Blood, Vol. 110, Issue 7, 2371-2380, October 1, 2007

Mutations in the cofilin partner Aip1/Wdr1 cause autoinflammatory disease and macrothrombocytopenia
Blood Kile et al. 110: 2371

Supplemental materials for: Kile et al

Files in this Data Supplement:

• Figure S1. Wild-type mRNA levels are decreased in mice carrying the rd allele of Wdr1 (PDF, 98.6 KB) -
  Real-time RT-PCR analysis of Wdr1 mRNA in adult tissues and CD41 positive cells sorted from bone marrow. A Taqman minor groove binder probe specific for the sequence deleted in the mutant transcript produced by cryptic splicing of the rd allele specifically detected wild-type message. n=4 mice per genotype. Results were normalized to GAPDH.
  
  
• Figure S2. The mutant form of Wdr1 is unstable and potentially misfolded (PDF, 325 KB) -
  (A) Pulse chase experiments. Plasmids encoding flag-tagged wild-type (i) or mutant forms of Wdr1 produced by cryptic splicing at the rd allele (Wdr1^{Δ346-347}) (ii) were transfected into 293T cells, which were then pulsed with S³⁵, and chased with cold media for the indicated time periods. Cells were lysed, immunoprecipitated with anti-flag antibodies, and separated by SDS-PAGE. Phosphoimaging (top panels) revealed that Wdr1^{Δ346-347} has a significantly reduced half-life. (B) Immunoprecipitation of transiently transfected 293T cells showed that a mouse anti-Wdr1 monoclonal antibody is capable of recognising both wild-type and Wdr1^{Δ346-347} by western blot, but only the wild-type by IP, suggesting that Wdr1^{Δ346-347} is misfolded.
  
  
• Figure S3. A gene trap insertion at the Wdr1 locus (PDF, 337 KB) -
  (A) The XN462 ES cell line contains a gene trap insertion in intron 2 of Wdr1 (B) Male mice that are compound heterozygous for the rd and the gene trap allele of Wdr1 are runted relative to littermates (i) and (ii), suffer a severe autoinflammatory disease characterised by pustular lesions on the lower ventrum (ii). Platelet counts in compound heterozygotes were profoundly reduced: 1363 ± 370 Wdr1^+/+, 136 ± 50 Wdr1^gt/rd; n=4.
  
  
• Figure S4. Lymphocytes are not required for thrombocytopenia or autoinflammatory disease in Wdr1^rd/rd mice (PDF, 507 KB) -
  (A) The rd mutation was crossed onto the Rag2-/- (B and T cell deficient) or Jh-/- (B cell deficient) backgrounds. In both cases, at 3 months of age, mice lacking mature lymphocytes had developed autoinflammatory lesions similar to Wdr1^rd/rd counterparts. (B) Peripheral blood platelet counts in 12 week old mice. Thrombocytopenia was observed in the absence of B and T cells. Data represent mean ± SD, n=6 mice of each genotype.
  
  
• Figure S5. Splenectomy does not increase platelet counts in Wdr1^rd/rd mice (PDF, 200 KB) -
  7 week old mice were subjected to splenectomy (Spl) or sham surgery (NT) and platelet counts monitored. Data represent mean ± SD, n=10 mice of each genotype.