APPENDIX FOR: 
“NSMCE2 suppresses cancer and aging in mice independently of its SUMO 
ligase activity” 
Ariana Jacome, Paula Gutierrez-Martinez, Federica Schiavoni, Enrico Tenaglia, Paula 
Martinez, Sara Rodriguez, Emilio Lecona, Matilde Murga, Juan Mendez, Maria A. 
Blasco & Oscar Fernandez-Capetillo 
TABLE OF CONTENTS 
1. Appendix Figure Legends 2-5 
2. Appendix References 6 
3. Appendix Figures S1-13 7-19 
4. Appendix Table S1 20 
NSMCE2 suppresses cancer and aging 2 
Appendix Figure S1. Expression of NSMCE2 in murine and human tissues  
(a) Western blot using the monoclonal anti-human NSMCE2 developed in this 
study in U2OS cells after transfection with 3 independent siRNA sequences. β-
actin was used as a loading control. (b) IHC in human tissues using the 
NSMCE2 antibody shown in (a). The expression of NSMCE2 is highest in 
proliferating areas such as lymph nodes or the proliferating areas of skin and 
ovary. In addition, NSMCE2 is distinctively high in spermatocytes. Scale bar 
indicates 100 µm. (c) Expression of NSMCE2 in mouse 13.5 dpc embryos using 
an independent polyclonal antibody against the murine protein. The expression 
of NSMCE2 is again highest in areas of intense proliferation (revealed by Ki67 
staining), such as the developing brain. The specificity of this antibody was 
validated by its lack of reactivity in NSMCE2 deficient cells (see main Figs. 2, 5). 
Scale bar indicates 100 µm. 
Appendix Figure S2. Induction of NSMCE2 foci by catalytic inhibition of 
topoisomerase activity 
The images illustrate the staining pattern of NSMCE2 and BRCA1 in wt MEF 
exposed to ICRF-193 (5 µM, 2 hrs), a catalytic inhibitor of TOP2A. Scale bar 
indicates 2.5 µm. 
Appendix Figure S3. SUMO ligase activity of the NSMCE2 C185S/H187A 
mutant 
(a) Outline of the process used to evaluate the SUMO ligase activity of wild type 
and C185S/H187A (NSMCESD) mutant proteins. Wt and mutant cDNAs 
expressing both proteins were cloned directly from RNA obtained from wt and 
Nsmce2SD/SD MEF, and subsequently expressed in HEK293T cells. Clones 
expressing similar amounts of wt and mutant NSMCE2 were selected. EGFP-
SUMO1 or Flag-SUMO2 were transfected into these clones, and were used to 
pull down SUMOylated proteins using immunoprecipitation. The SUMO ligase 
activity was evaluated by looking at NSMC2 autosumoylation. (b) The panels 
illustrate the SUMO ligase activity of the wt and SUMO protein when evaluated 
as explained in (a). Whereas NSMCE2 SUMOylation with SUMO2 is fully 
NSMCE2 suppresses cancer and aging 3 
absent on the NSMCESD mutant, some residual activity can be detected when 
using overexpressed SUMO1. However, these experiments are done on the 
context of endogenous NSMCE2 from HEK293T cells and this activity could 
derive from the endogenous protein. Nevertheless, we note that this is the very 
same mutation that has been used as a SUMO deficient strain (Andrews et al, 
2005; Branzei et al, 2006; Kegel et al, 2011; Pebernard et al, 2008; Takahashi 
et al, 2008).  
Appendix Figure S4. NSMCE2 foci formation in wt and Nsmce2SD/SD MEF 
High-Throughput Microscopy (HTM) mediated quantification of the number of 
NSMCE2 foci per cell in wt and Nsmce2SD/SD MEF in response to MMS (1 mM 
1hr; 16 hr recovery) or MMC (0,25 µM  1hr; 16 hr recovery). No significant 
differences between genotypes could be observed. 
Appendix Figure S5. MUS81-dependent SCE in NSMCE2-deficient MEF 
SCE events per metaphase on MEF from the indicated genotypes, which were 
obtained through genetic crosses of Nsmce2lox/lox and Mus81-/- mice. The 
Mus81 knockout strain used for these analyses has been described before 
(McPherson et al, 2004). Deletion of the conditional Nsmce2 allele was 
achieved by infection with a Cre-expressing Adenovirus (AdCre, kind gift of M 
Barbacid). Data are representative of 2 independent experiments. Error bars 
indicate s.d.; ***P<0.001. 
