Supplemental Information

From

The Atypical Ubiquitin Ligase RNF31 Stabilizes Estrogen Receptor α and

Modulates Estrogen-Stimulated Breast Cancer Cell Proliferation

Jian Zhu, Chunyan Zhao, Amirhossein Kharman-Biz, Ting Zhuang, Philip Jonsson,

Ning Liang, Cecilia Williams, Chin-Yo Lin, Yichun Qiao, Kazem Zendehdel, Staffan

Strömblad, Eckardt Treuter and Karin Dahlman-Wright,

Supplemental experimental procedures

Supplemental figure legends

1 Supplemental experimental procedures

Chromatin immunoprecipitation (ChIP) assays: ChIP assays were performed essentially as previously described41. MCF-7 cells were treated with vehicle or 10 nM

E2 for 30 minutes before crosslinking. The antibodies used were RNF31 antibody

(ab46322, Abcam), ERα antibody (HC-20, Santa Cruz Biotechnology) and rabbit IgG

(Santa Cruz Biotechnology). Primer sequences are given in Supplementary Table S1.

Immunofluorescence assay: MCF-7 cells were treated with E2 or vehicle for 30 min before being fixed with 4% paraformaldehyde in PBS for 10 min, permeabilized with

0.2% Triton X-100 for 5 min, and blocked by 5% BSA in PBS for 1 h. A rabbit anti-

RNF31 polyclonal antibody and mouse anti-ERα monoclonal antibodies were used, followed by Alexa Flour 647 (Invitrogen) anti-rabbit antibody and FITC-conjugated anti-mouse antibodies (Jackson ImmunoResearch, West Grove, PA). As negative controls, the samples were incubated with the secondary antibodies without primary antibodies. Images were acquired under conditions fulfilling the Nyquist criterion using Nikon A+ laser scanning confocal system with a 60X oil NA1.4 objective and pinhole size of 1.0 Airy Unit. The acquired pictures were further processed and assembled using ImageJ. For pcDNA3-myc-RNF31 and pcDNA3-ERα overexpression in HEK-293 cells, the general steps were as described above. The primary antibodies staining HEK-293 cells were mouse anti-Myc monoclonal antibody and rabbit anti-ERα polyclonal antibody.

Analysis of human breast tumor samples: Human samples were from the Iranian

2 Tumor Bank, Cancer Institute of Iran. Informal consent was obtained from all patients who donated tumor to the tumor bank. The national research Ethics Committee of I.R. of Iran and the Regional Research Ethics committee of Karolinska Institutet approved this study. QPCR was run using RNF31 primers (TaqMan Assay, Hs00215938_m1) and the Ubiquitin C (UBC) gene was selected as internal control.

Supplemental figure legends

Supplementary figure 1

A: Representative diagrams of the FACS analysis shown in Fig. 1C.

B: Representative diagrams of the FACS analysis shown in Fig. 1D

Supplementary figure 2

A: RNF31 depletion decreases ERα protein levels in MDAMB175 and T47D cells.

Cells were transfected with siRNF31 or siControl for 72 h. ERα and RNF31 levels were determined by Western blot analysis. GAPDH was used as internal control.

B: RNF31 depletion, by either of two single siRNAs or a pool, decreases ERα protein levels. Cells were transfected with siRNF31 SMART pool or independently either of two siRNAs from this pool (oligo 1 or oligo 2, respectively) or siControl for 72 h. The efficiency in knocking down RNF31 was decided by q-PCR (Right panel). ERα and

RNF31 levels were determined by Western blot analysis (Left panel). GAPDH was used as internal control.

3 Supplementary figure 3

A: Effect of RNF31 depletion on mRNA levels of two genes that were not regulated by ER (JUND, GAPDH).

B: Effect of RNF31 depletion on luciferase reporter activity. MCF-7 cells were transfected with siRNF31 or siControl together with ERE, LXR or NF-kB reporter construct. LXR luciferase reporter was used as negative control. ERE and NF-kB luciferase reporters were used as positive control. Luciferase activity was measured

48 h after transfection.

C: Effect of RNF31 depletion on LXR transactivation in response to LXR agonist,

GW3965.

D: RNF31 depletion reduces ERα mRNA levels. MCF-7 cells were transfected with siRNF31 or siControl. 48 h after transfection, cells were treated with 10 nM E2 or vehicle for 6 h. The mRNA expression levels of RNF31 and ERα were determined by qPCR from triplicate experiments.

Supplementary figure 4

A: Co-immunoprecipitation assays reveal association between RNF31 and ERα in

HEK293 cells. Cells were transfected with 1ug RNF31 plasmid and 1ug ERα plasmid.

Immunoprecipitation was performed after 24 h.

B, C: The 3 repeats of co-IP assays revealing associations between endogenous

RNF31 and ERα in MCF-7 cells are shown.

Supplementary figure 5

Quantification of data from Fig. 8A. The relative density of each protein was measured by ImageJ.

4 Supplementary figure 6

Localization of RNF31 and ER in HEK293 cells. Cells were transfected with 1ug

Myc-RNF31 and ERplasmid and stained after 24 h. Intracellular localization of

RNF31 (green) and ERα was determined by indirect immunofluorescence. Nuclei

(blue) were stained with DAPI. Shown are representative images.

Supplementary figure 7

RNF31 is not recruited to ER target gene promoters. Chromatin immunoprecipitation assays were performed with RNF31 antibody, ER antibodyor rabbit Ig G and quantified by q-PCR. Result evaluation was done in triplicate.

Supplementary table 1

Real time PCR primers and chromatin immuno-precipitation primers used in the study.

Supplementary table 2

The P value and R2 value from the correlation analysis between RNF31 and ERα target genes. The genes, which were significantly affected in cell line based microarray data, are used as the candidates. TCGA RNA sequence database and KM plot.com database are adopted to do this analysis respectively.

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