations of RAGE protein. All analyses were performed and can be found below the exported absorbance values (in blue). pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** 4 xlsx and xpt files with the label 5&6 day contain absorbance readings (either at 450nm or 540nm) corresponding to serum levels of sRAGE captured 5-6 days post injection with either FFB vehicle control or sRAGE-AAV9. Similarly, 4 files labeled 20 day and 4 files labeled 31 day contain absorbance readings (either at 450nm or 540nm) corresponding to serum levels of sRAGE captured either at 20 or 31 days post injection with either FFB vehicle control or sRAGE-AAV9. 1 pzfx file contains the corresponding plot and statistical analysis for these measurements.  #### File: Fig3J(sRAGE\_transgenic\_Transplants\_inMDX\_6Kcells).7z **Description:** Quantification of engrafted dystrophin+ muscle fibers following transplantation of 6000 SMPs. czi files correspond to stitched and unstitched images of individual replicates of muscle with transplanted SMPs taken via tiled confocal microscopy. Each czi file also has a corresponding image export folder containing individual channel images saved as tif files (Blue Channel - Nuclei; Red Channel - Dystrophin; Green Channel - Laminin). xml files contain cell counter markers from ImageJ used to point out and enumerate engrafted dystrophin+ muscle fibers. xlsx file contains compiled numbers of engrafted muscle fibers corresponding to each replicate in a given genotype group. Data was further compiled and analyzed for statistical significance using prism. Prism pzfx file is included as well. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. .czi files and corresponding exported .tif files were created and can be viewed with Zeiss Zen Black software. Files can also be opened and analyzed using imageJ. xml files can be opened by first opening the corresponding .tif file on ImageJ then going to plugins-&gt; analyze-&gt; cell counter-&gt; cell counter-&gt; load markers. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** For every biological replicate, there are 3 separate images corresponding to different depths in the muscle tissue. There are 6 different mouse lines profiled (AGER -/-, AGER +/-, AGER +/+, AGER s/-, AGER s/s, AGER s/+) and each line has 5 biological replicates. For example, the biological replicate AGER -/- 1 has 3 separate images corresponding to 3 separate depths in the same muscle (denoted by AGER* -/-* 1_1*, AGER -/- 1_*2*, and AGER -/-* 1_3) and each of these has their own czi file, stitched czi file, and exported folder containing separate channel images along with xml file containing the number of engrafted fibers (calculated as cell markers) per depth. 1 pzfx file and 1 xlsx file contains the corresponding counts, plots and statistical analysis for these measurements.  #### File: Fig1J\_K\_L\_M.zip **Description:** Quantification of body weight (J), grid hang time (K), exercise endurance (L), and serum glucose levels (M) in vehicle (FFB) and sRAGE-AAV9 treated, aged (18 months old) mice. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance via Student’s two-tailed unpaired t test. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** 2 pzfx files contain the corresponding plot and statistical analysis for these measurements. #### File: Fig2B\_C\_D\_H\_I\_\_J\_3.zip **Description:** In vivo kinetics of serum sRAGE levels in AAV9-sRAGE or vehicle (FFB) injected diabetic (Leprdb), atherosclerotic (apoe) and wild-type (WT) mice. Trajectory of serum glucose and weight in AAV9-sRAGE or vehicle (FFB) injected diabetic (Leprdb), atherosclerotic (apoe) and wild-type (WT) mice. xlsx files correspond to ELISA absorbance measurements of mouse RAGE (R&D #MRG00) captured via microplate reader of serum following AAV and vehicle injections. Readings at 540nm are subtracted from readings at 450nm. xlsx files have been analyzed based on standard curve measurements. Data was further compiled and analyzed for statistical significance using prism. Prism pzfx file is included as well. Data analyzed for statistical significance by two-way ANOVA with Tukey post hoc test or one-way ANOVA with Tukey post hoc test.  Raw data .xtp files were created using a Cytation 3 Plate Reader and can be opened and viewed using BioTek Gen5 Software for imaging & microscopy. All relevant measurements were exported to corresponding .xlsx files from the Gen5 Software and further annotated/analyzed on Microsoft excel. Briefly, absorbance values at 450nm (exported directly in blue highlight) were normalized by subtracting the zero standard, further corrected by subtracting the corresponding readings at 540nm, then estimated concentration values were derived based on the standard curve, and dilution factors were accounted for at the end to arrive at correct concentrations of RAGE protein. All analyses were performed and can be found below the exported absorbance values (in blue). pzfx files were created using GraphPad Prism and can be viewed using the same software. Glucose measurements were derived using a OneTouch® Ultra® 2 glucometer. **Contents:** Given the number of samples that needed to be analyzed, two separate ELISA plates were run. As such, you will find 4 xlsx and xpt files with the label plate 1 and 4 xlsx and xpt files with the label plate 2 that together contain all of the absorbance readings (either at 450nm or 540nm) corresponding to serum levels of sRAGE captured in diabetic (Leprdb) atherosclerotic (apoe) and wild-type (WT) mice post injection with either AAV9-sRAGE or vehicle (FFB). 