Appendix Figure S6. Segregation deficiencies observed on NSMCE2-
deficient MEF 
(A) Quantification of the various segregation deficiencies that can be observed 
by DAPI staining in Nsmce2+/l+ and Nsmce2lox/lox MEF treated or not with 4-OHT 
for 8 days. (B) Examples of the types of segregation defects that can be 
observed on NSMCE2-deficient MEF from (A). Scale bar (white) indicates 2.5 
µm. 
NSMCE2 suppresses cancer and aging 4 
Appendix Figure S7. Normal DSB-repair kinetics on NSMCE2 deficient 
MEF 
Overall dynamics of DSB repair were assessed through HTM-mediated 
quantification of the induction and disappearance of 53BP1 nuclear foci in 
response to IR (5 Gy). No significant differences between the dynamics of 
53BP1 foci can be observed between UQ-NSMCE2lox/lox MEF that had been 
treated (or not) with 4-OHT for 48 hrs previous to the exposure to IR. 
Appendix Figure S8. Impact of NSMCE2 deficiency on the developing 
thymus 
Pregnant females were treated at 14.5 dpc for 3 days with intraperitoneal 
injections of 4-OHT. The image represents the overall loss of cellularity and 
accumulation of mitotic cells (identified by their reactivity towards an 
phosphorylated histone H3 Ser 10 antobody) observed on the thymuses of 
Nsmce2lox/lox developing embryos. Some the observed mitotic figures presented 
evidences of improper chromosome segregation such as anaphase bridges (an 
example indicated by the black arrow is shown magnified). Scale bar indicates 
20 µm. 
Appendix Figure S9. 4-OHT-induced deletion of NSMCE2 in adult mice 
Western blot analysis of NSMCE2 levels on the indicated organs from 
Nsmce2+/+ and Nsmce2lox/lox animals carrying the UQ.CreERT2 transgene, 3 
months after being fed with a 4-OHT containing diet. β-ACTIN was used as a 
loading control. 
Appendix Figure S10. Survival of mice deleted for NSMCE2 in adults 
Kaplan-Meyer survival curves of Nsmce2+/+ and Nsmce2lox/lox animals carrying 
the UQ.CreERT2 transgene after being fed a 4-OHT containing diet that started at 
weaning. The p value was calculated with the Mantel-Cox long rank test. 
***P<0.001. 
NSMCE2 suppresses cancer and aging 5 
Appendix Figure S11. Accumulation of micronuclei in NSMCE2 deficient 
intestines 
Representative example of the accumulation of micronuclei that can be 
observed on the intestinal epithelia from Nsmce2+/+ and Nsmce2lox/lox animals 
carrying the UQ.CreERT2 transgene after being fed a 4-OHT containing diet that 
started at weaning for 3 months. γH2AX was used to facilitate the detection of 
micronuclei, as reported by (Crasta et al, 2012). Scale bar indicates 100 µm. 
Appendix Figure S12. Formation of SMC5/6 complexes on NSMCE deleted 
B cells 
Immunoprecipitation of SMC5 (and IgG, as a control) from a 48 hr culture of B 
lymphocytes of CD19.Cre transgenic Nsmce2+/+ and Nsmce2lox/lox animals. 
NSMCE2 and SMC6 levels on the immunoprecipitates were analysed by WB. 
The image of the ponceau staining is shown as a loading control. 
Appendix Figure S13. Absence of co-localization between NSMCE2 and 
BLM foci 
NIH3T3 cells were transfected with a plasmid expressing BLM-YFP (kind gift of 
I. Hickson). NSMCE2 (red) and BLM (green) foci were induced by treating cells 
with MMS (1 mM  1hr; 24 hr recovery). DAPI was used to stain DNA.  Scale bar 
indicates 2.5 µm. The images are representative of cells presenting both BLM 
and NSMCE2 foci.  
Appendix Table S1. Percentage of animals or embryos born from 
NSMCE2GT/+ x NSMCE2GT/+ crosses 
NSMCE2 suppresses cancer and aging 6 
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