Short Telomere Syndromes Cause a Primary T Cell Immunodeficiency

Journal of Clinical Investigation

Supplementary Data

Short telomere syndromes cause a primary T cell immunodeficiency

Christa L. Wagner1, Vidya Sagar Hanumanthu1, C. Conover Talbot Jr2, Roshini S. Abraham3, David Hamm4, Dustin L. Gable1, Christopher G. Kanakry1, Carolyn D. Applegate5, Janet Siliciano6, J. Brooks Jackson7, Stephen Desiderio2,8,9, Jonathan K. Alder1, Leo Luznik1,8, Mary Armanios1,5,7,8,9

Departments of Oncology1, Medicine6, Pathology7, and Molecular Biology & Genetics9 Institute for Basic Biomedical Sciences2 McKusick-Nathans Institute of Genetic Medicine5 Sidney Kimmel Comprehensive Cancer Center8 Johns Hopkins University School of Medicine Baltimore, MD 21287

Department of Laboratory Medicine and Pathology3 Mayo Clinic Rochester, MN 55905

Adaptive Biotechnologies4 Seattle, WA 98102

Supplementary Figure 1

A Mouse Bone Marrow 100 WT → WT WT → mTR-/-G4 1

0.01 Fraction (%) +

0.0001 Donor 0 Wk 4 Wk 8 Mouse Bone Marrow

B 91.5 8.95 KSL4.55 C 15 ***

) WT 3 12

Lin- (x10 - 8.5 74.3 12.2 9 lin + 92.2 9.22 KSL2.88

sca 6 + mTR-/-G4 c-kit 3 Lin- 7.8 70.9 17.0 Lin

c-kit 0 c-kit Sca-1 WT mTR-/-G4

Lin- 37.6 6.79 SLAM 2.0 D E ) 5.66 *** c-kit+ 3 WT (x10

+ 1.5 1.79 53.8 SLAM 62.9 c-kit - 1.0 Lin- 47.3 4.22 SLAM 5.79 c-kit+ CD48

-/- + mTR G4 0.5

1.11

50.0 CD150 47.4 SLAM SSC

SSC 0.0 CD48 Lin CD150 c-kit WT mTR-/-G4

Mouse Peripheral Blood WT → WT mTR-/-G4 → WT F 40 G 14 *** 12 * 30 *** 10 8 20 donor-derived donor-derived ** +

+ 6 ** 10 4

% Gr1 2 % CD3 0 0 04 812 04 812 Weeks Weeks Supplementary Figure 2

A Newborn Thymus 150 C Mouse Thymocytes

125 6.49 100 hi lo CD3 neg CD3 75 CD3 8.13 36.5 21.7 (% of WT) 50

Live Thymocyte Count 25 0 WT mTR-/-G4 SSC SSC CD3 Annexin B Adult Thymus 1.52 93.6 10.3 25.6 200 175 150 125 100 (% of WT) 75 4.61 0.25 26.8 25.6 50 Live Thymocyte Count CD8 CD44 25 CD4 CD25 0 WT mTR-/-G4

D E 8 FG + 30 * + DN1: CD44+CD25- 14

50 + DN2: CD44+CD25+ DN3: CD44-CD25+ 12 25 40 - - 6 DN4: CD44 CD25 10 Annexin 20 Annexin + -

* Annexin 8 8 8 30 + - + 4 15 4 4 4 6 hi

20 lo 10 neg 2 4 10 2 5 % CD3 % CD3hi 8+ Annexin+ % CD3hi 8+ % CD3

% CD3 0 0 0 0 DN1 DN2 DN3 DN4

WT mTR-/-G4 Supplemental Figure 3

ABC 6 * 4 * 3

3 4 2 L) L) L) μ μ μ / / / 3 3 2 3 (x10 (x10 2 (x10 1 1 White Blood Cells Abs Neutrophil Count Abs Lymphocyte Count Abs Lymphocyte 0 0 0 WT mTR-/-G4 WT mTR-/-G4 WT mTR-/-G4 DE F 15 20 1500 L) μ

12 L)

15 μ / 3 1000 9 10 6 500 5 Platelets (x10

3 Hemoglobin (g/dL) Red Blood Cells (M/ 0 0 0 WT mTR-/-G4 WT mTR-/-G4 WT mTR-/-G4 Supplementary Figure 4

Mouse Peripheral T cells A B WT (Adult) mTR-/-G4 (Adult) WT (Old)

3.59 59.0 4.00 69.8 4.29 50.9 -/- 1.0 WT mTR G4 0.8 T cells + 0.6 CD3 + 0.4

0.2 35.3 1.98 23.6 2.62 42.2 2.47 % EdU

0.0 PI Annexin V Unstimulated Stimulated - CD3/CD28 + CD3/CD28

Human Peripheral T cells C D Young Controls Short Telomere Older Adult 60 YC ST 4 4 4 10 1.56 15.6 10 8.24 25.8 10 7.93 51.0 50 3 3 3 10 10 10 T cells T

+ 40 2 2 2 30 10 10 10 CD3 + 1 20 101 101 10 10 % EdU 0 0 0 10 81.2 1.69 10 56.3 9.7 10 27.6 13.5 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 0 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 Unstimulated Stimulated PI Annexin V - CD3/CD28 + CD3/CD28 Supplementary Figure 5

A Controls (n=85)

β 20 Short Telomere (n=1)

2SD 2SD 15

10 1SD 1SD 1SD 1SD 5 2SD

T cells expressing V T 1SD

+ 1SD 1SD

0 1 2 3 4 1 2 8 9 1 2 1 6 6 7 8 2 % CD3 . . .3 1 1 .2 14 1 1 1 20 .3 2 23 5 5 5 7.1 7.2 13. 13 13. 21 Vβ Family

B Young Control (n=4)

30 30 30 30 YC1 YC2 YC3 YC4

20 20 20 20 Frequency Frequency Frequency 10 Frequency 10 10 10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 20 30 4 50 60 70 8 10 20 30 40 50 60 70 80 10 20 30 40 5 60 70 80 1 20 30 4 5 60 70 8

CDR3 Length

Short Telomere (n=4)

30 30 30 30 ST1 ST2 ST3 ST4

20 20 20 20 Frequency Frequency 10 10 Frequency 10 Frequency 10

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 20 30 40 50 60 70 8 10 20 3 40 50 60 70 80 1 20 3 40 50 6 70 80 1 20 30 4 5 60 70 8

CDR3 Length Supplemental Figure 6

15 14 Microarray study groups 13 and individual telomere lengths 12 11 10 9 8 Young Controls Older Adults 7 99% 6 90%

Telomere length (kb) Telomere 5 50% 4 3 10% Short Telomere 2 1% 1 0 0 10 20 30 40 50 60 70 80 90 Age (Years)

Peripheral blood mononuclear cells

FACS Sort CD8 TEMRAs

5 10

4 10

3 10

3 -10 TEMRA

3 3 4 5 -10 0 10 10 10 CCCR7 CD45RA Total RNA

Gene expression microarray

Gene expression pattern

Ingenuity Pathway Analysis

+ p16INK4a* Cell cycle: G1/S checkpoint regulation ?????

