Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Effects of FSH on phosphorylation and nuclear exclusion of FOXO1 in ovarian GCs. ( a ) The expression of FSHR, FOXO1, and phosphorylated FOXO1 (p-FOXO1) corresponding to the sites Thr 24 , Ser 248 , and Ser 311 in cultured GCs under treatment with FSH or/and LMB was determined via Western blotting by using the anti-FSHR, anti-FOXO1, and anti-p-FOXO1 corresponding to Thr 24 , Ser 248 , and Ser 311 , respectively. The β-actin was used as a loading control. All blots were cropped, and the gels were run under the same experimental conditions. ( b ) The expression levels of FSHR protein in cultured GCs under FSH or/and LMB treatment by Western blotting, as shown in ( a ). ( c ) Expression levels of FOXO1 under treatment the same as ( b ). ( d ) The expression levels of p-FOXO1 corresponding to the site Thr 24 . ( e ) The expression levels of p-FOXO1 corresponding to the site Thr 248 . ( f ) The expression levels of p-FOXO1 corresponding to the site Thr 311 . For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05. ( g ) The subcellular localizations of FOXO1 protein in cultured GCs under FSH or/and LMB treatment by immunofluorescence staining method. The red line segment at the bottom right corner of the image is the scale bar (Leica, 400×; scale bar = 5 µm).

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control, Immunofluorescence, Staining

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: The roles of activated PI3K/Akt signaling in the FSH-induced phosphorylation of FOXO1 in GCs. ( a ) The expression of FSHR and phosphorylated Akt (p-Akt) in cultured GCs under treatment with FSH or/and Ly294002 was determined by Western blotting. ( b ) The expression levels of FSHR protein in GCs under FSH or/and Ly294002 treatment are shown in ( a ). ( c ) The expression levels of p-Akt protein in GCs under the same treatment as ( b ). ( d ) The expression of FOXO1, p-FOXO1 corresponding to the phosphorylation site, Se r248 or Ser 311 , in cultured GCs under treatment with FSH or/and Ly294002 and LMB was examined by Western blotting, respectively. ( e ) The expression levels of FOXO1 protein in cultured GCs. ( f ) The expression levels of pFOXO1 corresponding to the Ser 248 site. ( g ) The expression levels of pFOXO1 corresponding to the Ser 311 site. ( h – j ) The expression levels of PKACA and acetylated FOXO1 (Ac-FOXO1) in cultured GCs under treatment with FSH or/and KH7 were tested by Western blotting, respectively. ( k – m ) The expression levels of the pFOXO1 proteins corresponding to the site, Ser 248 or Ser 311 , in cultured GCs under treatment with Ly294002 or/and TSA were determined by Western blotting, respectively. β-actin was used as a loading control. All blots were cropped, and the gels were run under the same experimental conditions. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Phospho-proteomics, Expressing, Cell Culture, Western Blot, Control

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Effects of FSH-induced FOXO1 nuclear exclusion on GC proliferation and apoptosis via PI3K/Akt signaling pathway. ( a ) The subcellular localizations of FOXO1 protein in cultured GCs under FSH or/and Ly294002 treatment by immunofluorescence assay. The red line segment at the bottom right corner of the image is the scale bar (Leica, 400×; scale bar = 5 µm). ( b ) The expression levels of BCL mRNA in cells under FSH or/and LMB treatment by RT-qPCR assay. ( c ) The expression levels of CASP3 mRNA under the same treatment as ( b ). ( d ) The expression levels of CCND1 mRNA. ( e ) The expression levels of PCNA mRNA. ( f – k ) The GC proliferation and apoptosis under FSH or/and LMB treatment by flow cytometry assay. All data are presented as means ± SEM. n = 3. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Cell Culture, Immunofluorescence, Expressing, Quantitative RT-PCR, Flow Cytometry, Control

Journal: Cells

Article Title: FSH-Induced Nuclear Exclusion of FOXO1 Mediated by PI3K/Akt Signaling Pathway in Granulosa Cells Is Associated with Follicle Selection and Growth of the Hen Ovary

doi: 10.3390/cells14231864

Figure Lengend Snippet: Crosstalk of PI3K/Akt and P62/Keap1/Nrf2 pathways in regulating GC proliferation and apoptosis mediated by FOXO1. ( a ) Expression levels of P62 mRNA under FOXO1 overexpression or/and P62 knockdown examined by RT-qPCR assay. NC: negative control, OE: overexpression, SR: siRNA. The mRNA expression was normalized to that of the 18S rRNA gene; the values of the bar graphs represent the mean ± SEM of 3 hens ( n = 3) from a representative experiment. ( b ) The expression levels of Keap1 mRNA under the same condition as ( a ). ( c ) The expression levels of Nrf2 mRNA under the same conditions as ( a ). ( d ) The expression levels of p62 mRNA under 740-Y-P or/and LMB treatment by RT-qPCR assay. ( e ) The expression levels of BCL2 mRNA under 740-Y-P treatment or/and P62 knockdown by RT-qPCR. ( f – h ) The expression level results of CASP3 , CCND1, and PCNA mRNA under the same conditions as ( e ). ( i – m ). GC proliferation and apoptosis under treatment with FSH or/and P62 knockdown by flow cytometry assay. All data are presented as the means ± SEM. n = 3. For each group, the superscript symbol above the bar indicates that the difference was significant compared to the control group, ** p < 0.01, * p < 0.05.

Article Snippet: Cells were set up as follows: control (PBS, Procell, Wuhan, China); FSH (Follicle-stimulating hormone, 10 ng/mL, 12 h; Selleck Chemicals, Houston, TX, USA); LMB (Leptomycin B, 10 ng/mL, 12 h; Targetmol, Boston, MA, USA) [ ]; Ly294002 (Ly294002, 25 μM, 2 h; Targetmol, Boston, MA, USA) [ ]; 740-Y-P (740-Y-P, 20 μM, 24 h; Houston, TX, USA) [ ]; KH7 (KH7, 25 μM, 1 h; Targetmol, Boston, MA, USA) [ ]; TSA (Trichostatin A, 1 μM, 2 h; Targetmol, Boston, MA, USA).

Techniques: Expressing, Over Expression, Knockdown, Quantitative RT-PCR, Negative Control, Flow Cytometry, Control