RepoMan localisation across the cell cycle.

(A) Immunofluorescence analysis of endogenous RepoMan in HeLa cells along the cell cycle. CDCA2 antibody was used to detect endogenous RepoMan. DNA was stained with DAPI. Scale bar 5μm(B) Time lapse of YPF-RepoMan in cells treated with thymidine for 24h and release in normal media. DNA was stained with sirDNA. YFP-RepoMan in Green and DNA in Red Scale bar 5μm

Endogenous RepoMan interacts with EGFP-PHD1.

(A) Asynchronous HeLa cells were transiently transfected with 1µg of EGFP-PHD1 expression vector and after 48h were fixed with 4% PFA and subjected to immunofluorescence using CDCA2 and GFP antibodies (endogenous RepoMan and EGFP-PHD1 respectively). DNA was stained with DAPI. The Images show the localization of endogenous RepoMan and EGFP-PHD1 along the cell cycle: interphase, prometaphase, metaphase, and anaphase. Scale bars represent 5 µm. (B) Short sequence similarity between HIF1α and RepoMan around prolines 564 and 604 (PYIP, PSIP) respectively. (C) Co-immunoprecipitation between EGFP-PHD1 and endogenous RepoMan. Asynchronous HeLa cells were transiently transfected with EGFP-PHD1. After 48h cells were lysated and subject to immunoprecipitation using GFP trap magnetic beads and analysing by western blot.

PHD1 regulates phosphorylation of H3T3 during prometaphase.

(A) Schematic model of the role of PP1-RepoMan during prometaphase. (B) Hela cells were transfected with sicontrol, or siRNA RepoMan. Cells were arrested in prometaphase with nocodazole 100ng/ml for 16h and released from the arrest for 1h in normal media before fixation and stained with pH3T3 and DAPI. (C) Cells were treated as in (B) and harvested for western blot analysis (D) Asynchronous or prometaphase arrested HeLa cells were treated with FG4592 50 µM for 2h or DMSO. In case of mitotic cells, they were treated 1h before the release and during the release for another hour. Cells were lysed and subject to western blot (E) Asynchronous or prometaphase arrested HeLa cells were treated with 100µM Fumarate for 1h. In case of mitotic cells, they were treated for 1h during the release. Cells were lysed and subject to western blot. (F). Immunofluorescence images of HeLa cells arrested in prometaphase. HeLa cells were transfected with sicontrol, siPHD1, siPHD2 (as in B) Prometaphase arrested cells were release for 1h before fixation. Images scale bars represent 5µm. (G) Graph display the normalized pH3T3 intensity of 30 cells per condition of 3 independent experiments. pH3T3 intensity of each condition (siPHD1 and siPHD2) was normalized to the intensity values of pH3T3 in the sicontrol. The average of 3 independent experiments is shown. Unpair t test p<0.0001 Control vs siPHD1 (H) Western blot analysis of HeLa cells treated as in (F) and including asynchronous cells transfected with the control siRNA.

P604 in RepoMan is required for correct localisation and function in mitosis.

(A) Western blot analysis of HeLa-YFP-RepoMan wt or the P604A mutant induction in presence of 1 µg/ml doxycycline. Cells were transfected with the siRNA of RepoMan and after 24h cells were induced with doxycycline for another 24h before harvested. Blots were developed with GFP, RepoMan and actin antibodies. (B) Immunofluorescence of HeLa-YFP-RepoMan wt or the P604A synchronized in prometaphase with nocodazole after siRM knockdown. Dox was used to induce the expression of the YFP proteins. Anti phH3T3, GFP antibodies were used, and DNA was stain with DAPI. (C) Quantification of (B) in cells treated with siRM and induced or not with doxycycline. The graph displays the distribution of phH3T3 on prometaphase cells. Graph represents the percentage of cells showing phH3T3 localization as foci (centromeric) or diffuse localization (along chromosomes arms). Average of 4 independent experiments with a total number of cells 46 for siRM (not induced), 46 for siRM + YFPRMwt and 52 for siRM + YFP-P604A (9-20 cells per condition per experiment).