1 prism file (sRAGE injection dbdb apoe) contains the plot and statistical analysis for these measurements. 1 other prism file contains the plot and statistical analysis for the measurements of serum glucose and weight recorded before and after the animals were administered either AAV9-sRAGE or Vehicle.  #### File: 3kTransplants\_sRAGE\_to\_MDX(Fig3I).7z **Description:** Quantification of engrafted dystrophin+ muscle fibers following transplantation of 3000 SMPs. czi files correspond to images of individual replicates of muscle with transplanted SMPs taken via confocal microscopy. Each czi file also has a corresponding image export folder containing individual channel images saved as tif files (Blue Channel - Nuclei; Red Channel - Dystrophin; Green Channel - Laminin). xml files contain cell counter markers from ImageJ used to point out and enumerate engrafted dystrophin+ muscle fibers. csv files contain quantification of counter markers corresponding to each replicate. .czi files and corresponding exported .tif files were created and can be viewed with Zeiss Zen Black software. Files can also be opened and analyzed using imageJ. xml files can be opened by first opening the corresponding .tif file on ImageJ then going to plugins-&gt; analyze-&gt; cell counter-&gt; cell counter-&gt; load markers. csv files can be opened via Microsoft Excel *Contents: For every biological replicate, there are 3 separate images corresponding to different depths in the muscle tissue. There are 6 different mouse lines profiled (AGER -/- 6 biological replicated, AGER +/- 5 biological replicates, AGER +/+ 10 biological replicates, AGER s/- 7 biological replicates, AGER s/s 5 biological replicates, AGER s/+ 5 biological replicates). For example, the biological replicate AGER -/- 1 has 3 separate images corresponding to 3 separate depths in the same muscle (denoted by AGER* -/- 1_1, AGER -/- 1_2*, and AGER -/-* 1_3) and each of these has their own czi file, and exported folder containing separate channel images along with xml file containing the number of engrafted fibers (calculated as cell markers) per depth and csv file with the number of cell markers.  #### File: Fig3B\_2.zip **Description:** ELISA measurement of serum sRAGE concentration in sRAGE transgenic mice, RAGE KO mice and WT mice. xlsx files correspond to ELISA absorbance measurements of mouse RAGE (R&D #MRG00) captured via microplate reader of serum from transgenic mice. Readings at 540nm are subtracted from readings at 450nm. xlsx files have been analyzed based on standard curve measurements. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. Absorbance measurements were exported to .xlsx files from the BioTek Gen5 Software and further annotated/analyzed on Microsoft excel. Briefly, absorbance values at 450nm (exported directly in blue highlight) were normalized by subtracting the zero standard, further corrected by subtracting the corresponding readings at 540nm, then estimated concentration values were derived based on the standard curve, and dilution factors were accounted for at the end to arrive at correct concentrations of RAGE protein. All analyses were performed and can be found below the exported absorbance values (in blue). pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** 4 xlsx and xpt files captured on 12/9/22 or 12/12/22 contain absorbance readings (either at 450nm or 540nm) corresponding to serum levels of sRAGE in transgenic animals (AGER -/-, AGER +/-, AGER +/+, AGER s/-, AGER s/s, AGER s/+). 1 pzfx file contains the corresponding plot and statistical analysis for these measurements.  #### File: Fig3C.zip **Description:** Confirmation of C-terminal RAGE expression in mice bearing at least one *Ager+* allele, and absence in mice bearing only *Agers or* Ager- alleles, via Western blot. raw jpg files show western blot images of membranes stained for RAGE protein (1:200, rabbit anti-RAGE, ab3611) and the corresponding ladder or GAPDH protein control and the corresponding ladder.  jpg files were generated via a BioRad Chemidoc XRS+ Imaging System with the corresponding Image Lab Software. Images can be opened with Preview or Adobe Software and can be analyzed using ImageJ.  **Contents:** 4 jpg files in total. 2 correspond to the western blot of c-terminal RAGE along with the corresponding ladder of this blot. 1 corresponding to the western blot of GAPDH and another corresponding to the ladder of this GAPDH blot. #### File: Fig1E\_F\_H\_I.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in vehicle (FBB) and sRAGE treated, adult and aged mice at 7 days after cryoinjury. zip files contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. xlsx file contains numeric representation of cross sectional area of regenerating muscle fibers from each replicate. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by Student’s two-tailed unpaired t test. Myofiber size distributions analyzed by Mann-Whitney U test.