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Figure S1. (A) Two consecutive sections of a breast tumour TMA were stained with the same concentration of specific FMRP affinity purified antibodies (Ferrari et al, 2007) or the corresponding affinity-purified pre-immune IgGs. Shown are representative images. The breast tumour core was scored positive for FMRP using the specific antibody and negative with the pre-immune purified IgG. (B) Western blot analysis of FMRP expression in WT and Fmr1 KO mouse brain using specific Fmrp/FMRP antibodies (Ferrari et al, 2007). The signal appears clear in WT extracts and absent in Fmr1 KO extracts, showing specificity. (C) Immunohistochemistry detection of FMRP in formalin-fixed and paraffin-embedded mouse brains using in parallel the specific FMRP antibodies (affinity-purified IgGs) and the affinity purified IgGs from the preimmune serum. The regions in the hippocampus known to contain FMRP are strongly highlighted showing the specificity of the anti-FMRP antibodies in immunohistochemistry. Scale bars: 500 μM (left panels) and 100 μM (right panels). (D) FMRP scoring on normal and tumour breast tissues on TMAs. Representative images of IHC for FMRP performed on different human breast tissues that show the range of FMRP staining (low = ≤1 to very high = >1). Scale bars = 100 μm.

Figure S2. Kaplan–Meier curves of metastasis free probability. Left curve, EMC-344, MSK-99, NKI-295 datasets correlating FMR1 mRNA levels with the probability of having metastasis to distal organs (lung excluded). FMR1 low levels are indicated in red while high levels in blue. Middle curve: same as the left curve using only the NKI-295 node-negative dataset. Right curve: same as left curve using only NKI-295 node positive dataset. Pts = total number of patients analysed (n = 569). p-values were calculated using the Log-rank test. n = number of patients; events = number of patients with distal metastasis. High levels of FMR1 mRNA do not correlate with an increased probability of lung metastasis to distant organs.

Figure S3. (A) FMRP expression in mouse breast tumour cell lines (4T1, TS/A, n = 5, p < 0.01). FMRP levels were analysed by Western blot using specific FMRP antibodies (Ferrari et al, 2007) and normalized for Vinculin and Comassie staining (not shown). Quantification is reported in the histogram as ratio FMRP/Vinculin where FMRP levels in 4T1 cells were considered 100%. (B) Silencing of Fmr1 in 4T1 cells. 4T1 cells were transiently transfected with five different shRNAs as well as with a scrambled shRNA (see Methods). Fmrp levels were detected by Western blot, normalized to Vinculin and values reported in the histogram as ratio Fmrp/Vinculin where Fmrp levels in CTR cells were considered 100%. (C) 4T1 cells silenced for FMRP. Cells were stably transduced with different combination of three shRNAs against the Fmr1 gene. Fmrp levels were detected by Western blot and normalized to Vinculin or total proteins (Coomassie staining, data not shown). Fmrp levels are expressed as a ratio to control cells (100%). (D) Same as panel (C) for the TS/A cell line. (E) Primary tumour growth after orthotopic injection of control (CTR) and Fmr1 silenced 4T1 cells in WT syngenic mice (n = 12, p = 0.09, two-way ANOVA applying the Bonferroni correction). The graph represents tumour volume as a function of time after the injection. (F) Primary tumour growth after orthotopic injection of control (CTR) and Fmr1 silenced TS/A cells in WT syngenic mice (n = 13, p = 0.23, two-way ANOVA applying Bonferroni correction). The graph represents tumour volume as a function of time after the injection.