P Rb CDK4/6 Rb Cylin E2F D1* E2F

1472 ? Rb G1/S cell cycle progression Supplementary Figure 7

A Short Telomere vs. Young Control

Upregulated Downregulated

Cyclins and Cell Cycle Regulation ...... Natural Killer Cell Signaling ...... Chronic Myeloid Leukemia Signaling ...... Chronic Myeloid Leukemia Signaling ...... p53 Signaling ...... Glucocorticoid Receptor Signaling ...... RAR Activation ...... RAR Activation ...... Cell Cycle: G1/S Checkpoint Regulation ...... Valine Degradation I ...... Estrogen-mediated S-phase Entry ...... DNA Double-Strand Break Repair by Non-Homologous End Joining .... Retinoic acid Mediated Apoptosis Signaling ...... Endometrial Cancer Signaling ...... Small Cell Lung Cancer Signaling ...... FAK Signaling ...... Protein Ubiquitination Pathway ...... DNA Double-Strand Break Repair by Homologous Recombination ...... Cell Cycle Regulation by BTG Family Proteins ...... Nur77 Signaling in T Lymphocytes ...... JAK/Stat Signaling ...... Glioblastoma Multiforme Signaling ...... Superpathway of Geranylgeranyldiphosphate Biosynthesis I TGF-ß Signaling ...... Neuroprotective Role of THOP1 in Alzheimer's Disease ...... Small Cell Lung Cancer Signaling ...... CTLA4 Signaling in Cytotoxic T Lymphocytes ...... Hereditary Breast Cancer Signaling ...... Role of CHK Proteins in Cell Cycle Checkpoint Control ...... PPARa/RXRa Activation ...... TR/RXR Activation ...... iCOS-iCOSL Signaling in T Helper Cells ...... Endoplasmic Reticulum Stress Pathway ...... B Cell Receptor Signaling ...... Type I Diabetes Mellitus Signaling ...... Molecular Mechanisms of Cancer ...... Mevalonate Pathway I ...... CD28 Signaling in T Helper Cells ...... Virus Entry via Endocytic Pathways ...... Neurotrophin/TRK Signalin...... 01234 012345 -log(P-value) -log(P-value)

B Older Adult vs. Young Control Upregulated Downregulated

Allograft Rejection Signaling ...... All-trans-decaprenyl Diphosphate Biosynthesis ...... Antigen Presentation Pathway ...... Heparan Sulfate Biosynthesis ...... OX40 Signaling Pathway ...... Glucocorticoid Receptor Signaling ...... Apoptosis Signaling ...... IGF-1 Signaling ...... Type I Diabetes Mellitus Signaling ...... Regulation of IL-2 Expression in Activated and Anergic T Lymphocytes Graft-versus-Host Disease Signaling ...... Citrulline Biosynthesis ...... iCOS-iCOSL Signaling in T Helper Cells ...... Role of JAK2 in Hormone-like Cytokine Signaling ...... IL-4 Signaling ...... Assembly of RNA Polymerase I Complex ...... Autoimmune Thyroid Disease Signaling ...... Branched-chain a-keto acid Dehydrogenase Complex ...... tRNA Charging ...... Heparan Sulfate Biosynthesis (Late Stages) ...... Death Receptor Signaling ...... Insulin Receptor Signaling ...... PKCθ Signaling in T Lymphocytes ...... Chondroitin Sulfate Biosynthesis ...... Cytotoxic T Lymphocyte-mediated Apoptosis of Target Cells Galactose Degradation I (Leloir Pathway) ...... Nur77 Signaling in T Lymphocytes ...... dTMP De Novo Biosynthesis ...... Small Cell Lung Cancer Signaling ...... Natural Killer Cell Signaling ...... Cdc42 Signaling ...... B Cell Receptor Signaling ...... Polyamine Regulation in Colon Cancer ...... PKCθ Signaling in T Lymphocytes ...... Myc Mediated Apoptosis Signaling ...... Dermatan Sulfate Biosynthesis ...... Tumoricidal Function of Hepatic Natural Killer Cells ...... Colanic Acid Building Blocks Biosynthesis ...... Calcium-induced T Lymphocyte Apoptosis ...... Xenobiotic Metabolism Signaling ...... 0 0 2468 123 -log(P-value) -log(P-value) Supplementary Figure 8 Young Control Short Telomere Older Adult

CD4+ T cells CD8+ T cells

CD4+CD95+ CD8+CD95+ AB100 * 100

80 80 T T cells T T cells + + 60 60 * * CD95 CD95 40 40 + + ** 20 20 % CD8 % CD4 0 0 Naïve CM EM Naïve CM EM TEMRA

CD4+PD1+ C 20 D 50 CD8+PD1+

15 40

T T cells * + T T cells + 30 10 PD-1 * PD-1 +

+ 20 5 10 % CD4 % CD8 0 0 Naïve CM EM Naïve CM EM TEMRA

EFCD8+CD28- 15 60 CD4+CD28- T T cells - 40 T T cells - 10 CD28 + CD28

+ 20 5 % CD8

% CD4 0 0 Total EM TEMRA Total EM

GHCD4+CD57+ CD8+CD57+ 12 60 10 50 T T cells T T cells + 8 + 40 6 30 CD57 CD57 + + 4 * 20 2 10 % CD4 % CD8 0 0 TotalNaïve CM EM TotalNaïve CM EM TEMRA Supplementary Figure Legends

Supplemental Figure 1. Depleted stem cell pools in late generation telomerase RNA null mice, mTR-/- G4. A. Bone marrow donor-derived engraftment after sublethal irradiation for congenic transplant experimental data shown in Figure 3A-C. B and C. Representative flow plots and quantification of lineage- c-kit+ Sca-1+ (KSL) population in wild-type (WT) and mTR-/- G4 mice. D and E. Representative flow plots and quantification of the bone marrow lineage- c-kit+CD150+CD48- (SLAM) population. For C and E, WT (n=10, 6-16 weeks, 7M/3F) and mTR-/- G4 mice (n=9, 6-16 weeks, 4M/5F). F and G. Peripheral blood engraftment after injection of wild-type (WT) or mTR-/- G4 into wild-type (WT) mice. Each datapoint represents the mean from 5 individual mice. Donor mice were male and recipients were female. Error bars represent s.e.m and ***P <0.001 (Mann-Whitney test).

