Supporting Information

Velarde et al. 10.1073/pnas.1505675112 SI Materials and Methods 1:750). Multiple photomicrographs of wound sections were taken Generation of Transgenic Mice. We contracted Ozgene (www. under 10× magnification and tiled in Adobe Photoshop. ozgene.com) to generate a LoxP flanked Sod2 allele. We modified the endogenous Sod2 in mouse embryonic stem cells by Tail Whole Mounts. Epidermal sheets were permeabilized [0.25% inserting a PGK/neo cassette between exons 3 and 4, which fa- fish skin gelatin (Sigma-Aldrich), 0.5% Triton X-100, and 20 mM cilitated selection of the correctly modified allele. LoxP sites were Hepes, pH 7.2] for 30 min at room temperature, then sequentially inserted 3′ of exon 1 and 5′ of exon 4, and Cre-mediated re- incubated with primary at 4 °C overnight and sec- combination removed exons 2 and 3 (Fig. S6F). We chose this re- ondary antibodies at 4 °C overnight with 4-h washes between gion for deletion because exon 3 is crucial for manganese binding incubations using 0.2% Tween 20 in PBS. Primary antibodies and homotetramer formation. Thus, recombination completely in- were rabbit anti-SOD2 (Assay Designs; 1:1,000) and rat anti- activates Sod2. We confirmed the correctly modified locus by se- CD49f (BD Pharmingen; 1:1,000). Secondary antibodies were quencing, and generated mice with the modified allele in a C57BL/ Alexa Fluor 555 donkey anti-rabbit (Molecular Probes; 1:750) 6J background. The allele was registered (www.informatics.jax.org/ and Alexa Fluor 488 donkey anti-rat (Molecular Probes; 1:750). tm1Smel mgihome/nomen/strains.shtml), and is termed Sod2 (S). Samples were mounted in Prolong Gold with DAPI (Life Technologies). Genotyping. Genotyping was done using Platinum Blue PCR SuperMix (Life Technologies). For Sod2, we used primer pairs Keratinocyte Isolation. Shaved mouse skin was collected, and the for neo (0.4 μM) and bl6 (0.4 μM). PCR conditions were 95 °C fat layer was removed. Skin was then washed and decontaminated for 5 min; 34 cycles at 95 °C for 1 min, 59.5 °C for 1 min, and with Hibiclens (Fisher Scientific) and Ca/Mg-free HBSS con- 72 °C for 30 s; and 72 °C for 5 min. For K14 mice, we used the taining 5× antibiotics (Life Technologies). Skin was floated on primer pairs Cre (0.4 μM) and GDF (0.28 μM) and the following 25 U/mL dispase mix (BD Biosciences) in Ca/Mg-free HBSS con- qPCR conditions: 94 °C for 2 min; 34 cycles at 94 °C for 1 min, taining 50 μg/mL gentamicin and incubated at 4 °C overnight. 58.5 °C for 1 min, and 72 °C for 30 s; and 72 °C for 10 min. We Epidermal cells were scraped from the dermis and processed for detected the recombined Sod2 by qPCR using SensiFast PCR assays. For flow cytometry, epidermal cells were incubated Probe (Bioline) and EvaGreen (Biotium; catalog no. 31000-T). with TrypLE for 8 min at 37 °C. After neutralization with 10% Genotyping for Sod2 deletion used the primer pairs LoxP chelexed FBS (BioRad) in Ca/Mg-free HBSS, cells were col- (0.5 μM) and total control sequence (1 μM), and the following lected by centrifugation (300 × g for 5 min), and floating cells qPCR conditions: 95 °C for 10 min, and 40 cycles at 95 °C for were aspirated, filtered through a 70-μm strainer and washed 10 s, 60 °C for 30 s, and 72 °C for 10 s. Melting curves of PCR twice with Ca/Mg-free HBSS. Keratinocytes were resuspended in products were monitored to ensure the absence of primer di- CnT-07 (Zenbio) for flow cytometry. mers. The copy number of recombined Sod2 alleles was nor- malized to the copy number of the total control sequence. RT-PCR. Skin samples were homogenized in QIAzol Lysis Reagent (Qiagen), and RNA was isolated using the RNeasy Mini Kit Picrosirius Red Staining. Samples were fixed in 10% buffered for- (Qiagen). cDNA was synthesized using the High- Capacity cDNA malin, embedded in paraffin, and cut into 7-μm sections. Sections Reverse Transcription Kit (Life Technologies). Then 1 μgof were then deparaffinized, stained with Weigert’shematoxylinfor RNA was amplified at 95 °C for 7 min and 50 cycles of 95 °C for 8 min, and washed before being stained with picrosirius red so- 5 s and 60 °C for 30 s. Transcripts were quantified by qPCR using lution [0.1% Direct Red 80 (Sigma-Aldrich) in saturated (1.3%) SensiFast Probe (Bioline), normalized to β-actin and α-tubulin, picric acid] for 1 h. Sections were washed with 0.5% acetic acid and reported as relative levels. Probes were obtained from the and dehydrated before mounting on coverslips. Slides were viewed Roche Universal Probe Library system. Primer sets were used at under polarized light using a Nikon Eclipse E800 microscope. To 0.1 μM (Tables S2 and S3). capture the complete wound site, multiple photomicrographs of different sections were taken at 4× magnification, then tiled in Flow Cytometry. Epidermal cells were incubated with purified rat Adobe Photoshop. anti-mouse CD16/CD32 Fc block (BD Pharmingen) at 1:100 for 20 min on ice, followed by incubation for 20 min on ice with the Immunofluorescence. Skin was embedded in frozen OCT and following primary antibodies: Sca1-FITC (e-Bioscience) at 1:100, processed as described. Wound site specimens were fixed in 10% CD49f/ItgA6-RPE (AbD Serotec) at 1:50, and CD34-Alexa Fluor buffered formalin, embedded in paraffin, and cut into 7-μm 660 (e-Bioscience) at 1:40. After a wash with PBS containing 1% sections. Sections were deparaffinized, microwaved, blocked, and BSA, cells were fixed with 1% formaldehyde in PBS before flow immunostained. Primary antibodies were rabbit anti-Ki67 anti- cytometry analysis with a BD FACSAria (BD Biosciences). Cor- body (Novus Biologicals, NB110-89717; 1:750 for OCT; Thermo responding isotype controls were used for compensation correction. Fisher Scientific, RM-9106-S0; 1:500 for paraffin-embedded), Cell types were analyzed using FlowJo analysis software. rabbit anti-PCNA (Cell Signaling, B00177; 1:1,000), rat anti-CD49f (BD Pharmingen, 555734; 1:750) and rabbit anti- Data Analysis. Data are presented as mean ± SEM and were loricrin (Covance, PRB-145P; 1:1,000). Secondary antibodies subjected to statistical analysis using Student’s t test or two-way were Alexa Fluor 555 donkey anti-rabbit (Molecular Probes; ANOVA (with Bonferroni post hoc analysis), as indicated in the 1:750) and Alexa Fluor 488 donkey anti-rat (Molecular Probes; figure legends. P < 0.05 was considered statistically significant.

