Research Paper Volume 12, Issue 24 pp 25528—25546

miRNA-122-5p stimulates the proliferation and DNA synthesis and inhibits the early apoptosis of human spermatogonial stem cells by targeting CBL and competing with lncRNA CASC7

Figure 4. Influence of CBL silencing on the proliferation, DNA synthesis, and early apoptosis of human SSC line. (A) Real-time qPCR showed changes of CBL mRNA by CBL siRNA1, CBL siRNA2, and CBL siRNA3 in human SSC line. (B, C) Western blots revealed changes of CBL protein by CBL siRNA1, CBL siRNA2, and CBL siRNA3 in human SSC line. * denoted statistically significant differences (p< 0.05) of CBL siRNA1-, 2-, and 3-treated human SSC line compared with the control siRNA. (D) CCK-8 assays showed the growth curve of human SSC line treated with control siRNA and CBL siRNA1 for 5 days. * indicated statistically significant differences (p<0.05) in human SSC line between control siRNA and CBL siRNA1. (E) Western blots illustrated the changes of PCNA protein in human SSC line at day 3 after transfection of control siRNA and CBL siRNA1. (F) The relative protein level of PCNA in human SSC line at day 3 after transfection of control siRNA and CBL siRNA1 through normalization to the signals of their loading control ACTB. * showed statistically significant differences (p< 0.05) in human SSC line between control siRNA and CBL siRNA1. (G, H) EDU incorporation assay demonstrated the percentages of EDU-positive cells in human SSC line affected by control siRNA and CBL siRNA1. Scale bars= 50 μm. * indicated statistically significant differences (p<0.05) between CBL siRNA1-treated human SSC line and the control siRNA. (I, J) APC Annexin V and PI staining and flow cytometry depicted the percentages of early apoptosis in human SSC line transfected with control siRNA and CBL siRNA1. * displayed statistically significant differences (p<0.05) between CBL siRNA1-treated human SSC line and the control siRNA.