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

Journal: Journal of Virology

Article Title: Venezuelan Equine Encephalitis Virus Capsid Protein Forms a Tetrameric Complex with CRM1 and Importin ?/? That Obstructs Nuclear Pore Complex Function ▿

doi: 10.1128/JVI.02554-09

Figure Lengend Snippet: Identification of the NES in VEEV capsid protein. (A) Sequence alignment of H68 peptides derived from different New World alphaviruses with a consensus NES sequence (NES), functional NES of cyclic-AMP (c-AMP)-dependent kinase inhibitor (PKI) (51) and supraphysiological NES (S1) (13). The sequence alignment was performed using ClustalW. Conservative hydrophobic amino acids in the NES are shown in red. Amino acids that are identical between different members of the New World alphaviruses are shaded in green. Asterisks indicate identical residues; colons indicate conserved substitutions; periods indicate semiconserved substitutions. WEEV, western equine encephalitis virus. (B) BHK-21 cells were coinfected with packaged replicons expressing VEEV capsid-GFP and 4×Tomato-3×NLS and treated with leptomycin B at 2 h postinfection as described in Materials and Methods. The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope. Scale bars, 20 μm. (C) (Top) Amino acid alignments of mutated H68-GFP fusions. Conserved hydrophobic amino acids are indicated in red, and introduced point mutations are shown in blue. (Bottom) Representative confocal images of cells expressing 4×Tomato-3×NLS and mutant proteins. Scale bars, 20 μm. (D) Box plot demonstrating the nuclear/cytoplasmic distribution of 4×Tomato-3×NLS when expressed alone and the distribution of the same protein when coexpressed with mutant peptide-GFP. A small but statistically significant increase in nuclear-reporter accumulation was detected for single-amino-acid mutants. The double mutant no longer affected nuclear accumulation of 4×Tomato-3×NLS. (E) Box plot demonstrating the nuclear/cytoplasmic distributions of H68-GFP and its mutants. The increase in nuclear accumulation of the mutant-peptide-GFP fusions was correlated with their reduced efficiencies in nuclear import inhibition. The P values were calculated using the Mann-Whitney test (n = 30 for all experiments).

Article Snippet: The images were acquired after 4 h of leptomycin B treatment on a Zeiss LSM510 confocal microscope.

Techniques: Sequencing, Derivative Assay, Functional Assay, Western Blot, Expressing, Microscopy, Mutagenesis, Inhibition, MANN-WHITNEY

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Ankle1 shuttles between nucleus and cytoplasm. a Schematic representation of Ankle1’s domain organization depicting predicted ankyrin repeats, the LEM domain and a GIY-YIG nuclease domain. Putative nuclear export sequences (NES1, NES2) and nuclear localization sequences (NLS1, NLS2), identified in silico are indicated. b Immuno-fluorescence analysis of ectopic Ankle1-V5 in U2OS cells without or following a 3 h treatment with 50 nM leptomycin B, an inhibitor of CRM1-mediated export. Cells were stained with antibodies to V5, and DNA with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: In Silico, Fluorescence, Staining

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Localization of Ankle1 fragments containing different domains and export and import signals. Localization of GFP-tagged Ankle1 truncation constructs ectopically expressed in U2OS ( a ) and HeLa ( b ) cells was determined by confocal fluorescence microscopy. Molecular weights and schematic representations of domain organization of respective truncation protein constructs are indicated. Cells were fixed after 3 h of mock or leptomycin B treatment. DNA was counterstained with DAPI. Scale bar: 10 μm

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Construct, Fluorescence, Microscopy

Journal: BMC Cell Biology

Article Title: Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals

doi: 10.1186/s12860-016-0102-z

Figure Lengend Snippet: Mutation analyses identify NES2 and NLS2 as the predominant sequences controlling nucleo-cytoplasmic shuttling of Ankle1. a , b , d , e U2OS cells were transiently transfected with Ankle1-V5 carrying point mutations in NLS or NES sequences and either mock-treated or treated with leptomycin B for 3 h and processed for confocal immunofluorescence analyses using antibodies to V5 and DAPI to detect DNA. Scale bars: 10 μm. c Mean fluorescence intensities in nuclei and cytoplasm of cells expressing wild-type Ankle1-V5, Ankle1-NES1mut-V5 or Ankle1-NES2mut-V5 were measured in original unprocessed digital images prior to contrast/brightness adjustment and nucleus to cytoplasm signal ratios were calculated. Data were obtained from three independent experiments and analyzed using Student’s t -test. Ankle1-NES1, P = 0.002; Ankle1-NES2, P = 5.4E-21; n = 50; 15–17 cells each from three independent experiments

Article Snippet: Inhibition of CRM1-dependent nuclear export was performed using 10 ng/mL leptomycin B (Enzo Life Sciences, Lausen, Switzerland) for three hours.

Techniques: Mutagenesis, Transfection, Immunofluorescence, Fluorescence, Expressing