Velarde et al. www.pnas.org/cgi/content/short/1505675112 1of8 Fig. S1. Generation of keratinocyte-specific conditional Sod2-deficient mice. (A) Diagram of primers designed to detect deletion (Deleted) or presence (Intact) of exons 1–3oftheSod2 gene and an unmodified control gene region (Total) by PCR. (B–D) Representative PCR gel showing amplification of DNA with deleted or intact Sod2 sequences from whole dorsal skin (B), separated epidermis and dermis (C), and tail, heart, liver, toe, intestinal jejunum, and lung (D)ofK14S mice after corn oil/vehicle (veh) or tamoxifen (TAM) treatment. (E) Average percent recombination at the Sod2 locus, determined by qPCR, using genomic DNA isolated from the epidermis and dermis of 22-mo-old K14S and K14R mice, treated with corn oil (vehicle control, veh; n = 8) or tamoxifen (TAM; n = 8) at age 4 mo. (F and G) Sod2 mRNA levels determined by qPCR of RNA isolated from the epidermis (F) and dermis (G)ofK14S and K14R mice, treated with veh (n = 5) or TAM (n = 5) at age 4 mo. Mean ± SEM values with asterisks indicate significant differences at P < 0.05 relative to vehicle control by Student’s t test. (H) Representative photomicrographs of skin of 8-mo-old K14S mice, treated with veh or TAM at age 4 mo, stained for cytochrome c oxidase (COX; brown) activity. (Upper) Hair follicles outlined by dashed lines. (Lower) Skeletal muscle beneath the SC layer. (I) Quantification of succinate dehydrogenase (SDH) and COX staining scored by ranking the staining intensities. The highest intensity was assigned the highest ranked value, and mean averages were calculated. Two blinded researchers independently ranked the intensities. Asterisks indicate significant differences at P < 0.05 by Student’s t test.

