Supplementary Figure S1

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1 Supplementary Figure S1 Luciferase ctivity (%) HeLa E2 OHT Figure S1. SENP2 represses estradiol-dependent transcriptional activity in HeLa cells. HeLa cells were transiently transfected with 5 ng prl-cmv-renilla as an internal control, 2 ng psg5-erα and5ngel+reportervectortogetherwith increasing amounts of expression vector. Cells were treated with vehicle (), estradiol (1-8 M) or OHT (1-8 M) for 16 h. Luciferase values were normalized for transfection efficiency with an internal renilla luciferase control, and expressed as a percentage of the activity obtained with cells only transfected with the reporter gene and treated with vehicle. The figure corresponds to a single assay representative of at least three independent experiments.

2 Supplementary Figure S2 ERα (ESR1) Relative mrn levels E2 E2 E2 ERα GFP ctin Figure S2 : SENP2 does not affect ERα expression. MCF7 cells were transiently transfected with either or expression vectors. Cells were treated for 16 h with vehicle () or estradiol (1-8 M), 24 h after transfection. () mrn extracts were subjected to real time PCR assays, using a ERα probe. Values are expressed as relative units and are plotted as the mean ± SD of three independent experiments. () Whole-cell lysates were then subjected to Western blotting analysis with the anti-gfp, the anti-er or the anti-ctin antibodies.

3 Supplementary Figure S ERβ E PR R C R D ERRγ Luciferase ctivity (%) R1881 Luciferase ctivity (%) Figure S3. SENP2 differentially modulates nuclear receptors-dependent transcriptional activity. HeLa cells were transiently transfected with expression vector, 5 ng of prl-cmv-renilla as an internal control, 2 ng of psg5-erβ and5ngofel+reporter vector (), 2 ng of psg5-pr and 5 ng of pfc31 reporter plasmid (), 2 ng of pcmv-r and5ngofpfc31reporterplasmid(c)or2ngofpsg5-errγ and5ngofel+reporter plasmid (D). Cells were treated with vehicle (), E2 (1-8 M) (), R52 (1-8 M) () or R1881 (1-8 M) (C) for 16 h. Luciferase values were normalized for transfection efficiency with an internal renilla luciferase control, and expressed as a percentage of the activity obtained with cells only transfected with the reporter gene. The figure corresponds to a single assay representative of at least three independent experiments.

4 Supplementary Figure S SENP2 Relative mrn levels ps2 (TFF1) E2 25 PR (PGR) Relative mrn levels Relative mrn levels Relative mrn levels Cyclin D1 (CCND1) * Relative mrn levels Cathepsin D (CTSD)

5 Supplementary Figure S4 C pm-senp2 (ng) SiCtrl SiHDC pm-n8 (ng) SiCtrl SiHDC3 Figure S4. SENP2 represses estradiol-dependent transcriptional activity in T47D cells.. T47D cells were transiently transfected with 15ng of, 5 ng of prl-cmv-renilla as an internal control and 5 ng of EL+ reporter vector. Cells were treated with vehicle () or E2 (1-8 M) for 16 hours. Luciferase values were normalized for transfection efficiency with an internal renilla luciferase control, and expressed as a percentage of the activity obtained with -transfected cells treated with vehicle. The figure is expressed from a single assay representative of at least three independent experiments.. T47D cells were transiently transfected with either the or the espression vector and treated with vehicle () or estradiol (E2 1-8 M). mrn extracts were subjected to real-time PCRassays with primers specific to SENP2, ps2, PR, cyclin D1 and cathepsin D. C. T47D cells were first transfected with 7 pmol sictrl or sihdc3 as indicated and then with 5 ng prl- CMV-renilla and 25 ng L8G5 reporter plasmids, 12.5 ng LexVP16 and 15ng pm-senp2 as indicated. Luciferase values are expressed as percentages of the activity in the presence of sictrl.values are expressed in relative units ans plotted as means ± SD of three independant experiments. Student s t test was used for statistical analysis: *p<.1 and p<.1.

