FIG. 5.

Effect of Bhlhe40, Hoxc4, and Tec siRNA on rat SSC function. A) Bhlhe40, Hoxc4, and Tec siRNA significantly decreased expression of Bhlhe40, Hoxc4, and Tec in cultures of rat EPCAM⁺ clump-forming germ cells 18 h after transfection. Expression of Rps2 was not changed by Bhlhe40, Hoxc4, or Tec siRNA, indicating that no off-target effects were present. B) Bhlhe40 and Hoxc4 siRNA did not significantly change the number of donor cells in cultures of Rat GFP⁺ EPCAM⁺ clump-forming germ cells at either 4 or 8 days after transfection. Cultures treated with Tec siRNA had more GFP⁺ cells than negative control-treated cultures at 4 days post-siRNA; however, this difference was not present after 8 days. C) Bhlhe40, Hoxc4, and Tec siRNA did not significantly change the percentage of apoptotic cells (Nexin Positive) in cultures of Rat GFP⁺ EPCAM⁺ clump-forming germ cells at either 4 or 8 days after transfection. D) Bhlhe40, Hoxc4, and Tec siRNA significantly reduced the number of SSCs in cultures of EPCAM⁺ clump-forming germ cells 8 days after transfection as demonstrated by the SSC transplantation assay. E) Photomicrographs of representative testes transplanted with negative control or gene-specific siRNA demonstrate the data depicted in D. Bhlhe40, Etv5 (Fig. 3), and Hoxc4 siRNA transplants were conducted at the same time with the same negative control; therefore, the same negative control image was used for all three treatments. Bar = 2 mm. *Statistically significant differences; error bars represent the SEM.