Foxl2, Sumo-1 and Ubc9 are expressed in mouse ovary.

<p><i>A.</i> Pias1, Ubc9 and Sumo-1 are expressed in 4 week mouse ovaries: 4-week old mouse ovaries were lysed and analysed by immunoblotting with anti- Pias1, Ubc9, and Sumo-1 specific antibodies. 50 ug of protein were loaded in each lane. <i>B.</i> Co-Localization of Foxl2, Sumo-1 and Ubc9 in 4 week-old mouse ovary: Hybridization with anti-Foxl2 (red) and anti-Ubc9 (green) or anti-Sumo-1 (green) antibodies. Yellow spots in the 40× magnification show the co-localization of Foxl2 with Ubc9 and Sumo-1, especially in primordial and primary follicle granulosa cells. Immunofluorescence analysis was performed using a Leica DMIRE2-TCS-SL Confocal Laser Scanning microscope (from 488 to 633 excitation wavelength).</p

FOXL2 interacts with sumoylation machinery, is sumoylated and co-localize with SUMO-1 and PIAS1.

<p><i>A., B.</i> FOXL2 interacts with PIAS1 and UBC9: COS-7 cells were co-transfected with pCRUZ-HA-FOXL2 and pCRUZ-myc-PIAS1 (A), or with pCRUZ-myc-FOXL2 and pCRUZ-HA-UBC9 (B) or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody and analysed by western blotting with anti-FOXL2, anti-PIAS1 and anti-UBC9 antibodies. Mock IP consisted in an immunoprecipitation with only protein-A beads, without antibody. Expression of all proteins was also analysed in total lysates (input). <i>C.</i> FOXL2 is sumoylated in transfected COS-7 cells: COS-7 cells were transfected with pCRUZ-myc-FOXL2 (0.5 µg) alone or with pCRUZ-HA-SUMO-1 (4.5 µg), or with HA and myc empty vectors (−). Lysates were immunoprecipitated with anti-myc agarose conjugated antibody, and analysed by western blotting using anti-FOXL2 antibody (upper panel) or anti-SUMO-1 antibody (lower panel). Both antibodies recognized band(s) of about 105-160-kDa indicated by brackets (<b>[</b>), not present in the transfection of FOXL2 alone. <i>D.,E.</i> FOXL2 is sumoylated <i>in vivo</i> in physiological conditions: (D) Immunoprecipitation was done using anti-FOXL2 antibody on α-T31 cell lysate and the eluate analyzed by western blotting using anti-FOXL2 antibody (left panel) or anti-SUMO-1 antibody (right panel). Both antibodies recognized band(s) of about 105–160-kDa indicated by brackets (<b>[</b>). The asterisk indicates the FOXL2 signal from the previous hybridization. (E) 4-week old mouse ovaries were lysed with or without NEM, 60 µg of protein was loaded on SDS PAGE, electrophoresed, and then immunoblotted. Western blotting with an anti-Foxl2 antibody showed a 45 kDa band corresponding to native Foxl2 and a slower migrating band of about 105–160 kDa, also recognised by anti-Sumo-1 antibody (not shown). <i>F.</i> FOXL2 and SUMO-1 co-localize in the nucleus: pCRUZ-myc-FOXL2 and pCRUZ-HA-SUMO-1 were co-transfected into COS7 cells, and immunofluorescence was performed using anti-myc and anti-HA antibody. In red (Alexa 633) is shown FOXL2, in green (Alexa 488) SUMO-1. The yellow colour indicates co-localization, and is particularly seen in spots resembling PML bodies. <i>G.</i> Wild type FOXL2 co-localizes with PIAS1 in the nucleus: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. The intracellular distribution of FOXL2 (red) and PIAS1 (green) was detected by indirect immunofluorescence with mouse anti-myc and rabbit anti-HA primary antibodies and Alexa Fluor 633 anti-mouse and Alexa Fluor 488 anti-rabbit secondary antibodies. <i>H.</i> PIAS1 enhances FOXL2 sumoylation: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 and pCRUZ-HA-PIAS1. And lysed with or without NEM. Lysates were immunoblotted with anti-FOXL2 antibody and densitometric analysis was performed on sumoylated band using Image J software. Densitometric analysis is reported compared to that in lane 1.</p

Identification and characterization of putative FOXL2 sumoylation sites.

<p><i>A.</i> Mutations of K114, K150 and KFULL results in a strong reduction of the largest sumoylated band: COS 7 cells were transfected with pCRUZ-myc-FOXL2 wild type or in the mutated forms, with and without pCRUZ-HA-SUMO-1. 48 h after transfection cells were lysed and 50 ug of protein were analysed by western blotting with anti-myc antibody. The largest sumoylated band is absent in K114, K150, and K-FULL. <i>B.</i> Mutation of K114 or K150 affects FOXL2 cellular localization: pCRUZ-myc-FOXL2 (WT, K25R, K87R, K114R, K150R, FULL) and pCRUZ- HA-SUMO-1 were co-transfected in COS-7 cells. The intracellular distribution of FOXL2 (red) and SUMO-1 (green) was detected by indirect immunofluorescence with mouse anti-myc and rabbit anti-HA primary antibodies and Alexa Fluor 633 anti-mouse and Alexa Fluor 488 anti-rabbit secondary antibodies. Yellow spots show the overlap (co-localization) of the two signals. <i>C., D.</i> Mutation of putative sumoylation sites results in changes of FOXL2 transcriptional activity: (C) COS-7 cells were co-transfected with pGL3-StAR luciferase reporter vector and wild-type or mutated FOXL2. Reporter Luciferase activity expressed from the StAR promoter was measured. Luciferase activity is reported as relative activity. It is the mean of at least 4 independent experiments, normalized to the reporter gene <i>Renilla</i>, encoded by a pTRLK vector, used as an internal control, and compared to promoter activity alone. Significance was estimated using Student's t-Test, (*) p<0.01, (**)<0.001, (***)<0.0001, (****)<10⁻⁷<i>(D)</i> COS-7 cells were co-transfected with pGL3-StAR luciferase reporter vector, and pCRUZ-myc-FOXL2 wild-type or pCRUZ-myc-FOXL2-KFULL, with or without pCRUZ-HA-SUMO-1. The mutation of all 4 putative sumoylation sites (FOXL2-KFULL) leads to a level of FOXL2 inhibition independent of the addition of SUMO-1, whereas wild-type mediated inhibition is increased by SUMO-1. Significance was estimated using Student's t-Test and reported in the chart.</p

