DEV02697 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure 1 -

  Fig. S1. Specificity of antibodies. (A) Cell extract was prepared from E14.5 male gonads, resolved by SDS-PAGE and subjected to western analysis with anti-Nanos2 antiserum before purification (left), flow through (center) and eluate from affinity column conjugated with GST-Nanos2. (B) Cell extract was prepared from E12.5 male gonads, resolved by SDS-PAGE and subjected to western analysis with preimmune serum (left) or purified anti-Nanos3 antiserum (right). Arrowheads indicate positions of endogenous Nanos2 (A) and Nanos3 (B).

• Supplemental Figure 2 -

  Fig. S2. Detection of apoptosis. Sections prepared from E18.5 male gonads from Nanos2^+/−, Nanos2^−/− and Nanos2^−/− with FLAG-Nanos3 transgene were subjected to immunological detection with anti-cleaved caspase3 antibody (Cell Signaling) followed by Alexa-488 conjugated goat-anti-rabbit IgG together with TRA98 followed by Alexa-594 conjugated goat anti-rat IgG. Blue, DAPI staining. Scale bars, 100 μm. White arrowheads indicate apoptotic germ cells.

• Supplemental Figure 3 -

  Fig. S3. Gene dosage effect of Nanos3 in the absence of Nanos2. Sections were prepared from testes of Nanos2^+/−Nanos3^+/− (A,D,G), Nanos2^−/−Nanos3^+/+ (B,E,H) and Nanos2^−/−Nanos3^+/− (C,F,I) at E18.5 (A-C), P4 (D-F) and P8 (G-I). The number of germ cells was compared by TRA104 antibody staining. In the Nanos2-null testis, the number was decreased at E18.5, but transiently increased at P4. However, the removal of single dosage of the Nanos3 gene in addition to Nanos2 results in the lack of any increase at P4. (J) Graphical representation of the number of germ cells (TRA104-positive cells) per 10 tubules from sections of Nanos2^+/−Nanos3^+/− (open bars), Nanos2^−/−Nanos3^+/+ (grey bars) and Nanos2^−/−Nanos3^+/− (solid bars) testes at E18.5, P4 and P8. The values represent mean values ± s.e.m. Scale bars, 100 μm.