Table 1.
Sequences of primer pairs for quantitative real-time PCR.
Figure 1.
Detection of inhibin α-subunit expression in the mouse anterior pituitary.
A: Inhibin α-subunit expression in the mouse anterior pituitary gland using immunohistochemical methods. Immunohistochemical analysis of inhibin α subunit expression in the anterior pituitary gland by inhibin monoclonal antibody [A1] and PBS [A2], magnification is ×40. B: Inhibin α subunit expression in the cultured mouse anterior pituitary cells using indirect immunofluorescence. Indirect immunofluorescence analysis of inhibin α subunit expression in the anterior pituitary cells by inhibin monoclonal antibody [B1] and PBS [B2], magnification is ×40.
Figure 2.
Inhα RNAi recombinant plasmids expression in the anterior pituitary cells.
Three Inhα RNAi recombinant plasmids were transfected in anterior pituitary cells named as pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative, respectively for 24 h, 48 h or 72 h, the best efficient of which was selected for further investigation.
Figure 3.
Detection of INH α subunit in the anterior pituitary cells after 48 h transfected with pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative, respectively. A:
Transcription levels of INH α gene in the anterior pituitary cells transfected with pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative respectively. The statistical differences were tested using one-way ANOVA. Asterisk [*] means P<0.05, two asterisks means P<0.01. B: INHα subunit expression were detected by western blotting in the anterior pituitary cells after 48 h transfected with pshRNA-1, pshRNA-2, pshRNA-3 and pshRNA-negative respectively.
Figure 4.
Cell cycle analysis of mouse pituitary cells by flow cytometry.
The results of cell cycle examination demonstrated that the number of cells in S phase was significantly decreased in pShRNA2 group [p<0.05, compared with pShRNA-negative and PBS groups], indicating S phase arrest.
Table 2.
Analysis of Cell Cycle by Flow Cytometry in the anterior pituitary cells 48-2, pshRNA-negative and PBS [Mean ± SEM, n = 3].
Table 3.
Measurement of mouse pituitary cell apoptosis.
Figure 5.
Levels of mRNA and protein expressions of apoptosis related genes were detected by q-PCR and western blotting.
A: The mRNA levels of apoptosis related genes were examined by q-PCR. The results demonstrated that Caspase-3 and Bcl-2 was significantly decreased in pshRNA2 group 48 h post-transfection [p<0.05, compared with pshRNA-negative and PBS groups] but the levels of Bax and p53 gene was unlatered [p>0.05]. B: The protein levels of apoptosis related genes were detected by western blotting. The results showed that Caspase-3, Bax and Bcl-2 genes was up-regulated by inhibin gene silencing. But p53 gene was not affected by inhibin silencing.
Figure 6.
Effects of inhibin silencing on INHβB, FSHβB, LHβB, GnRHR and β-glycan.
The levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were quantified by real-time PCR and western-blotting among pshRNAi-2, pshRNAi-negative and PBS groups, respectively. A: The mRNA levels of INHβB, FSHβB, LHβB, GnRHR and β-glycan were examined by q-PCR. Compared with pshRNA-negative and PBS groups, the mRNA levels of INHβB, FSHβB, LHβB and GnRHR were up-regulated [P>0.05] in APCs transfected with pshRNAi-2. However,mRNA levels of INHβB and GnRHR were significantly up-regulated, whereas mRNA level of β-glycan was significantly down-regulated. B: The proteins levels of FSH, LH, ACVB, INHB, GnRHR and β-glycan were detected by western blotting. The results showed that FSH, LH, ACVB, INHB, GnRHR proteins were up-regulated by inhibin gene silencing. But the protein of β-glycan was down-regulated by inhibin silencing.
Figure 7.
Concentration of FSH in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.
The results indicated that the FSH concentration 48-transfection was higher in pshRNA-2 group than those of pshRNA-negative and PBS groups respectively.
Figure 8.
Concentration of LH in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.
The results indicated that the LH concentrations in the group were not affected by inhibin gene.
Figure 9.
Concentration of INHB and ACVB in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.
A: Detection of the INHB concentration was done by RIA kit. Compared with pshRNAi-negative and PBS groups, the INHB concentrations was decreased significantly at 24 and 48 h after transfection with pshRNAi-2. B: Similarly, compared with pshRNAi-negative and PBS groups, the ACVB concentrations was increased significantly at 24 and 48 hours after transfection with pshRNAi-2.