Supplemental Figure 2. A and B. Live thymocyte counts from wildtype (WT) and mTR- /-G4 newborn (n=12/genotype, sex undetermined because of early age) and adult mice (all female, n=4/group, 19-20 weeks, representative of 2 experiments), respectively. C. Gating strategy for quantifying intra-thymic CD3neg, CD3lo and CD3hi populations. The flowplots shown are representative from a WT newborn mouse. The CD3neg CD4-CD8- (DN) population was analyzed with CD25 vs. CD44 gating to determine the relative populations of DN1-4 subsets. D. Comparison of thymocyte apoptosis rates in CD3neg CD4- CD8- (double negative, DN) populations 1, 2, 3 and 4 as defined by their cell surface markers shown in the left upper corner (n=9 mice/group, 18-21 weeks, all female). E. Apoptotic fraction of CD3lo CD4+ CD8+ (double positive, DP) thymocytes in adult mice. F and G. Apoptotic fraction of CD3hi CD4+ and CD3hi CD8+ thymocytes. For E-G, n=4 mice/group, 20-22 weeks, all female and the data are representative of two independent experiments. For panels D-G, the apoptotic fraction was quantified as the total Annexin V+ population. Error bars represent s.e.m and *P <0.05 (Mann-Whitney test).

Supplemental Figure 3. Lymphopenia in short telomere mice. A-F. Complete blood counts of wild-type (WT, n=10, 7M/3F, 6-16 weeks) and mTR-/-G4 mice (n=9, 4M/5F, 6- 16 weeks) showing abnormally low absolute (Abs) lymphocyte counts. Fig. 3I and J show the absolute CD4 and CD8 counts, respectively. Error bars represent s.e.m and *P<0.05 (Mann-Whitney test).

Supplemental Figure 4. Mouse and human EdU incorporation with T cell stimulation and representative flow plots of Annexin V staining. A. EdU incorporation in isolated CD3+ T cells stimulated with CD3/CD28 ex vivo and quantified at 48h. Unstimulated cells were also included as a negative control. WT and mTR-/-G4 (n=3/genotype). The experiment was replicated twice with similar results and data shown are for one experiment. B. Representative flow plots from peripheral mouse splenocyte- derived isolated CD3+ T cells, analyzed 48 hours after stimulation with CD3/CD28 antibodies. C. EdU incorporation in isolated human CD3+ T cells stimulated with CD3/CD28 ex vivo and quantified at 48h. YC represent Young Controls (n=7, 3M/4F) and ST represents short telomere patients (n=5, 1M/4F) studied under both unstimulated and stimulated conditions. The data are from two independent experiments. D. Representative flow plots of CD3+ T cells derived from the 3 groups shown in panel C. They show Annexin V staining at 48 hours after stimulation with CD3/CD28. For B and D, the total Annexin V+ PIneg (early) plus Annexin V+ PIlo (late) apoptotic populations are quantified in Figures 3K and L, respectively. Error bars represent s.e.m.

Supplemental Figure 5. Raw TCR-Vβ flow cytometry data and complementarity- determining region 3 (CDR3) length by immunoSEQ. A. Example of raw analysis performed to generate the data shown in Fig. 4A. The percentage of CD3+ T cells expressing each of 24 Vβ proteins in a patient carrying a TERT mutation is plotted relative to data from 85 controls. The error bars in the controls represent +/- 1 standard deviation (SD). This analysis was performed for each of the Young Controls, Short Telomere and Older Adult cases studied in Fig. 4A. B. CDR3 length distribution is graphed from data generated by immunoSEQ (Adaptive Technologies, Seattle) on sorted CD8+ T cells from 4 young controls and 4 short telomere subjects (2M, 2F for each).

Supplemental Figure 6. Design and analysis of microarray experiment. At the top, the telogram shows actual lymphocyte telomere length data relative to the age-adjusted nomogram for each of the 12 individuals studied across the three groups. Telomere length + was measured by flow cytometry and FISH. CD8 TEMRA cells were sorted by multi- color flow cytometry using the gating scheme shown. All the short telomere individuals were asymptomatic at the time of lymphocyte procurement. RNA was amplified, reverse- transcribed, and subjected to transcriptional profiling using the Affymetrix GeneChip® PrimeView™ Human Gene Expression Array. The differentially expressed genes were then input into the Ingenuity Pathways Analysis (IPA) platform.

Supplemental Figure 7. Top 20 up- and down-regulated pathways derived from Ingenuity Pathways Analysis (IPA) from differentially expressed genes in sorted + + CD8 terminally differentiated effector memory CD45RA (TEMRA) cells. A. Comparison of short telomere patients with young controls. Up- and down-regulated pathways are shown respectively sorted by the –log(P-value). B. The top 20 up- and down-regulated pathways of differentially expressed genes in older adults compared to young controls similarly sorted by P-values. For these analyses, the most highly differentially expressed genes, defined as a minimum of 2 standard deviations, increased or decreased in each of short telomere and older adult subjects relative to the mean for the young control group. P-values were calculated using the Fisher’s exact test (one-sided). There was no overlap across the comparisons in the top 20 up-regulated pathways. Three down-regulated pathways were shared: natural killer cell signaling, glucocorticoid receptor signaling and B cell receptor signaling. The list of the differentially expressed genes for each pathway is included in Supplementary Tables 3 and 4, for the short telomere vs. young control and older adult vs. young control comparisons, respectively.

Supplemental Figure 8. Immunophenotype of peripheral T cells from telomerase mutation carriers (ST) compared to age-matched young controls (YC) and older adults (OA). A and B. CD95 expression in CD4+ and CD8+ T cell subsets, respectively. C and D. PD-1 expression on CD4+ and CD8+ T cell subsets, respectively. E and F.