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** AGED AGER +_+ Cryo 7dpi zip contains images of regenerating muscle fibers from old (18 month old) animals (5 biological replicates per treatment) treated with either vehicle (FFB) or sRAGE-AAV9. Each biological replicate has 4 images corresponding to 4 fields of view in the regenerating muscle. AGER +_+ Cryo 7dpi zip contains images of regenerating muscle fibers from middle aged (7-8 month old) animals (7 FFB treated replicates and 8 sRAGE-AAV9 treated replicates) treated with either vehicle (FFB) or sRAGE-AAV9. Each biological replicate has 4 images corresponding to 4 fields of view in the regenerating muscle. 1 pzfx file and 1 xlsx file contains the corresponding cross sectional area, plot and statistical analysis for these measurements.  #### File: Fig3F.zip **Description:** Representative H&E stained images of regenerating muscle from *Ager+/+, Agers/+ and* Agers/s mice subjected 7 days previously to cryoinjury. Representative images provided either in tif or vsi format.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. **Contents:** Representative images correspond to regenerating muscle from AGER +/+, AGER s/+ and AGER s/s animals. #### File: Fig3D.zip **Description:** Enumeration of the mean CSA of muscle fibers in uninjured sRAGE transgenic mice, RAGE KO mice and WT mice. Folders contain tif and vsi files corresponding to two fields of view per replicate (taken at 10X magnification). zip files correspond to 200 ROIs corresponding to individual muscle fibers per tif image. csv files contain the cross sectional area quantification for each of the 200 muscle fibers per tif image. xlsx file contains numeric representation of cross sectional area of uninjured muscle fibers from each replicate. pzfx file contains numeric representation of cross sectional area of uninjured muscle fibers from each replicate and analyzed for statistical significance. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs (scale.tif image used to set the scale for quantification). ROIs are saved as .zip files and .csv files contain quantification of ROI cross sectional area. ImageJ was used to create and can be used to view .zip files by first opening the corresponding .tif file on ImageJ then separately opening the corresponding .zip file. csv files can be viewed on Microsoft Excel. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** Images from uninjured muscle of AGER +/- and AGER s/- animals were captured and analyzed on 12/9/22 and are contained in that folder. Images from uninjured muscle of AGER -/-, AGER +/+, AGER s/+ and AGER s/s animals were captured and analyzed on 12/13/18 and are contained in that folder. Each biological replicate has 2 separate images corresponding to 2 fields of view in the uninjured muscle. There are 3 biological replicates per genotype. 1 pzfx file contains the corresponding plot and statistical analysis for these measurements.  #### File: Fig3I\_J.zip **Description:** Quantification of engrafted dystrophin+ muscle fibers following transplantation of 3000 (I) or 6000 (J) SMPs. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by one-way ANOVA with Tukey post hoc test. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** 2 pzfx files contain the corresponding plot and statistical analysis for these measurements.  #### File: Fig2E\_F\_K\_L.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in vehicle (FFB) and AAV9-sRAGE treated, diabetic (Leprdb), atherosclerotic (apoe) and wild-type (WT) mice at 7 days after cryoinjury. zip files contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. zip and csv files corresponding to individual .tif images contain ROIs of regenerating muscle fibers and the corresponding numerical measure of the cross sectional area per ROI. xlsx file contains compiled numeric representation of cross sectional area of regenerating muscle fibers from each replicate. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by one way ANOVA with Tukey post hoc test or Student’s two-tailed unpaired t test. Myofiber size distributions analyzed by Mann-Whitney U test and Kruskal-Wallis test.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs which were saved as .zip files and enumerated in .csv files for each tif image. ImageJ was used to create and can be used to view .zip files by first opening the corresponding .tif file on ImageJ then separately opening the corresponding .zip file. csv files can be viewed on Microsoft Excel. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents:** APOE_cryo.zip contains images of cryo-injured muscle corresponding to biological replicates of atherosclerotic (apoe) mice injected with either AAV9-sRAGE (6 replicates) or vehicle (FFB) control (6 replicates). Each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle. dbdb_cryo.zip contains images of cryo-injured muscle corresponding to biological replicates of diabetic (Leprdb) mice injected with either AAV9-sRAGE (6 replicates) or vehicle (FFB) control (6 replicates). Each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle. 1 xlsx file contains the corresponding compiled cross sectional area for all of these animals.  #### File: Fig3G\_H.