Velarde et al. www.pnas.org/cgi/content/short/1505675112 2of8 Fig. S2. Collagen formation, cell proliferation and apoptosis in wounds of young K14S mice. (A) Representative photomicrographs of picrosirius red staining of the wound area of 8-mo-old veh- or TAM-treated K14S mice at 2–10 d after injury. To capture the complete wound site, multiple photomicrographs of different sections were taken at 4× magnification, then tiled in Adobe Photoshop. (B) Representative photomicrographs of PCNA (green) immunofluorescence in wound areas of 8-mo-old veh- or TAM-treated K14S mice at 6 d after skin injury (veh and TAM, n = 5). Nuclei are stained by DAPI (blue). (C) Representative photomicrographs and quantitative measurement of Ki67 (green) immunofluorescence in wound areas of 8 mo old veh- and TAM-treated K14S mice at 6 d after skin injury (veh and TAM, n = 5). Nuclei are stained by DAPI (blue). Cell layers above the dashed lines indicate the epidermis. Mean ± SEM values with asterisks indicate significant differences at P < 0.05 by Student’s t test. (D) mRNA levels of associated with proliferation and expressed predominantly in epidermal basal cells, determined by qPCR, in wound areas of 8-mo-old veh- or TAM-treated K14S mice, at 2 d (veh and TAM n = 3) and 6 d (veh n = 5; TAM n = 7) after injury. Means ± SEM with asterisks indicate differences at P < 0.05 by two-way ANOVA followed by Bonferroni post hoc analysis. (E) Representative photomicrographs of TUNEL- positive (green) immunofluorescence in wound areas of 8-mo-old veh- or TAM-treated K14S mice, at 2 d and 6 d after skin injury (veh and TAM, n = 5). Nuclei are stained by DAPI (blue). Tissues treated with DNase I served as a positive control (Pos Ctrl). The TUNEL assay was performed using the DeadEnd Fluorometric TUNEL System (Promega).

Velarde et al. www.pnas.org/cgi/content/short/1505675112 3of8 Fig. S3. and histology of healing wounds in young K14S mice. (A) mRNA levels of inflammatory cytokines, determined by qPCR, in wound areas of 8-mo-old veh- and TAM-treated K14S mice, at 2 d (veh and TAM, n = 3) and 6 d (veh, n = 5; TAM, n = 7) after injury. Mean ± SEM values with asterisks indicate differences at P < 0.05 by two-way ANOVA followed by Bonferroni post hoc analysis. (B and C) Representative photomicrographs of H&E staining of the wound area of 8-mo-old veh- and TAM-treated K14S mice at 6 d (B) and 10 d (C) after injury (veh and TAM, n = 5). Stratum basale (SB, arrows) are identified by strong nuclear staining (blue) at the bottom of the wound site. Stratum granulosum (SG, asterisks) are cells with granules in the cytoplasm. Stratum spinosum (SS) are cells with low nuclear staining above the SB layer. Stratum corneum (SC) is the outermost layer.