Percentage of total and T cells subsets without CD28 expression on CD4+ and CD8+ T cells, respectively. Per convention, CD28 loss occurs in the terminally differentiated populations of CD4+ and CD8+ T cells, respectively, and these were the populations studied. Total refers to all CD4+ and CD8+ T cell sub-populations, including the naïve populations, respectively. G and H. CD57 expression on total and CD4+ and CD8+ T cell subsets, respectively. For A-H, YC (n=5-6, 2M/3-4F), ST (n=6-7, 1-2M/5F), and OA (n=5, 3M/2F). Error bars represent s.e.m, *P<0.05 (Student’s t-test, two sided). CM, + Central Memory; EM, Effector Memory; TEMRA, Effector Memory, CD45RA .

Supplementary Table 1. Demographics of subjects studied*

Young Controls Short Telomere Older Adults

(n=22) (n=16) (n=12)

Mean Age (y) 25.8 20.8 73.3 Range (14-33) (1-39) (61-82)

Sex Male 12 8 7 Female 10 8 5

Mean deltaT (kb)** -0.40 -3.63 -0.37 Range (-2.0, 1.1) (-4.51, -2.36) (-2.26, 1.36)

*These demographics correspond to the subjects for whom telomere length is shown in Fig. 2a,b. Notably, thte Young Controls listed here include 4 additional healthy individuals for whom telomere length data were not available.

**deltaT refers to the difference of lymphocyte telomere length from the age-adjusted median as measured by flowFISH.

Supplementary Table 2. Mutations of short telomere syndrome subjects studied (n=16)

First Clinical Mode of Age (y) M/F Mutation Prior reports(1-7) phenotype Inheritance

0.7 M Enterocolitis DKC1 Ala308Gly de novo Alder et al. 2018

6 M Aplastic anemia TR del375-377 AD Alder et al. 2011

8 F Aplastic anemia TERT Phe71Leu AD Alder et al. 2018

10 M Enteropathy DKC1 IVS1+592C>G X-linked Knight et al. 2001

Sinopulmonary X-linked 15 M Classic DC n/a infections

AD Armanios et al. 17 F Asymptomatic* TERT Lys902Asn 2005

20 M Asymptomatic TERT Gly135Glu AD Gorgy et al. 2015

CMV pneumonitis AD 20 F TR U80A Alder et al. 2018 P. jiroveci

22 M Aplastic anemia Classic DC AD n/a

23 M Aplastic anemia TERT Gly135Glu AD Gorgy et al. 2015

23 F Asymptomatic TERT Gly135Glu AD Gorgy et al. 2015

30 F Asymptomatic* TR del375-377 AD Alder et al. 2011

32 F Asymptomatic TR del375-377 AD Alder et al. 2011

34 F Asymptomatic TR C204G AD Fogarty et al. 2003

34 M Aplastic anemia DKC1 Leu317Phe de novo Marrone et al. 2013

39 F Aplastic anemia TR C204G AD Fogarty et al. 2003

Abbreviations AD, autosomal dominant; CMV, cytomegalovirus; DC, dyskeratosis congenita

*These two individuals were asymptomatic at the time of procurement of lymphocytes but subsequently developed herpes zoster infection

1. Alder, J.K., Hanumanthu, V.S., Strong, M.A., DeZern, A.E., Stanley, S.E., Takemoto, C.M., Danilova, L., Applegate, C.D., Bolton, S.G., Mohr, D.W., et al. 2018. Diagnostic utility of telomere length testing in a hospital-based setting. Proc Natl Acad Sci U S A. 2. Alder, J.K., Guo, N., Kembou, F., Parry, E.M., Anderson, C.J., Gorgy, A.I., Walsh, M.F., Sussan, T., Biswal, S., Mitzner, W., et al. 2011. Telomere length is a determinant of emphysema susceptibility. Am J Respir Crit Care Med 184:904-912.

3. Fogarty, P.F., Yamaguchi, H., Wiestner, A., Baerlocher, G.M., Sloand, E., Zeng, W.S., Read, E.J., Lansdorp, P.M., and Young, N.S. 2003. Late presentation of dyskeratosis congenita as apparently acquired aplastic anaemia due to mutations in telomerase RNA. Lancet 362:1628-1630. 4. Marrone, A., and Mason, P.J. 2003. Dyskeratosis congenita. Cell Mol Life Sci 60:507-517. 5. Gorgy, A.I., Jonassaint, N.L., Stanley, S.E., Koteish, A., DeZern, A.E., Walter, J.E., Sopha, S.C., Hamilton, J.P., Hoover-Fong, J., Chen, A.R., et al. 2015. Hepatopulmonary syndrome is a frequent cause of dyspnea in the short telomere disorders. Chest. 6. Armanios, M., Chen, J.L., Chang, Y.P., Brodsky, R.A., Hawkins, A., Griffin, C.A., Eshleman, J.R., Cohen, A.R., Chakravarti, A., Hamosh, A., et al. 2005. Haploinsufficiency of telomerase reverse transcriptase leads to anticipation in autosomal dominant dyskeratosis congenita. Proc Natl Acad Sci U S A 102:15960-15964. 7. Knight, S.W., Vulliamy, T.J., Morgan, B., Devriendt, K., Mason, P.J., and Dokal, I. 2001. Identification of novel DKC1 mutations in patients with dyskeratosis congenita: implications for pathophysiology and diagnosis. Hum Genet 108:299-303.

+ Supplementary Table 3A. Canonical pathways for up-regulated genes in sorted CD8 TEMRA cells from individuals with short telomeres compared to young controls

-log(P- Ingenuity Canonical Pathway R*** Molecules value)** E2F6,E2F4,HDAC5,SUV39H1,CCNE1,CD Cyclins and Cell Cycle Regulation 3.34E00 1.43E-01 KN2A,PPP2R5D,CCNA2,RB1,HDAC3,PP P2CA

E2F6,E2F4,HDAC5,NFKB1,GAB2,PIK3C2 Chronic Myeloid Leukemia Signaling 3.22E00 1.3E-01 A,SUV39H1,CDKN2A,RB1,HDAC3,STAT5 B,AKT2

