Concentration of INHB and ACVB in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.

<p>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.</p

Effects of inhibin silencing on INHβB, FSHβB, LHβB, GnRHR and β-glycan.

<p>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. <b>A:</b> 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.</p

Detection of inhibin α-subunit expression in the mouse anterior pituitary.

<p>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.</p

Inhα RNAi recombinant plasmids expression in the anterior pituitary cells.

<p>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.</p

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:

<p>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>B:</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.</p

Concentration of FSH in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.

<p>The results indicated that the FSH concentration 48-transfection was higher in pshRNA-2 group than those of pshRNA-negative and PBS groups respectively.</p

Cell cycle analysis of mouse pituitary cells by flow cytometry.

<p>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.</p

Analysis of Cell Cycle by Flow Cytometry in the anterior pituitary cells 48-2, pshRNA-negative and PBS [Mean ± SEM, n = 3].

<p>All results were evaluated by One-way ANOVA.</p><p>[*] indicates level of significance in columns [P<0.05].</p

Measurement of mouse pituitary cell apoptosis.

<p>There were significantly less apoptotic cells and more vital cells in the pShRNA-2 group compared to those of pShRNA-negative and PBS group respectively. [Means±SE, n = 3].</p><p>All results were evaluated by One-way ANOVA,</p><p>[*] P<0.05</p><p>[**] P<0.01 indicates level of significance in columns.</p

Concentration of LH in the culture medium released by the mouse anterior pituitary cells at the different times after transfection.

<p>The results indicated that the LH concentrations in the group were not affected by inhibin gene.</p