Figure 6.

LRP6 Knockdown Inhibits the Proliferation and Synthesis Function of Human Sertoli Cells

(A) CCK-8 assay showed the growth curve of human Sertoli cells after transfection of control siRNA or LRP6 siRNA-3 for 1–5 days. (B) Western blots demonstrated the expression of PCNA after 48 hr of transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (C). β-actin served as a loading control of proteins. (D) EDU incorporation assay showed the EDU-positive cells in human Sertoli cells at 48 hr after transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (E). Cell nuclei were counterstained with Hoechst 33342. The percentages of EDU-positive cells were counted out of 500 total cells from three independent experiments. (F) Western blots demonstrated GDNF, SCF, BMP4, FGF2, and CXCL12 proteins in human Sertoli cells at 48 hr after transfection of control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (G). β-actin served as a loading control of proteins. (H) CCK-8 assay showed the growth curve of human spermatogonial stem cell line that cultured with culture medium collected daily from Sertoli cells transfected with control siRNA or LRP6 siRNA-3. (I) Western blots demonstrated PCNA protein in human spermatogonial stem cell line after 72 hr of culture with culture medium collected daily from Sertoli cells transfected with control siRNA or LRP6 siRNA-3. Results of three independent assays were concluded in (J). β-actin served as a loading control of proteins. *p < 0.05; **p < 0.01. Scale bars, 20 μm (D).