CSNK1D,PIK3C2A,MED1,HIF1A,JMY,CD p53 Signaling 2.98E00 1.22E-01 KN2A,RB1,TP53INP1,THBS1,AKT2,PMAI P1,TNFRSF10B

REL,ADCY4,NFKB1,RARA,PRKAR2B,CS NK2B,MED1,AKT2,CSNK2A1,SMAD7,SN RAR Activation 2.86E00 9.83E-02 W1,RELB,RXRA,TNIP1,STAT5B,RAC1,GT F2H1

E2F6,E2F4,HDAC5,SUV39H1,CCNE1,CD Cell Cycle: G1/S Checkpoint Regulation 2.83E00 1.43E-01 KN2A,RB1,HDAC3,MAX

Estrogen-mediated S-phase Entry 2.49E00 2.08E-01 E2F6,E2F4,CCNE1,CCNA2,RB1

Retinoic acid Mediated Apoptosis IRF1,RARA,PARP11,ZC3HAV1,TNKS2,R 2.48E00 1.38E-01 Signaling XRA,CASP3,TNFRSF10B

PIAS3,NFKB1,PIK3C2A,SUV39H1,CCNE1 Small Cell Lung Cancer Signaling 2.47E00 1.27E-01 ,RXRA,RB1,MAX,AKT2

DNAJC6,UBE2L3,USP22,DNAJB7,USP14, UBE2V1,DNAJB12,UBE2N,BIRC3,USP36, Protein Ubiquitination Pathway 2.45E00 8.27E-02 USP46,PSMC2,HLA- A,USP12,HSPA5,PSMA2,HLA- C,DNAJB4,HSPH1,PSMB2,PSMD2

Cell Cycle Regulation by BTG Family E2F6,E2F4,CCNE1,PPP2R5D,RB1,PPP2 2.45E00 1.71E-01 Proteins CA

PIAS3,GNAQ,NFKB1,SOCS5,PIK3C2A,S JAK/Stat Signaling 2.42E00 1.25E-01 OCS4,SOCS6,STAT5B,AKT2

Superpathway of Geranylgeranyldiphosphate Biosynthesis I 2.38E00 2.5E-01 HMGCS1,HMGCR,FNTB,HADHB (via Mevalonate)

Neuroprotective Role of THOP1 in HLA-A,MME,PRKAR2B,HLA- 2.15E00 1.5E-01 Alzheimer's Disease C,PNOC,GNRH1

CTLA4 Signaling in Cytotoxic T HLA-A,PIK3C2A,CLTA,PPP2R5D,HLA- 2.05E00 1.1E-01 Lymphocytes C,CLTB,AKT2,CTLA4,PPP2CA

Role of CHK Proteins in Cell Cycle E2F6,E2F4,RFC1,TLK2,PPP2R5D,HUS1, 2.04E00 1.27E-01 Checkpoint Control PPP2CA

LDLR,PIK3C2A,MED1,HIF1A,RXRA,BCL3, TR/RXR Activation 1.95E00 1.06E-01 HDAC3,AKT2,THRB

Endoplasmic Reticulum Stress Pathway 1.94E00 1.9E-01 HSPA5,CASP3,MBTPS1,EIF2AK3

IRF1,NFKB1,HLA-A,SOCS5,HLA- Type I Diabetes Mellitus Signaling 1.92E00 9.9E-02 DMA,SOCS4,CASP3,HLA- C,SOCS6,RIPK1

Mevalonate Pathway I 1.88E00 2.5E-01 HMGCS1,HMGCR,HADHB

HLA-A,PIK3C2A,CLTA,DNM2,HLA- Virus Entry via Endocytic Pathways 1.83E00 1.01E-01 C,CLTB,RAC1,TFRC,ITGA5

+ Supplementary Table 3B. Canonical pathways for down-regulated genes in CD8 TEMRA cells from individuals with short telomeres compared to young controls

-log(P- Ingenuity Canonical Pathway R*** Molecules value)** PAK2,PIK3R3,AKT3,RRAS2,CD244,LILRB 1,KIR3DL1,KLRB1,KLRC3,PIK3C2B,FCER Natural Killer Cell Signaling 4.11E00 2.08E-01 1G,KLRC1,SOS1,FCGR2A,NCR3,PIK3CA, CD300A,KLRC2,SH3BP2,PRKCB,PTPN6, FCGR3A/FCGR3B

CTBP1,TGFB3,HDAC2,CDK4,PIK3R3,AK T3,RRAS2,RBL2,HDAC8,PIK3C2B,IKBKB, Chronic Myeloid Leukemia Signaling 3.6E00 2.07E-01 SOS1,MYC,PIK3CA,CTBP2,TGFBR1,MD M2,HDAC4,CDK6

HSPA1A/HSPA1B,MAPK14,AKT3,CREB1, PRKAA1,SMARCA2,NR3C2,PRKACB,NC OA3,FOS,A2M,POLR2L,FKBP5,PIK3CA,D USP1,CD3D,CREBBP,TGFBR1,NFAT5,G Glucocorticoid Receptor Signaling 3.53E00 1.52E-01 TF2E1,TAF15,TGFB3,PIK3R3,RRAS2,NC OA1,SMAD2,PPP3CB,IKBKB,PIK3C2B,HL TF,POLR2C,PPP3CA,SOS1,BCL2,IL5,GT F2H5,FOXO3,PRKAB2,GTF2H1

MAPK14,PML,PTEN,AKT3,SMARCA2,NS D1,PRKACB,FOS,PIK3CA,DUSP1,PRKCB ,CREBBP,PNPLA4,TGFB3,PIK3R3,RDH1 RAR Activation 3.46E00 1.68E-01 1,NCOA1,CSNK2B,SMAD2,RXRB,HLTF,P RMT1,PARP1,PDPK1,SMAD5,PRMT2,GT F2H5,ADCY9,GTF2H1

HIBCH,HIBADH,AUH,ALDH6A1,DBT,BCK Valine Degradation I 3.34E00 3.89E-01 DHB,HADHA

DNA Double-Strand Break Repair by Non- PARP1,RAD50,XRCC1,PRKDC,XRCC4,M 3.19E00 4.29E-01 Homologous End Joining RE11A

CTNNA1,MYC,PIK3R3,PDPK1,AKT3,PTE Endometrial Cancer Signaling 2.92E00 2.31E-01 N,PIK3CA,RRAS2,LEF1,PIK3C2B,FOXO3, SOS1

ASAP1,PAK2,PTEN,PIK3R3,AKT3,TLN1,R FAK Signaling 2.63E00 1.86E-01 RAS2,VCL,PIK3C2B,GIT2,ACTA2,SOS1,P DPK1,CAPN5,PIK3CA,ARHGAP26

