Mutational analysis of <i>SRY</i> and <i>WT1</i>.

<p>(A) wild type (upper panel, control) and mutated sequence (lower panel, patient) of <i>SRY</i>. (B) schematic representation of the SRY protein. The K128R mutation resides in the HMG domain, just before the cNLS. (C) <i>In vitro</i> luciferase assays of SRY-WT (wild-type) and SRY-K128R (mutant) in HEK293T cell line. Cells were co-transfected with TESCO-E1b-<i>Luc</i>, SF1 and WT or mutant SRY to assess for activation of TESCO. The mean percentages of fold change of luciferase activity of TESCO-E1b-<i>luc</i> over the empty vector, relative to WT SRY levels from six independent assays (each performed in triplicate) are shown. Error bars represent standard error of the mean (SEM). (D) pcDNA3-FLAG-SRY wild-type (WT, 2 µg) or pcDNA3-FLAG-SRY mutant (K128R, 2 µg) were transiently transfected into HEK293T cells using Fugene 6. Exogenous SRY (WT or K128R) expression was detected using a FLAG antibody and a green fluorescent Alexa-488 dye coupled secondary antibody. Nuclei were stained with 4′,6-diamino-2-phenylindole (DAPI). Both wild type and mutant SRY show strong nuclear staining. (E) SRY fluorescence was quantified as previously described <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040858#pone.0040858-Argentaro1" target="_blank">[35]</a>. Nuclear accumulation of SRY (WT or K128R) expressed as fluorescence in the nucleus over that in the cytoplasm (Fn/c) were background fluorescence has been subtracted. Measurements represent the average of 3 independent transfections. Results are relative to WT transfected cells (Fn/c given value of 100%). The number of cells analysed is n = 111 (WT) and n = 121 (K128R). Error bars represent the standard error of mean values. Two-tail t-Test of unpaired sample means was performed between WT transfected cells and mutant transfected cells and showed no significant differences. P = 0.49. (F) mutated sequence (upper panel, patient) and wild type sequence (lower panel, control) of <i>WT1</i>, showing the heterozygous +4C&gt;T change.</p

Immunohistochemical staining of the left GB lesion.

<p>(A) representative hematoxylin and eosin (HE) staining. The germ cells present in the GB stain positive for OCT3/4 (B, brown staining), TSPY (C, red staining), and SCF (D, red staining). Supportive cells in the GB stain positive for FOXL2 (E, brown staining), while SOX9 (F) is negative. All slides are counterstained with hematoxylin. Magnification 100x for all.</p