Velarde et al. www.pnas.org/cgi/content/short/1505675112 4of8 Fig. S4. Wound closure in aging K14S mice. (A) Average wound size (mean ± SEM) after injury by an 8-mm dermal punch biopsy in 11-mo-old K14S mice, treated with veh or TAM (veh, n = 13; TAM, n = 11) at age 4 mo. Percent wound size refers to the wound area relative to the initial wound area × 100. (B) Representative photomicrographs of H&E staining of skin of K14S mice at 4, 7, and 10 mo after veh (Left) or TAM (Right) treatment (veh and TAM, n = 5). + (C and D) Heat map (C) and mRNA levels (D) of genes expressed predominantly in basal interfollicular epidermis (IFE), suprabasal IFE, K14 stem cells, and involucrin (Ivl) committed progenitor cells, analyzed by qPCR, in skin from K14S mice at 4 mo and 11–20 mo after treatment with veh or TAM.

Velarde et al. www.pnas.org/cgi/content/short/1505675112 5of8 Fig. S5. Epidermal stem cells in old K14S mice. Representative histographs (A) and quantification (B) of flow cytometry analysis of epidermal cells stained for CD49f, CD34, and Sca1, isolated from the skin of 14-mo-old K14S mice treated with veh or TAM at age 4 mo (n = 5 for each). Populations of bulge stem cells + + − − − − (CD49fhi/CD34 ), suprabasal bulge stem cells (CD49flo/CD34 ), junctional zone stem cells (CD49fhi/CD34 /Sca1 ), isthmus stem cells (CD49flo/CD34 /Sca1 ), basal − + − + interfollicular epidermal cells (CD49fhi/CD34 /Sca1 ), and suprabasal interfollicular epidermal cells (CD49flo/CD34 /Sca1 ) are labeled.

Velarde et al. www.pnas.org/cgi/content/short/1505675112 6of8 Fig. S6. Cellular senescence in K14S mice and human keratinocytes. (A) Representative photomicrographs of skin of 8-mo-old K14S mice treated with veh or + TAM at age 4 mo, stained for SA-βgal activity (blue) and nuclei (red). The blue line indicates surface region with SA-βgal cells; the red line, surface region of total epidermal cells. (B) Average percentage of 14 mo old K14S mice with detectable levels (Ct <40) of p16INK4a mRNA in isolated epidermal samples, 10 mo after veh or TAM treatment. (C) mRNA levels of wound healing-related growth factors, as determined by qRT-PCR of RNA isolated from wound areas of 8-mo- old veh- or TAM-treated K14S mice, treated at age 4 mo at 2 d (Upper)or6d(Lower) after skin injury. Mean ± SEM values with asterisks indicate significant differences at P < 0.05 by Student’s t test. (D) Ki67 (red) and CD49f (green) staining by immunofluorescence and nuclear staining by DAPI (blue) of skin from acetone- or TPA-treated 8-mo-old K14S mice, treated with veh or TAM at age 4 mo. (E) Human keratinocytes treated with 100 nM rotenone for 7 d (n = 3) and analyzed for morphology by bright field microscopy. (F) Targeting strategy for inactivating Sod2 by Cre-lox recombination. The Sod2 locus is shown in black, the five exons of Sod2 are shown in dark blue, the PGK/neo cassette is shown in light blue and orange, and the loxP insertion sites are marked in red.

Table S1. Primer sequences used for genotyping Gene name Forward primer sequence Reverse primer sequence

Neo 5′-AGGCTATTCGGCTATGACTGGGG-3′ 5′-TGGATACTTTCTCGGCAGGAGC-3′ Bl6 5′- TCTGGACAAACCTGAGCCCTAAG-3′ 5′-CCTGAACACATTCTCTATTCCTCCC-3′ Cre 5′-CCCGCAGAACCTGAAGATGTT-3′ 5′-CGGCTATACGTAACAGGGTG-3′ Gdf 5′-AAGCCCTCAGTCAGTTGTGC-3′ 5′-AAAACCATGAAAGGAGTGGG-3′ LoxP 5′-GCGGTCGTGTAAACCTCAATAGAG-3′ 5′-AAAAAACCAACAATCGGGGC-3′ Total control 5′- GAGGGGCCCTGATTACTCC-3′ 5′- GAAACCCTGGAGACTTTCCTC-3′

Velarde et al. www.pnas.org/cgi/content/short/1505675112 7of8 Table S2. Mouse primer sequences used for qPCR Gene name Forward primer sequence Reverse primer sequence UPL probe no.