DNA Double-Strand Break Repair by 2.32E00 3.57E-01 POLA1,RAD52,RAD50,ATRX,MRE11A Homologous Recombination

MAP2K5,HDAC2,CD28,APAF1,HLA- Nur77 Signaling in T Lymphocytes 2.32E00 2.17E-01 DQA1,CD3D,BCL2,PPP3CB,FCER1G,PP P3CA

Glioblastoma Multiforme Signaling 2.23E00 1.52E-01 PLCB1,CDC42,CDK4,PTEN,PIK3R3,AKT3 ,PDGFD,RRAS2,LEF1,PIK3C2B,RHOQ,S

OS1,APC,FZD4,MYC,PIK3CA,MDM2,PLC L2,WNT4,CDK6,TSC1,NF1

TGFB3,MAPK14,CDC42,RRAS2,SMAD2, TGF-β Signaling 2.18E00 1.72E-01 SMURF2,ACVR2A,SOS1,FOS,SMAD5,BC L2,CREBBP,TGFBR1,ACVR1,RUNX2

CDK4,PTEN,PIK3R3,AKT3,APAF1,PIK3C Small Cell Lung Cancer Signaling 2.17E00 1.83E-01 2B,IKBKB,RXRB,MYC,PIK3CA,SKP2,BCL 2,CDK6

HDAC2,CDK4,PTEN,PIK3R3,AKT3,RAD5 0,RRAS2,HDAC8,SMARCA2,PIK3C2B,HL Hereditary Breast Cancer Signaling 2.17E00 1.61E-01 TF,POLR2C,POLR2L,PIK3CA,CREBBP,H DAC4,CDK6,MRE11A

PLCB1,TGFB3,MAPK14,RRAS2,SMAD2,P RKAA1,IKBKB,ACVR2A,MED24,PRKACB, PPARα/RXRα Activation 2.15E00 1.45E-01 CLOCK,SOS1,NCOA3,ACOX1,IL18RAP,A P2A2,PRKCB,CREBBP,TGFBR1,PLCL2,A CVR1,ADCY9,PRKAB2,GPD2

PTEN,PIK3R3,AKT3,HLA- DQA1,PPP3CB,PIK3C2B,IKBKB,FCER1G, iCOS-iCOSL Signaling in T Helper Cells 2.05E00 1.63E-01 PPP3CA,PDPK1,CD28,PIK3CA,TRAT1,C D3D,CAMK2G,NFAT5

MAPK14,CDC42,PAG1,PTEN,PIK3R3,AK T3,RRAS2,CREB1,PPP3CB,PIK3C2B,IKB B Cell Receptor Signaling 2.05E00 1.43E-01 KB,EGR1,GAB1,PPP3CA,SOS1,FCGR2A, PDPK1,PIK3CA,PIK3AP1,CAMK2G,PRKC B,CREBBP,PTPN6,NFAT5

MAPK14,CDC42,AKT3,APAF1,PTCH1,RH OQ,GAB1,PRKACB,APC,FOS,HHAT,PIK3 CA,PRKCB,CREBBP,XIAP,TGFBR1,CDK6 ,PRKDC,NF1,CTNNA1,PLCB1,TGFB3,PA Molecular Mechanisms of Cancer 2.02E00 1.23E-01 K2,CDK4,PIK3R3,RRAS2,SMAD2,LEF1,PI K3C2B,NLK,SOS1,FZD4,MYC,RALBP1,S MAD5,BCL2,CAMK2G,RALA,RASGRF2,W NT4,MDM2,HIPK2,ADCY9,CDC25B

CDC42,PIK3R3,AKT3,HLA- DQA1,PPP3CB,PIK3C2B,IKBKB,FCER1G, CD28 Signaling in T Helper Cells 1.99E00 1.57E-01 PPP3CA,FOS,ARPC5,PDPK1,CD28,PIK3 CA,CD3D,PTPN6,NFAT5

FOS,MAP2K5,CDC42,PIK3R3,PDPK1,PIK Neurotrophin/TRK Signaling 1.97E00 1.79E-01 3CA,RRAS2,CREB1,PIK3C2B,CREBBP,G AB1,SOS1

+ Supplementary Table 4A. Canonical pathways for up-regulated genes in sorted CD8 TEMRA cells from older adults with normal telomere length compared to young controls

-log(P- Ingenuity Canonical Pathway R** Molecules value)* HLA-DPB1,HLA-DOA,HLA-DQA1,HLA- DQB1,HLA-DRB4,HLA-DRA,HLA-A,HLA- Allograft Rejection Signaling 6.15E00 3.5E-01 DRB1,HLA-DMA,TNF,HLA-DMB,HLA- DPA1,HLA-C,FAS

HLA-DPB1,HLA-DOA,CD74,HLA- DQA1,HLA-DRB4,HLA-DRA,HLA-A,HLA- Antigen Presentation Pathway 6.09E00 3.71E-01 DRB1,HLA-DMA,HLA-DMB,HLA- DPA1,HLA-C,CIITA

HLA-DPB1,CD3E,HLA-DOA,HLA- DQA1,HLA-DQB1,HLA-DRB4,HLA- OX40 Signaling Pathway 5.2E00 2.98E-01 DRA,HLA-A,HLA-DRB1,MAP2K4,HLA- DMA,HLA-DMB,HLA-DPA1,HLA-C

MAP4K4,BCL2L11,CAPN1,MAP3K14,IKB KB,BAD,BIRC3,PARP1,CAPN3,MAP2K4,B Apoptosis Signaling 5.13E00 2.27E-01 CL2A1,TNF,RPS6KA1,CASP3,DFFB,FAS, BID,CASP8,MAPK3,SPTAN1

IRF1,CD3E,HLA-DOA,MYD88,HLA- DQA1,HLA- DQB1,MAP3K14,HSPD1,IKBKB,HLA- Type I Diabetes Mellitus Signaling 4.72E00 2.08E-01 DRA,HLA-A,HLA-DRB1,MAP2K4,HLA- DMA,TNF,HLA-DMB,CASP3,HLA- C,FAS,BID,CASP8

HLA-DRB1,HLA-A,HLA-DOA,HLA- DMA,HLA-DQA1,HLA- Graft-versus-Host Disease Signaling 4.7E00 3.08E-01 DQB1,TNF,KIR2DL1/KIR2DL3,HLA- DMB,HLA-C,FAS,HLA-DRA

