A and B) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without FOXL2 and SENP2 overexpression (each 20 ng/well). C and D) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without FOXL2 and UBC9-DN overexpression (each 20 ng/well). Each value is representative of six biological replicates. Error bars represent the standard error of the mean (SEM). Statistical significance in Student t-tests: n.s.: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001. All experiments were repeated at least three times with consistent results.

A and B) Presence of nuclear structures displaying high FOXL2-GFP concentration in a climate of high SUMOylation. Micrographs of COS-7 cells (A) and KGN cells (B) expressing FOXL2-GFP, the tripartite SUMO-FOXL2-GFP fusion or NLS-GFP, as indicated. Left column: GFP. Right column: Hoechst 33342 DNA staining. First line shows a representative “normal” cell, where FOXL2 displays its most common localisation, superimposable to DNA. Second and third lanes show representative cells with “FOXL2 nuclear bodies” where FOXL2 is enriched in subnuclear structures clearly distinct from chromatin structures. Fourth lane (only in A) shows a typical cell stained with nuclear localised GFP. Scale bar: 5 µm. Scale bar is valid for all micrographs. (C) Quantification of cells presenting subnuclear structures in COS-7 expressing SUMO-FOXL2-GFP, FOXL2-WT-GFP or FOXL2-KFULL-GFP with or without overexpression of SUMO1 or SENP2. At least 300 GFP-positive cells were scanned per condition (in groups of 50 to estimate the standard deviations). Error bar: SEM. Letters a, b, c, d refer to statistical categories in a Student's t-test. Conditions with different letters are statistically different with p<0.05.

A) Western blot following anti-FLAG immunoprecipitation of COS-7 lysates and SDS-PAGE. Cells were transfected with a mock vector (pcDNA3.1) or FOXL2-FLAG. Independent immunoblots were performed using anti-FOXL2 C-terminus antibody, mouse anti-acetyl-Lysine (Cell Signaling Technologies), mouse anti-phospho-Serine, anti-phospho-Threonine and anti-phospho-Tyrosine (Sigma-Aldrich), as indicated on the left of each image. B) Schematic representation of FOXL2 post-translational modifications identified by mass spectrometry. Immunoprecipitated FOXL2, in the form of a crude eluate or the isolated 51–52 kD band revealed by Coomasie staining, was digested using trypsin or chymotrypsin and analyzed using ESI-MS-MS with an Orbitrap analyser. Databases were searched with acetylation of lysines and phosphorylation of serines, threonines and tyrosines set as dynamic modification in addition of modification of cysteines by a carbamidomethyl or n-ethylmaleimide groups and oxidation of methionine that were due to sample preparation. FOXL2 sequence is represented by a green and red bars, the green color representing residues present in at least one high confidence peptide, whereas other residues are in red. The positions of the phosphorylated and acetylated residues in the FOXL2 sequence, based on the database search, are represented. Manual inspection revealed that the modification on serine 4 or tyrosine 5 is more likely a sulfation than a phosphorylation. Lower bar represents the position of the forkhead and poly-Alanine domains.

A and B) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without FOXL2-WT or FOXL2-KFULL overexpression (20 ng/well). Each value is representative of six biological replicates. Error bars represent the SEM. Statistical significance in Student t-tests: n.s.: non-significant, *: p<0.05, **: p<0.01, ***: p<0.001 C) Western blot of whole cell extracts of KGN cells transfected with or without FOXL2-V5 and myc-UBC9-DN (each 1 ug/well in a 6 well plate), using pcDNA3.1 as a mock vector. Lic- (expressing a fusion protein His-Tag∶S-Tag∶HSV-Tag∶His-Tag) was used as an internal transfection control (500 ng/well). First panel: FOXL2. Second panel: Lic- (anti-HSV). Third panel: UBC9-DN (anti-myc). Below second panel is indicated the relative intensity of the FOXL2 band compared to the Lic- band. D) Western blot of whole cell extracts of KGN cells transfected for 24 h with or without FOXL2-WT-V5 or FOXL2-KFULL-V5 (2 µg/well in a 6-well plate). Lic- (500 ng) was used as a transfection control. First panel: FOXL2. Second panel: Lic- (anti-HSV). Below second panel is indicated the relative intensity of the FOXL2 band compared to the Lic- band. All experiments were repeated at least three times with consistent results.