Actb 5′-CTAAGGCCAACCGTGAAAAG-3′ 5′-ACCAGAGGCATACAGGGACA-3′ 64 Ccna2 5′- TGCAAACTGTAAGGTTGAAAGC-3′ 5′- TGTAGAGAGCCAAGTGGAAGG-3′ 67 Ccnb1 5′-TGCATTTTGCTCCTTCTCAA-3′ 5′-CAGGAAGCAGGGAGTCTTCA-3′ 45 Ccnd1 5′-GAGATTGTGCCATCCATGC-3′ 5′-CTCCTCTTCGCACTTCTGCT-3′ 67 Cd36 5′- TTGAAAAGTCTCGGACATTGAG-3′ 5′- TCAGATCCGAACACAGCGTA-3′ 6 Cdc20 5′- GAGTGCTGTGGATGTGCATT-3′ 5′- TCAGCTCCTTATAGTGGGGAGA-3′ 58 Cdca5 5′- TCAAGACTTGTAGTGTCCCTGGTA-3′ 5′- CTCCAGAGTCAGGCTCAACA-3′ 67 Cdkn2a(p16)5′-AATCTCCGCGAGGAAAGC-3′ 5′-GTCTGCAGCGGACTCCAT-3′ 91 Cenpe 5′- CGCCTCCCAGTCTGTTGT-3′ 5′- TCACTGTCCCGTGTGGAAG-3′ 58 Cenpp 5′- GCAGAATGCTGCAAAACG-3′ 5′- CAAAGATTCCCACTCCTCAGA-3′ 7 Dgat2 5′- GCTGGTGCCCTACTCCAAG-3′ 5′- GCTTGGGGACAGTGATGG-3′ 9 Dll1 5′- GGGCTTCTCTGGCTTCAACT-3′ 5′- CACTTGGCACCGTTAGAACA-3′ 103 Dsc1 5′- TGGTCCACCTTTTCAGTTCC-3′ 5′- ATGGCACGTTTACCATCCTG-3′ 11 Elovl7 5′- CAGTGTCCCCCAGGTAAGTG-3′ 5′- CACAAACCCTACAACCAGTGAC-3′ 1 Il1a 5′- TTGGTTAAATGACCTGCAACA-3′ 5′- GAGCGCTCACGAACAGTTG-3′ 52 Il1b 5′- TGTAATGAAAGACGGCACACC-3′ 5′- TCTTCTTTGGGTATTGCTTGG-3′ 78 Il1r2 5′-GCAAGAAGCAGCAAGGTACA-3′ 5′-CCGCACCAACTTCCTGAG-3′ 1 Itga2 5′- AGGGGAGCAAATATTCAGCA-3′ 5′- CCAACTTGTGCCATTTCCAT-3′ 63 Itga3 5′- GAGCTGTGGTTGGTGCTTG-3′ 5′- GCACTTCCACAAGAGGAGGAT-3′ 45 Itga6 5′- GACCAGTGGATGGGAGTCAC-3′ 5′- TGCACACGTCACCACTTTG-3′ 21 Itgb1 5′- CTGCTTCTAAAATTGAGATCAGGA-3′ 5′- TCCATAAGGTAGTAGAGATCAATAGGG-3′ 41 Ivl 5′- GGATCTGCCTGATCAAAAGTG-3′ 5′- CAGCTGCTGCTTTTGTGG-3′ 71 Kif11 5′- ACAATGGAAGGTGAAAGGTCA-3′ 5′- TGGAATTATACCAGCCAGAGGA-3′ 52 Kif14 5′- GGGCAGAGGTCTCTGAACTG-3′ 5′- GCAAGCACGGTAGTTCTCCT-3′ 58 Klf9 5′- CTCCGAAAAGAGGCACAAGT-3′ 5′- GCGAGAACTTTTTAAGGCAGTC-3′ 76 Krt1 5′- TTTGCCTCCTTCATCGACA-3′ 5′- GTTTTGGGTCCGGGTTGT-3′ 62 Krt10 5′- CGTACTGTTCAGGGTCTGGAG-3′ 5′- GCTTCCAGCGATTGTTTCA-3′ 95 Krt6b 5′- CACATTTGGTGCTTCATGCT-3′ 5′- CCAGGCAACTCAGAGACAGA-3′ 52 Lass4 5′- TTCCCAGTGGCTCTGGTC-3′ 5′- GGCAAGGCCACAAATCTCT-3′ 76 Lgr6 5′- TGTGCCAACAGCTGCCTA-3′ 5′- AGGCTGGGTAACTCCTCGAT-3′ 1 Lor 5′-GGTTGCAACGGAGACAACA-3′ 5′- CATGAGAAAGTTAAGCCCATCG-3′ 11 Lrig1 5′- ACAGCTGCCCCACATACAAC-3′ 5′- GGGATGGTAGGCTGTGTCA-3′ 21 Pdgfa 5′-GTGCGACCTCCAACCTGA-3 5′-GGCTCATCTCACCTCACATCT-3′ 52 Pdgfb 5′-CGGCCTGTGACTAGAAGTCC-3′ 5′-GAGCTTGAGGCGTCTTGG-3′ 32 S100a3 5′- AGCAACAGCAGCAGTGTGAG-3′ 5′- TGGAAGGTGCACACGATG-3′ 1 Sod2 5′-CCATTTTCTGGACAAACCTGA-3′ 5′- GACCCAAAGTCACGCTTGATA-3′ 67 Tgfb1 5′-TGGAGCAACATGTGGAACTC-3′ 5′-CAGCAGCCAATTACCAAG-3′ 72 Tgfb2 5′-CTTACCCTAAGCGAGAAAGTGC-3′ 5′- GCACCCTTCCCTAGCTTCTC-3′ 31 Tgfb3 5′- GCAGACACAACCCATAGCAC-3′ 5′- GGGTTCTGCCCACATAGTACA-3′ 1 Tgm1 5′- GCCCTTGAGCTCCTCATTG-3′ 5′- CCCTTACCCACTGGGATGAT-3′ 10 Tnf 5′- TCTTCTCATTCCTGCTTGTGG-3′ 5′- GGTCTGGGCCATAGAACTGA-3′ 49 Tuba1a 5′-CTGGAACCCACGGTCATC-3′ 5′-GTGGCCACGAGCATAGTTATT-3′ 88 Vegf 5′-AAAAACGAAAGCGCAAGAAA-3′ 5′-TTTCTCCGCTCTGAACAAGG-3′ 1