FOXL2 stability is increased by SUMO-1 and is not dependent on proteasome-mediated degradation.

<p><i>A.</i> Sumoylation regulates FOXL2 stability: COS-7 cells were co-transfected with pCRUZ-myc-FOXL2 (0.5 µg) and increasing amounts of pCRUZ-HA-UBC9 or pCRUZ-HA-PIAS1(3.5–4.5 µg) or pCRUZ-HA-SUMO-1 (4.5 µg). Lysates treated with and without NEM were analysed by immunoblotting with anti-FOXL2 antibody. The bracket shows slower migrating bands of about 105–160 kDa. Densitometric analysis was performed on sumoylated band using Image J software and is reported compared to that in lane 1. <i>B.</i> FOXL2 stabilization is mediated by SUMO-1 in a dose-dependent manner: COS7 were transfected with 500 ng of pCRUZ-myc-FOXL2 and increasing amounts of pCRUZ-HA-SUMO-1 (0, 1, 2, 3, 4 µg). Immunoblotting against myc shows that FOXL2 increases with the augmentation of SUMO-1. In the lower panel anti-actin is used as loading control. <i>C.</i> FOXL2 stability is not dependent on ubiquitination and proteasome-mediated degradation: COS7 cells were transfected with pCRUZ-HA-FOXL2 alone and with pCRUZ-HA-SUMO-1 in quadruplicate. After 12 h of transfection, cells were lysed (t0) or treated with cycloheximide (inhibitor of protein biosynthesis), cycloheximide and MG132 (proteasome-inhibitor) or neither. After 36 h cells were lysed and 50 ug of protein were blotted and hybridization performed with anti-myc antibody.</p

PIAS1 and UBC9 in contrast to FOXL2, activate <i>StAR</i> promoter.

<p>COS-7 cells were co-transfected with pGL3-StAR luciferase reporter vector, and increasing amounts (1.5, 3 µg) of PIAS1 or UBC9, with or without FOXL2 (1 µg). Relative luciferase activity is compared to that of promoter activity alone. Luciferase activity is reported as relative activity, as the mean of at least 4 independent experiments, normalized to the reporter gene <i>Renilla</i>, encoded by pTRLK vector, used as an internal control, and compared to promoter activity alone. Statistical significance was estimated with Student's t-Test, p-values <0.05 are significative, (*) p<0.01, (**) p<0.001.</p

Sumoylation sites prediction.

<p><i>A.</i> The SUMOplot™ Analysis Program (<a href="http://www.abgent.com/sumoplot.html" target="_blank">http://www.abgent.com/sumoplot.html</a>) was used to predict and score sumoylation sites within the FOXL2 protein (upper panel). <i>B.</i> Sequence alignment of the FOXL2 region surrounding the putative sumoylation sites using the ClustalW2 Web based tool (right panel). In grey is highlighted the forkhead domain. In pink the SUMO consensus sequences K25, K87, K114, K150, with higher score, in blue those with lower scores (K36, K48, K54).</p

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary-1

or testis by at least two-fold. Functional categories of genes were assembled from GO annotations and from PubMed.<p><b>Copyright information:</b></p><p>Taken from "Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary"</p><p>http://www.biomedcentral.com/1741-7007/6/24</p><p>BMC Biology 2008;6():24-24.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426674.</p><p></p

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary-4

Nes in ovary; (C) three-dimensional heatmap of expression of the same genes in ovary; (D) experimental design for males; (E) two-dimensional heatmap of expression of 3000 most significant genes in testis (819 genes overlapped with ovary); (F) three-dimensional heatmap of expression of the same genes in testis.<p><b>Copyright information:</b></p><p>Taken from "Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary"</p><p>http://www.biomedcentral.com/1741-7007/6/24</p><p>BMC Biology 2008;6():24-24.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426674.</p><p></p

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary-3

Hs in ovary and testis, respectively. (C), (D) Proportion of genes that were over-expressed either in ovary or testis, respectively, by at least two-fold among genes that were increased or decreased by CR at specific age from 6 to 24 months.<p><b>Copyright information:</b></p><p>Taken from "Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary"</p><p>http://www.biomedcentral.com/1741-7007/6/24</p><p>BMC Biology 2008;6():24-24.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426674.</p><p></p

Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary-2

or testis by at least two-fold. Functional categories of genes were assembled from GO annotations and from PubMed.<p><b>Copyright information:</b></p><p>Taken from "Effects of aging and calorie restriction on the global gene expression profiles of mouse testis and ovary"</p><p>http://www.biomedcentral.com/1741-7007/6/24</p><p>BMC Biology 2008;6():24-24.</p><p>Published online 3 Jun 2008</p><p>PMCID:PMC2426674.</p><p></p