Premature Aging in Mice Deficient in DNA Repair and Transcription Jan de Boer, Jaan Olle Andressoo, Jan de Wit, Jan Huijmans, Rudolph B. Beems, Harry van Steeg, Geert Weeda, Gijsbertus T. J. van der Horst, Wibeke van Leeuwen, Axel P. N. Themmen, Morteza Meradji, Jan H. J. Hoeijmakers

Supplemental Table 1. Summary of Aging Phenotype TTD mice.
	Wildtype	TTD
Life span: 50% mortality mark	>2 year	~ 7 months, for survival curve see (1) 1)
Body weight	Normal, obesity in mid life, later loss of weight	Mild growth retardation, later in life more severe with fatty tissue hypoplasia and cachexia
Osteoporosis	Normal at age 14 months	Reduced to 54% of wt, kyphosis of spinal column2)
Artherosclerosis	Normal 2)	Normal
Red blood cell count	Normal	84% of wild-type, reduced Hb values
Organs	Normal	Normal, except enlarged spleen (anemia?)
Blood chemistry	Normal	Reduced values of branched-chain amino acids
Fertility             Male                        Female	Normal Normal	Normal (at least to 7 months) Initially fertile (reduced), early atrophy of ovary.
Hair greying 3, 4)	Sporadic	Frequent 1), melanocyte depletion
Skin 3)	Normal	Sebaceous gland hyperplasia
Neurology	Normal	Increased frequency of mild tremors, no overt myelination defect
Sarcopenia	Normal	Increased levels of 1-methyl histidine
1) Influenced by the genetic background; in pure C57/Bl6 life span is significantly longer; 2) Mice in general rarely show artherosclerosis; 3) No correlation apparent with degree of cachexia; 4) At the age of ~14 months 5/5 TTD mice (with sufficient hair) showed pronounced greying, in contrast to 0/22 age-matched wt mice (p < 0,001), at >2 years a significant fraction of wt Bl6 mice shows occasional gray hair.

Supplemental Table 2. Blood analysis of TTD mice.
	wild-type mice	TTD mice	p-value1)
red blood cell count (1012/L)	10.3	8.7	< 0.01
hemoglobin (mmol)	9.4	8.12)	< 0.01
hematocrit (L/L)	0.56	0.47	< 0.01
mean cell volume (fL)	54.5	54.3	ns
ns: not significant 1)No significant difference between wt and TTD mice was seen at the age of 3-4 weeks 2)Reduced levels of haemoglobin were recently also observed in TTD patients (2)

Supplemental Table 3. Analysis of serum of TTD mice.
component [Mol]	wild-type mice	TTD	p-value
Valine	222	169	< 0.05
Isoleucine	103	69	< 0.01
Leucine	164	118	< 0.05
1-me-histidine	15.7	21.2	0.05
Phenylalanine	78	60	< 0.05
Proline	166	139	ns
Arginine	130	120	ns
Asparagine	25	27	ns
albumine	0.40	0.48	ns
glucose	1.34	1.04	ns
creatinine	63.4	54.6	ns
urea	11.4	14.7	ns
ns: not significant, this includes all other amino acid concentrations, as well LDH levels (3)

Supplemental Table 4. Recovery of XPA/TTD double mutant embryos and mice.
	Found	Expected	# analysed1)
XPA/TTD E13.5	13	9.5	56
XPA/TTD E18.5	10	15.5	69
XPA/TTD newborn (7-10 days)	12	32	223
1) Total number of offspring analysed (including mutants, heterozygotes and wt)

2. Experimental procedures

Establishment and culturing of cells
Mouse embryo's of the required genotypes (13.5 days after gestation) were used for derivation of primary mouse embryo fibroblasts (MEFs) (1). We used established lines from primary MEFs that survived after crisis.

Determination of DNA repair parameters
Cellular survival after exposure to the indicated UV-C dose (J/m²) was assayed using the ³H-Thymidine incorporation method (4). For experiments with paraquat (1,1'-Dimethyl-4,4'-bipyridinium dichloride, Sigma) cells were seeded at 20-40% confluency and split on each of the three subsequent days. Then, equal numbers of cells were seeded on gelatin-coated plates (1,6 x 10⁴ cells per well of a 6-well plate) and cultured in the presence or absence of paraquat. Medium was refreshed every 24 hours. Cell survival was measured after 68 hours using the ³H-thymidine incorporation method (4).

Determination of blood parameters
Blood cell values were analyzed using a Sysmen F800 apparatus (Toa Medical Electronics) and general blood content values via spectrophotometry on an Elan Autoanalyzer (Eppendorf Merck). Amino acids in plasma were measured by ion exchange chromatography on a Pharmacia Biochrome 20 amino acid analyser with ninhydrin detection. Organic acids in urine were extracted with ethylacetate, dried and converted into methylesters by diazomethane. Measurements were performed by gaschromatography-mass spectrometry (Fisons MD-800).

Radiological analysis of bone mineralization
Radiographs with a two-fold magnification were taken in dorso-frontal and lateral direction. A special X-ray system, developed for human mammography (CGR Senograph 500T) was operated at 30 kV and 32 mAS. A molybdeen focus (0.1 mm) was utilised, with focus-film distance 65 cm and focus-object distance 32.5 cm. Kodak X-ray films (MIN-R MA 18 x 24 cm) were used in combination with a Dupont Cronex low-dose mammography-intensifying screen. Mice were sedated during the radiographic procedure. Mineral density was quantified by scanning the radiographs (DuoScan Agfa). Using Imagequant the number of pixels in a defined area of the skull or of the complete sixth tail vertebra was determined. For histological examination, dissected tissues fixed in 10% formal saline were processed and embedded in paraffin and stained with hematoxylin and eosin, or formalan (melanin staining) using routine procedures.

3. References

1. J. de Boer et al., Mol. Cell 1, 981 (1998).

2. V. Viprakasit et al., Hum. Molec. Genet. 10, 2797 (2001).

3. Our unpublished results.

4. J. de Boer et al., Cancer Res. 59, 3489 (1999).