CD3E,HLA-DOA,PIK3C2A,HLA- DQA1,HLA- iCOS-iCOSL Signaling in T Helper Cells 3.89E00 1.94E-01 DQB1,CHP1,IKBKB,NFATC1,AKT2,BAD,H LA-DRA,HLA-DRB1,PIK3CD,ITPR3,HLA- DMA,CAMK2G,HLA-DMB,RAC1,SHC1

RPS6KB2,HLA-DOA,PIK3C2A,HLA- DQA1,HLA- IL-4 Signaling 3.62E00 2.11E-01 DQB1,JAK3,NFATC1,AKT2,HLA- DRA,HLA-DRB1,PIK3CD,HLA-DMA,HLA- DMB,SYNJ2,SHC1

HLA-DRB1,HLA-A,HLA-DOA,HLA- Autoimmune Thyroid Disease Signaling 3.6E00 2.78E-01 DMA,HLA-DQA1,HLA-DQB1,HLA- DMB,HLA-C,FAS,HLA-DRA

WARS2,CARS2,MARS,HARS,CARS,YAR tRNA Charging 3.39E00 2.63E-01 S2,FARSB,AARS2,WARS,QARS

MAP4K4,MAP3K14,IKBKB,ACTA2,BIRC3, TNFRSF10B,PARP1,PARP11,MAP2K4,ZC Death Receptor Signaling 3.35E00 1.87E-01 3HAV1,TNF,CASP3,DFFB,FAS,BID,CASP 8,SPTAN1

CD3E,HLA-DOA,PIK3C2A,HLA- DQA1,HLA- DQB1,MAP3K14,CHP1,IKBKB,NFATC1,M PKCθ Signaling in T Lymphocytes 3.33E00 1.76E-01 AP3K13,HLA-DRA,HLA- DRB1,PIK3CD,MAP2K4,HLA- DMA,CAMK2G,HLA-DMB,RAC1,MAPK3

Cytotoxic T Lymphocyte-mediated CD3E,HLA-A,CASP3,HLA- 3.31E00 3.08E-01 Apoptosis of Target Cells C,DFFB,FAS,BID,CASP8

CD3E,HLA-DRB1,HLA-DOA,HLA- Nur77 Signaling in T Lymphocytes 3.28E00 2.39E-01 DMA,HLA-DQA1,HLA-DQB1,CHP1,HLA- DMB,CASP3,NFATC1,HLA-DRA

CCNE2,PIAS3,CDK4,PIK3C2A,CDK2,IKB Small Cell Lung Cancer Signaling 3.1E00 1.97E-01 KB,MAX,AKT2,PIK3CD,SUV39H1,SKP2,T RAF1,RB1,BID

HLA-DPB1,CD3E,HLA-DOA,HLA- DQA1,HLA-DQB1,MYL6B,HLA- DRB4,WASL,APC,CFL2,HLA-DRA,HLA- Cdc42 Signaling 3.07E00 1.64E-01 A,HLA-DRB1,MAP2K4,HLA- DMA,ITGB1,HLA-DMB,HLA-DPA1,HLA- C,EXOC7

OAZ1,OAZ2,PSMF1,SAT2,MAX,AZIN1,AP Polyamine Regulation in Colon Cancer 3.05E00 3.18E-01 C

PIK3CD,PIK3C2A,MAP2K4,CDKN2A,CAS Myc Mediated Apoptosis Signaling 2.92E00 2.07E-01 P3,FAS,YWHAE,BID,AKT2,SHC1,BAD,CA SP8

Tumoricidal Function of Hepatic Natural SERPINB9,CASP3,DFFB,FAS,BID,M6PR, 2.8E00 2.92E-01 Killer Cells CASP8

CD3E,PRKCD,HLA-DRB1,HLA- Calcium-induced T Lymphocyte Apoptosis 2.74E00 2.08E-01 DOA,ITPR3,HLA-DMA,HLA-DQA1,HLA- DQB1,CHP1,HLA-DMB,HLA-DRA

HLA-DPB1,HLA-DOA,HLA-DQA1,HLA- DQB1,HLA-DRB4,HLA-DRA,HLA-A,HLA- Allograft Rejection Signaling 6.15E00 3.5E-01 DRB1,HLA-DMA,TNF,HLA-DMB,HLA- DPA1,HLA-C,FAS

+ Supplementary Table 4B. Canonical pathways for down-regulated genes in CD8 TEMRA cells from older adults with normal telomere length compared to young controls

-log(P- Ingenuity Canonical Pathway R*** Molecules value)** All-trans-decaprenyl Diphosphate 2.39E00 1E00 PDSS2,PDSS1 Biosynthesis

CHST2,HS3ST3B1,HS2ST1,GLCE,NDST1 Heparan Sulfate Biosynthesis 2.3E00 1.73E-01 ,B3GALT6,UST,B3GAT2,EXTL2

HSPA1A/HSPA1B,CREB1,TAF4B,NR3C2, PRKAG2,POLR2L,GTF2F1,CDKN1C,NFA T5,MAPK8,NRIP1,MAP3K7,TGFB3,CHUK, Glucocorticoid Receptor Signaling 2.18E00 1.05E-01 PIK3R3,RRAS2,SMAD2,HLTF,POLR2C,P PP3CA,HSPA4,PPP3R1,BCL2,IL5,PRKAG 1,ICAM1,PRKAB2

PIK3R3,IRS2,SOCS5,RRAS2,PRKAG2,CS IGF-1 Signaling 2.12E00 1.35E-01 NK2A1,IGF1R,NEDD4,SOCS7,IGFBP7,M APK8,PRKAG1,FOXO1

Regulation of IL-2 Expression in Activated MALT1,TGFB3,CD28,CHUK,RRAS2,PPP3 2.08E00 1.45E-01 and Anergic T Lymphocytes R1,SMAD2,NFAT5,MAPK8,PPP3CA,VAV3

Citrulline Biosynthesis 1.95E00 3.75E-01 ALDH18A1,ARG1,GLS

Role of JAK2 in Hormone-like Cytokine 1.85E00 1.88E-01 SH2B3,SOCS7,IRS2,SOCS5,HLTF,EPOR Signaling

