DEV005884 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Loss of PDS5B in Pds5B^−/− MEFs and E18.5 brain. (A) Relative amounts of Pds5B mRNA from wild-type and Pds5^−/− MEFs using qRT-PCR normalized by GAPDH mRNA level. Error bars represent standard deviation (STD). * P<0.001, Student’s unpaired t-test. Note undetectable Pds5B mRNA in Pds5B^−/− MEFs. (B) Western blot of protein lysates prepared from MEFs probed with antibodies to PDS5B and β-tubulin. Note undetectable PDS5B protein in Pds5B^−/− MEFs lysates. (C) Western blot of E18.5 whole-brain tissue lysates probed with antibodies to PDS5B and β-tubulin. Note undetectable PDS5B protein in Pds5B^−/− brain compared with a high concentration of PDS5B in wild-type brain.

• Supplemental Figure S2 -

  Fig. S2. Loss of PDS5B does not affect cell proliferation and apoptosis in embryonic palatal shelves. (A) Proliferation index measured by BrdU incorporation in palatal shelves at E13.5 n=3 for each genotype and all cells designated in B and C (>200 cells in palatal shelf right of the dashed line) were counted for each animal. Error bars represent STD. (B,C) BrdU staining (BrdU: brown; hematoxylin: blue) of palatal shelves from E13.5 Pds5B^+/− (B) and Pds5B^−/− (C) embryos. (D,E) TUNEL staining (TUNEL: red; bisbenzimide: blue) of palatal shelves from E13.5 wild-type (D) and Pds5B^−/− (E) embryos. TUNEL-positive cells in other tissues (arrows in D and E). Note that cells in palatal shelves below the dashed line have no apoptosis in both wild-type and Pds5B^−/− embryos. Scale bars: 50 μm.

• Supplemental Figure S3 -

  Fig. S3. Analysis of sympathetic chain ganglia and colonic innervation in Pds5B-deficient mice. (A,B) TOH whole-mount staining of neonatal wild-type (A) and Pds5B-deficient (B) mice demonstrated normal formation of sympathetic ganglia chains in both. (C,D) AChE whole-mount staining of P0 gut shows distal colon aganglionosis of Pds5B-deficient mice (D) compared to wild type (C). Clustered neuronal cell bodies marked by asterisks in C and residual nerve fibers denoted by arrows in D.

• Supplemental Figure S4 -

  Fig. S4. Histological analysis of testicular defects in Pds5B-deficient mice. (A,B) H&E staining of E16.5 testes of wild-type and Pds5B^−/− mice. Note the decreased number of germ cells in Pds5B^−/− testicular cords (arrows in B). (C,D) H&E-stained sections of 6-week testicular transplants of wild-type (C) and Pds5B^-/- (D) mice. Note absence of germ cells in Pds5B^−/− transplanted testes (D). (E,F) GCNA1 (red) and BrdU (green) double staining of E12.5 testes from wild-type and Pds5B^−/− mice. Note the decreased number of dividing germ cells in the Pds5B^−/− embryonic testes. White dashed circles outline the embryonic testes. Scale bar: 50 μm.