6 Supplementary Figure S5 SENP2 R+/M- ERα R+/M- SENP2 R+/G- HDC3 R+/G- C SENP2 - actin D shctrl shsenp shctrl shsenp2 Dots per cell SENP2 SENP2 Figure S5. In situ proximity ligation assay control experiments.. Each primary antibody against SENP2 and ER was incubated with anti-rabbit PLUS probe and anti-mouse MINUS probe showing no background signal between the two probes and each primary antibody.. Each primary antibody against SENP2 and HDC3 was incubated with anti-rabbit PLUS probe and anti-goat MINUS probe showing no background signal between the two probes and each primary antibody. C. SENP2 rabbit primary antibody and actin mouse primary antibody showed no specific interaction between actin and SENP2. The corresponding probes of anti-rabbit PLUS and anti-mouse MINUS were used in the assay. D. SENP2 expression level was downregulated in shsenp2 MCF7 cells as shown by in situ proximity ligation assay using SENP2 rabbit primary antibody recognized by anti-rabbit PLUS and MINUS probes.

7 Supplementary Figure S6 SENP2 wt (C466S) (W375) ER SUMO1-ER ER SUMO1 GFP SENP2 Input GST SUMO1 wt 35 S SENP2 (C466S) (W375) Figure S6. Characterization of SENP2 catalytic mutants () ERα desumoylation assay. COS7 cells were transfected with 1 µg of expression vector encoding ER, His-SUMO1, Flag-PIS1 and increasing amounts (,5 to 2 µg) of expression vectors encoding or catalytically inactive mutants - SENP2(C466S) and (W375) as indicated. Cells were treated for 16 h with E2 (1-7 M), 24h after transfection. Transfected cells were lysed in the presence of NEM and immunoblotted with the anti- ER monoclonal antibody 1C6 (top), the anti-sumo1 antibody (33-24) (middle) or the anti-gfp antibody (bottom). () Interaction between SENP2 catalytic mutants and SUMO1. GST pull-down assays were carried out using bacterially expressed GST and GST-SUMO1 and 35 S-labeled SENP2, SENP2(C466S) and SENP2(W375). Input represents 1% of the material used for each assay.

8 Supplementary Figure S7 35 E Figure S7. SENP2 represses estradiol-dependent transcriptional activity of non sumoylated ER. HeLa cells were transiently transfected with 5 ng prl-cmvrenilla as an internal control, 2 ng psg5-erα(kv) and 5 ng EL+ reporter vector together with increasing amounts of expression vector. Cells were treated with vehicle () or estradiol (1-8 M) for 16 h. Luciferase values were normalized for transfection efficiency with an internal renilla luciferase control, and expressed as a percentage of the activity obtained with cells only transfected with the reporter gene and treated with vehicle. The figure corresponds to a single assay representative of at least three independent experiments.

9 Supplementary Figure S8 mrn expression (%) sirn Ctrl * HDC3 C * * SiCtrl SiHDC * SiCtrl SiHDC3 * * pm-senp2 pm-n8 Figure S8. HDC3 mediates SENP2 intrinsic repression. () MCF7 cells were transfected with either 7 pmol sictrl or sihdc3. Total RN was extracted 48 h after transfection and 2 µg submitted to reverse transcription. The qpcr was performed with primers specific to HDC3 sequence and data are expressed as the percentage of HDC3 mrn in the presence of sictrl. () MCF7 cells were first transfected with 7 pmol either sictrl or sihdc3 as indicated on day 1 and then with 5 ng prl-cmv-renilla and 25 ng L8G5 reporter plasmids, 12.5 ng LexVP16 and increasing concentration of SENP2-fused Gal4DD expression plasmids on day 2. Luciferase values were expressed as the percentage of activity in the absence of transfected Gal4DD-SENP2 vector. (C). MCF7 cells were transfected as described in except Gal4DD- N8 vectors replaced Gal4DD-SENP2 vectors. Luciferase values were expressed as the percentage of activity in the absence of transfected Gal4DD-N8 vector. Figures are representative of at least three independent experiments. Student t test was used for statistical analysis: *p <.1, p <.1 and *p <.5.

10 Supplementary Figure S9 Proliferation (%) MCF7 shctrl shsenp2 Proliferation (%) MCF7 (W375) ( N8) Figure S9. SENP2 affects MCF7 cell proliferation. Proliferation of MCF7 cells stably transfected or not with shctrl, shsenp2,,, (W375) or (ΔN8) was measured over 6 days, under treatment. Changes in impedance on the E-plate (Roche) were displayed as a cell index value (CI). CI values of quadruplicates were plotted and normalized to the last value before addition of. Relative proliferation values were expressed as a percentage of the normalized CI obtained at day (D). Figures are representative of at least three independent experiments.