Table S3. Human primer sequences used for qPCR Gene name Forward primer sequence Reverse primer sequence UPL probe no.

ACTB 5′- CCAACCGCGAGAAGATGA -3′ 5′- TCCATCACGATGCCAGTG -3′ 64 CCNA2 5′- CCATACCTCAAGTATTTGCCATC -3′ 5′- TCCAGTCTTTCGTATTAATGATTCAG-3′ 67 CCNB1 5′- CCAGTGCCAGTGTCTGAGC-3′ 5′- TGGAGAGGCAGTATCAACCA-3′ 21 CD36 5′- CCTCCTTGGCCTGATAGAAA-3′ 5′- GTTTGTGCTTGAGCCAGGTT-3′ 9 CDKN2A(p16)5′- GAGCAGCATGGAGCCTTC -3′ 5′- CGTAACTATTCGGTGCGTTG -3′ 67 DGAT2 5′- GAGGGGTCTGGGAGATGG-3′ 5′- TTGGACCTATTGAGCCAGGT-3′ 55 KLF9 5′- CTCCGAAAAGAGGCACAAGT-3′ 5′- CGGGAGAACTTTTTAAGGCAGT-3′ 76 LGR6 5′- TGTGCCAGCTTCTTCAAGG-3′ 5′- GGGCCTTTTTGAAGACTCCT-3′ 41 S100a3 5′- TCTGGTTCAGGTTCCTGACTG-3′ 5′- TGGAAGGTGCACACGATG-3′ 50 TUBA1A 5′- CTTCGTCTCCGCCATCAG -3′ 5′- TTGCCAATCTGGACACCA -3′ 58

Velarde et al. www.pnas.org/cgi/content/short/1505675112 8of8