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in wild-type, knockout and transgenic mice at 7 days after cryoinjury. Folders contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. zip and csv files corresponding to individual .tif images contain ROIs of regenerating muscle fibers and the corresponding numerical measure of the cross sectional area per ROI. xlsx file contains compiled numeric representation of cross sectional area of regenerating muscle fibers from each replicate. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by one way ANOVA with Tukey post hoc test. Myofiber size distributions analyzed by Kruskal-Wallis test.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs (scale.tif image used to set the scale for quantification). ROIs were saved as .zip files and enumerated in .csv files for each tif image. ImageJ was used to create and can be used to view .zip files by first opening the corresponding .tif file on ImageJ then separately opening the corresponding .zip file. csv files can be viewed on Microsoft Excel. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software. **Contents:** Each folder corresponds to separate experiments carried out on different dates all corresponding to cryo-injurys of muscle from transgenic animals (AGER -/-, AGER +/-, AGER +/+, AGER s/-, AGER s/s, AGER s/+). For every folder, each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle.  #### File: FigS1.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in vehicle (FFB) and sRAGE treated, adult (7-8 months old) and aged (18 months old) mice at 7 days after BaCl2 induced injury. Zip files contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs. **Contents: **AGED AGER +_+ Bacl2 7dpi zip contains images of regenerating muscle fibers from old (18 month old) animals (5 biological replicates per treatment) treated with either vehicle (FFB) or sRAGE-AAV9. Each biological replicate has 4 images corresponding to 4 fields of view in the regenerating muscle. AGER +_+ Bacl2 7dpi zip contains images of regenerating muscle fibers from middle aged (7-8 month old) animals (7 FFB treated replicates and 8 sRAGE-AAV9 treated replicates) treated with either vehicle (FFB) or sRAGE-AAV9. Each biological replicate has 4 images corresponding to 4 fields of view in the regenerating muscle. #### File: FigS2.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in vehicle (FFB) and AAV9-sRAGE treated, diabetic (Leprdb), atherosclerotic (apoe) and wild-type (WT) mice at 7 days after BaCl2-induced injury. Zip files contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by Student’s two-tailed unpaired t test. Myofiber size distributions analyzed by Mann-Whitney U test.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs. pzfx files were created using GraphPad Prism and can be viewed using the same software.  **Contents: **B&C contains dbdb_Bacl2.zip which contains images of Bacl2-injured muscle corresponding to biological replicates of diabetic (Leprdb) mice injected with either AAV9-sRAGE (6 replicates) or vehicle (FFB) control (6 replicates). Each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle. E&F contains APOE_Bacl2.zip which images of Bacl2-injured muscle corresponding to biological replicates of atherosclerotic (apoe) mice injected with either AAV9-sRAGE (6 replicates) or vehicle (FFB) control (6 replicates). Each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle. #### File: FigS3.zip **Description:** Enumeration of the mean CSA and distribution of regenerating (centrally nucleated) muscle fibers in wild-type, knockout and transgenic mice at 7 days after Bacl2-induced injury. Folders contain tif and vsi files corresponding to four fields of view per replicate. The four images encompass the area of injured muscle captured via Hematoxylin and eosin staining. zip and csv files corresponding to individual .tif images contain ROIs of regenerating muscle fibers and the corresponding numerical measure of the cross sectional area per ROI. pzfx file contains data compiled and analyzed for statistical significance using prism. Data analyzed for statistical significance by one way ANOVA with Tukey post hoc test. Myofiber size distributions analyzed by Kruskal-Wallis test.  .vsi and corresponding .tif files were created using Olympus cellSens software and can be opened using the same software or ImageJ for .tif files. ImageJ was used to derive cross sectional area values based on muscle cell ROIs (scale.tif image used to set the scale for quantification). ROIs were saved as .zip files and enumerated in .csv files for each tif image. ImageJ was used to create and can be used to view .zip files by first opening the corresponding .tif file on ImageJ then separately opening the corresponding .zip file. csv files can be viewed on Microsoft Excel. xlsx files were generated and can be viewed on Microsoft Excel. pzfx files were created using GraphPad Prism and can be viewed using the same software. **Contents: **Each folder corresponds to separate experiments carried out on different dates all corresponding to Bacl2-injurys of muscle from transgenic animals (AGER -/-, AGER +/-, AGER +/+, AGER s/-, AGER s/s, AGER s/+). For every folder, each biological replicate has 4 separate images corresponding to 4 fields of view in the injured muscle. 1 pzfx file contain the corresponding plot and statistical analysis for these measurements.  ## Code/software Microsoft Excel, GraphPad Prism, ImageJ, Olympus cellSens, Zeiss Zen Black, BioTek Gen5, BioRad Image Lab Software