Assembly of RNA Polymerase I Complex 1.79E00 3.33E-01 POLR1B,TAF1A,POLR1C

Branched-chain α-keto acid 1.65E00 5E-01 DLD,DBT Dehydrogenase Complex

Heparan Sulfate Biosynthesis (Late CHST2,HS3ST3B1,HS2ST1,GLCE,NDST1 1.65E00 1.56E-01 Stages) ,UST,EXTL2

PIK3R3,IRS2,RRAS2,PDE3B,PRKAG2,GA Insulin Receptor Signaling 1.54E00 1.11E-01 B1,GYS2,PPP1R3D,STXBP4,MAPK8,CRK ,PRKAG1,SYNJ1,FOXO1

CHST2,HS3ST3B1,HS2ST1,NDST1,B3GA Chondroitin Sulfate Biosynthesis 1.47E00 1.43E-01 LT6,UST,B3GAT2

Galactose Degradation I (Leloir Pathway) 1.45E00 4E-01 GALE,GALK2

dTMP De Novo Biosynthesis 1.45E00 4E-01 DHFR,TYMS

PIK3R3,KIR2DL2,RRAS2,KLRC2,PRKD3, Natural Killer Cell Signaling 1.44E00 1.13E-01 KIR3DL1,PAK1,KLRB1,KLRC3,KLRC1,SY NJ1,VAV3

B Cell Receptor Signaling 1.41E00 1.01E-01 MAP3K7,PIK3R3,CHUK,RRAS2,CREB1,E GR1,GAB1,PPP3CA,MALT1,PPP3R1,PIK

3AP1,MAPK8,NFAT5,MAP3K4,SYNJ1,FO XO1,VAV3

MALT1,MAP3K7,PIK3R3,CD28,CHUK,RR PKCθ Signaling in T Lymphocytes 1.39E00 1.11E-01 AS2,PPP3R1,NFAT5,MAPK8,MAP3K4,PP P3CA,VAV3

CHST2,HS3ST3B1,HS2ST1,NDST1,B3GA Dermatan Sulfate Biosynthesis 1.35E00 1.35E-01 LT6,UST,B3GAT2

Colanic Acid Building Blocks Biosynthesis 1.34E00 2.31E-01 MPI,GALE,GALK2

ABCB1,MAP2K5,CHST2,HS3ST3B1,MAP 3K7,GSTO2,PIK3R3,CYP3A7,HS2ST1,RR Xenobiotic Metabolism Signaling 1.32E00 9.2E-02 AS2,PRKD3,GSTM3,ALDH18A1,NQO1,U ST,ALDH3A2,PPP2R2B,MAPK8,MAP3K4, NDST1,ALDH8A1,CUL3,NRIP1

All-trans-decaprenyl Diphosphate 2.39E00 1E00 PDSS2,PDSS1 Biosynthesis

* P-value was calculated by Fisher’s exact test (right-tailed). **R refers to the ratio of the number of genes in the indicated pathway divided by the total number of genes that make up that pathway.

Supplementary Table 5. Human Flow Cytometry Antibodies Utilized (n=26)

Marker Fluorochrome Clone Manufacturer

CD3 AF700 UCHT1 Biolegend CD3 APC UCHT1 BD Biosciences CD3 FITC UCHT1 Biolegend CD4 AF488/FITC RPA-T4 BD Biosciences CD4 BV605 OKT-4 BioLegend CD4 PE-CY5 RPA-T4 Biolegend CD4 PerCP/Cy5.5 RPA-T4 eBioscience CD8 AF700 OKT-8 eBioscience CD8 PE-CF594 RPA-T8 BD Biosciences CD8 V450 RPA-T8 BD Biosciences CD8a FITC RPA-T8 Biolegend CD8a PE RPA-T8 eBioscience CD19 BV421 HIB19 Biolegend CD19 BV650 HIB19 Biolegend CD28 PE-CY7 CD28.2 BD Biosciences CD31 FITC WM59 eBioscience CD31 PE WM59 Biolegend CD45RA APC/CY7 HI100 Biolegend CD56 PE-CF594 B159 BD Biosciences CD56 PE-CY7 HCD56 Biolegend CD57 FITC HCD57 Biolegend CD95 PE-CF594 DX2 BD Biosciences CD197 (CCR7) BV421 G043H7 Biolegend CD197 (CCR7) PE 3D12 eBioscience CD197 (CCR7) V450 150503 BD Biosciences CD279 (PD-1) APC ebioJ105 eBioscience

Supplementary Table 6. Mouse Flow Cytometry Antibodies Utilized (n=18)

Marker Fluorochrome Clone # Manufacturer

CD3 AF700 17A2 Biolegend CD3e PE 145-2C11 eBioscience CD4 FITC RM4-5 Biolegend CD8a APC 53-6.7 Biolegend CD8a BV605 53-6.7 Biolegend CD19 PECF594 1D3 BD Biosciences CD19 PECY7 6D5 Biolegend CD25 BV421 BC61 Biolegend CD44 PECY7 IM7 eBioscience CD45.1 AF700 A20 Biolegend CD45.1 PE A20 eBioscience CD45.2 PECY7 104 BD Biosciences CD45.2 PerCP-Cy5.5 104 Biolegend CD48 PECY7 HM48-1 BD Biosciences CD117 (c-kit) APC 2B8 eBioscience CD150 (SLAM) PE TC15-12F12.2 Biolegend Lineage panel: CD3/GR1/CD11b/ FITC n/a Biolegend CD45R (B220)/Ter-119 Ly-6a/e (sca-1) PE E13-161.7 Biolegend

13 mouse T cells Full unedited gel for Figure 5G IR total splenocytes

1:2000 Actin The same gel was stripped and re-probed for Kap1. 1:500 pKap1 Two colors Black and white

8 lanes used for manuscript 8 lanes used for manuscript

kDa WT WT G4 G5 Old Old G4, 0.5G4, Gy 1 GyG4, 2 Gy WT WT G4 G5 Old Old G4, 0.5G4, Gy 1 GyG4, 2 Gy 250 150 100 p-Kap1 75 50

37 Actin

25 20

mouse T cells IR total splenocytes

1:2000 Actin 1:1000 Kap1

Two colors Black and white 8 lanes used for manuscript 8 lanes used for manuscript

kDa WT WT G4 G5 Old Old G4, 0.5G4, Gy 1 GyG4, 2 Gy WT WT G4 G5 Old Old G4, 0.5G4, Gy 1 GyG4, 2 Gy 250 150 100 Kap1 75 50

37 Actin

25 20