RepoMan P604 hydroxylation is required for the recruitment of B56ψ in prometaphase cells.

(A) Schematic representation of RepoMan Proline 604 proximity to B56-PP2A binding site (LSPIxE). (B) Proximity ligation assay. Graph represents the number of PLA foci per cell in the indicated conditions (monastrol arrested or asynchronous). Quantified PLA foci are located in the YFP area (RepoMan localisation) of the mitotic cells (phSer10 positive cells) Endogenous RepoMan was knocked down using siRM. Median is shown in red. Statistical analysis was performed applying an unpaired t-test and the p value comparing wt vs mutant upon monastrol treatment is <0.0001. Number of cells per condition in the same order than is shown are: 229,301,237,71,130 and 139. Before PLA reaction cells were fixed with PFA 4% and stained with Anti-GFP + B56ψ antibodies or only GFP antibody was used as a negative control. After PLA reaction cells were stain with anti phH3Ser10 as a mitotic marker (C) Co-immunoprecipitation between YFP-RepoMan wt or P604A mutant with endogenous B56ψ. Endogenous RepoMan was knocked down using siRM. 16h after transfection YFP RepoMan was induced with 1 µg/ml doxycline and cells were synchronized in prometaphase. Cells lysates were subject to immunoprecipitation using GFP trap magnetic beads and analysing by western blot. (D) Western blot analysis of HeLa-YFP-RepoMan wt or P604A / mCherry B56ψ cell line. Cells were depleted of endogenous RepoMan and the indicated YFP-RepoMan variants were induced with dox. GFP, CDCA2 (RepoMan) and actin as a loading control were utilized. (E) Immunofluorescence showing the recruitment of mCherry B56ψ through YFP-RepoMan wt or P604A at ectopic foci on chr 1 in prometaphase arrested cells. GFP, m-cherry and Flag antibodies were used to detect YFP, B56ψ and dCas9 respectively. DAPI show DNA, Graphs represent B56ψ levels (F) and GFP levels(G). Average of 3 independent experiments, 58 (wt) 60 (P604A) cells. Scale bar 5µm. Mann-Whitney test was applied P value <0.0001.

P604 of RepoMan is required for normal mitotic progression.

(A) Schematic representation of the experimental design. (B) Representative Immunofluorescence of YFP-RepoMan arrested in prometaphase with monastrol. Cells were released for 30 min. Monopolar or bipolar cells are shown for either RepoMan wt or P604A. Cells were fixed and stained with MT, GFP antibodies and DAPI shows the DNA. Scale bar 5µm. (C) Quantification of chromosome alignment in bipolar spindles after release of 1h from monastrol arrest. The graph represents the percentage of aligned chromosomes in bipolar spindles respect to the total mitotic cells from 3 independent experiments 100 mitotic cells were analysed per experiment/condition. Unpaired t test unaligned wt vs mut p= 0.0105 (D) Time lapse of YFP-RepoMan wt or P604A cells arrested in prometaphase with monastrol and released into fresh media. (YFP-RepoMan) green and DNA (Magenta). Representative images are shown scale bar 5μm. (E) The graph shows the percentage of normal vs defective mitosis over the of total mitotic cells (58 cells RepoMan wt and 56 cells for RepoMan P604A) from three independent experiments. Unpaired t test p=0.0119 wt vs mut.

Schematic model of RepoMan hydroxylation during prometaphase.

PHD1 hydroxylates RepoMan at proline 604. During prometaphase this modification is important for the binding of PP2A-B56γ to RepoMan. PP2A-B56γ has a crucial role in the loading of RepoMan to chromatin during prometaphase, leading PP1-RepoMan to dephosphorylates phH3T3 from chromosome arms. The resulted enrichment on the phH3T3 at the centromere assures the correct localisation of the CPC (chromosomal passenger complex) important for the proper mitosis progression.