A and B) FOXL2 colocalization with PML, SUMO1 and SP100 in nuclear bodies. COS-7 cells overexpressing SUMO-FOXL2-GFP, FOXL2-WT-GFP, FOXL2-KFULL-GFP or NLS-GFP (as indicated, green color) were stained with an anti-PML antibody (panel A, red) or an anti-SUMO1 anti-body (panel B, first three lines, red) In the fourth line of panel B, COS-7 cells overexpressing FOXL2-WT-GFP and SP100A-HSV were stained with an anti-HSV antibody. Representative cells with FOXL2 enriched in nuclear bodies are shown. DNA was stained using Hoechst 33342 (in blue). Scale bar: 5 µm. Scale bar is valid for all micrographs. (C) Interaction between FOXL2 and SP100. SP100-HSV was expressed alone (lane 1) or co-expressed with V5-tagged FOXL2-WT (lane 2) or KFULL (lane 3). FOXL2 was precipitated using anti-V5-conjugated affinity resin for western blot analysis. Upper panel shows a 50 kDa band detected with anti-FOXL2 antibody. Second panel shows a 70 kDa band detected with an anti-HSV antibody. Third and fourth panels display a western blot analysis of the lysates, with anti-FOXL2 and anti-HSV antibodies, respectively. (D) Quantification of cells displaying subnuclear structures in COS-7 expressing SUMO-FOXL2-GFP or FOXL2-WT-GFP with or without overexpression of SUMO1, PML-IV-RFP, PML-V-RFP or SP100A-HSV. At least 300 GFP-positive cells were scanned per condition (in groups of 50 to estimate the standard deviations). Error bar: SEM. Letters a, b, c, d refer to statistical categories in a Student's t-test. Conditions with different letters are statistically different with p<0.05. (E) PML localisation in COS-7 or KGN cells overexpressing PML-V-RFP. Green: anti-PML directed against all isoforms of PML. Red: PML-V-RFP. Blue: DNA stained with Hoechst 33342. In non-transfected cells, PML is mostly nucleoplasmic with a few dots (1–5 in COS-7 cells, 5–20 in KGN cells) indicative of PML NBs. With PML-V-RFP expressed, PML NBs are much enlarged and the proportion of nucleoplasmic PML is decreased. Scale bar: 10 µm. Scale bar is valid for all micrographs. F and G) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without FOXL2 and PML-V-RFP overexpression (each 20 ng/well). Each value is representative of six biological replicates. Error bars represent SEM. Statistical significance in Student t-tests: n.s.: p>0.05, *: p<0.05, **: p<0.01, ***: p<0.001. H) Western blot of whole cell extracts of KGN cells transfected with or without FOXL2-V5 and PML-V-RFP (2 µg/well in a 6-well plate), using pcDNA3.1 as a mock vector. Lic- (expressing a fusion protein His-Tag∶S-Tag∶HSV-Tag∶His-Tag) was used as a transfection control (500 ng). First panel: FOXL2. Second panel: Lic- (anti-HSV). Third panel: PML-V (anti-HA). Below second panel is indicated the relative intensity of the FOXL2 band compared to the Lic- band. All experiments were repeated at least three times with consistent results.

A and B) Luciferase assays in KGN cells transfected with Per2-luc and pSODluc-3340 (40 ng/well), respectively, with or without overexpression of wild-type FOXL2 or various FOXL2 alleles (20 ng/well), each one corresponding to a substitution of a potentially modified residue by an amino-acid simulating the non-modified residue. Each value is representative of six biological replicates. Error bars represent the SEM. Statistical significance in Student t-tests, compared to FOXL2-WT: *: p<0.05. Hyperactivities of FOXL2-S326A on Per2-luc and FOXL2-K366R on pSODluc-3340 compared to FOXL2-WT were seen only in one experiment out of four. C) Quantification of cells presenting subnuclear structures in COS-7 expressing wild-type FOXL2-GFP with or without overexpression of SUMO1 or SENP2, or the same FOXL2-GFP alleles as above. At least 200 GFP-positive cells were scanned per condition (in groups of at least 50 cells to estimate the standard deviations). Error bar: SEM. Statistical significance in Student t-tests, compared to FOXL2-WT: *: p<0.05.