Fig. 3: Suppression of FLC differentiation by Tmsb10 KD.
From: Tmsb10 triggers fetal Leydig differentiation by suppressing the RAS/ERK pathway

a The experimental procedure for reconstruction of fetal testes. W-EGFP cells (E16.5) were plated and treated with siRNA for 24 h. Whole testicular cells prepared from wild-type fetuses (E16.5) were mixed with siRNA-treated W-EGFP cells to reconstruct testes. The detailed procedure for testis reconstruction and culture is described in the Materials and Methods. b A representative image of the reconstructed testis. Testicular tubules (tu) formed, and FLCs with strong EGFP staining were observed in the interstitial regions of the reconstructed testes. c The reconstructed testes were cultured in the presence (+) or absence (−) of SAG for 21 days. The reconstructed testes were examined under a fluorescence microscope to measure EGFP fluorescence. Scale bar = 100 µm. d The EGFP-positive cells (indicated by the relative EGFP-positive area) in the reconstructed testes above were analyzed quantitatively after incubation for 21 days. The cells were cultured in the absence (−, open bar) or presence of SAG (+, gray bar). n = 3. ***p < 0.001. e Representative images of the reconstructed testes using W-EGFP cells treated with the following siRNAs: control (siCnt), Tmsb10 (siT10), Tmsb4x (siT4x), and Ad4BP/SF-1 (siAd4). Scale bar = 100 µm. f The EGFP-positive cells in the reconstructed testes above were analyzed quantitatively. Letters a, b, and c denote significant differences between the cell groups treated with siCnt, siT10, siT4x, and siAd4. n = 3. p < 0.001. g Testes were reconstructed using W-EGFP cells treated with siCnt or siT10. They were cultured for 21 days in the presence of SAG. Scale bar = 100 µm. h The EGFP-positive cells in the reconstructed testes above were analyzed quantitatively. n = 3. ***p < 0.001. i W-EGFP cells were treated with siCnt (open bar) or siT10 (light blue bar). Expression of Gli1 in the W-EGFP cells was examined by qRT-PCR. The data were normalized by Rn18s and are presented as means ± SEM. n = 3. **p < 0.01.