Figure S4. (A) IHC analysis of Fmrp expression in primary tumours generated by orthotpic injection of CTR or Fmr1 shRNA TS/A cells. Shown is an overview (left and right panels) or a detail (middle panel) of FMRP-stained primary tumours created after injection of control (left and middle panels) or Fmr1-silenced TS/A cells (right panel). The tumour margin is marked by a black arrow and also shown in the enlarged detail. (B) Fmr1 mRNA expression levels in tumours generated with shRNA versus Fmr1 shRNA (n = 5, p < 0.05, Student's t-test). (C) FMRP levels in normal and primary tumour tissues by Western blotting analysis. Lanes 1–3, protein extracts from breast tissues (mammary fat pad); lanes 4–11, 8 different murine breast tumours. FMRP levels were analysed by Western blot using specific FMRP antibodies (Ferrari et al, 2007) (left upper panel), the signal was normalized for Coomassie staining of the membrane (left lower panel). The right panel reports the quantification of the signals intensity (p < 0.01, Student's t-test). (D) Lung metastasis generated after orthotopic injection of control or Fmr1 silenced TS/A cells were analysed for FMRP expression levels. Black arrows on left panels point to the metastasis, one of them is enlarged on the right panel. Scale bars: 200 µm (left panels) and 20 µm (right panels).

Figure S5. (A) Fmrp and GFP expression in 4T1 cells. 4T1 cells were transiently transfected using a recombinant plasmid containing the Fmr1 shRNA (3 or 4) under the U6 promoter and the GFP in a separate transcription unit. Fmrp was detected by immunofluorescence. Scale bars: 20 µm. (B) Cancer cell survival in the bloodstream. Mice were injected intravenously (I.V.) with Fmr1 silenced and control 4T1 cells, blood was collected 6 and 24 h after the injection (n = 5), and analysed as in panel B. (C) Lung metastasis formation in I.V. injected mice (n = 5).

Figure S6. (A) 4T1 cell morphology after Ca2+ deprivation. Upper panels: phase-contrast images of 4T1 cells expressing CTR shRNA and CTR vector after 18 min of EDTA treatment, lower panels: 4T1 cells expressing Fmr1 shRNA and GFP-FMR1. All insets show time = 0. Scale bar: 50 µm. (B) Cell area in CTR and Fmr1 shRNA cells (n = 18.590, p < 0.01) (C) Transendhotelial migration assay of CTR shRNA and Fmr1 shRNA 4T1 cells (n = 5, p < 0.01). Histograms show the quantification of migrating cells. (D) 3D tumour cell spheroids invasion assay. 4T1 cells, stably transduced with CTR shRNA and Fmr1 shRNA 4/5 and 3/5 (unpublished observations), were embedded in a collagene type I gel and imaged after 24 h. White dotted line indicates the spheroid body. Left panels indicate the quantification of the different parameters analysed (n = 15 per condition, p < 0.001, p < 0.001, Student's t-test). Scale bar = 200 µm.

Figure S7. FMRP immunoprecipitation from 4T1 cells with specific FMRP antibodies (Ferrari et al, 2007) and affinity purified pre-immune IgGs visualized by Western blotting.

Figure S8. Immunohistochemistry of FMRP, E-cadherin and Vimentin performed on human non-metastatic (BC) and metastatic (mBC) breast cancer (upper panels). Lower panel shows a correlation between FMRP, E-cadherin and Vimentin in BC and mBC (n = 14).

Figure S9. mRNA stability assay in 4T1 CTR and Fmr1 shRNA cells. RNA was isolated at the indicated time points after Actinomycin D treatment and the stability of Fn1, Jag1, Mmp9, Serpine1 and Egfr mRNAs was analysed by RT-qPCR (n = 5, * p < 0.05, ** p < 0.01).

Figure S10. Translational efficiency analysis in 4T1 CTR and Fmr1 shRNA cells. Quantification of the translational efficiency of Histone H3.3, Mtap1b, Cav2, Dsp, Krt14 and Mitf mRNAs reported as ratio of P over NP (2−[ΔCt(P)–ΔCt(NP)]) (n = 4, * p < 0.05, ** p < 0.01).

Figure S11. (A) E-cadherin and Vimentin mRNA levels in 4T1 CTR and Fmr1 siRNA (Ambion (ID 10919) cells detected by RT-qPCR (n = 3, p < 0.05). (B) Translational efficiency analysis in 4T1 CTR and Fmr1 siRNA cells. Upper panel, polysome-mRNPs distribution on a sucrose gradient. Low left panel, fractions 1–5 corresponding to translating polysomes (P) and fractions 6–10 corresponding to silent mRNPs (NP) were pooled. Low right panel, quantification of the translational efficiency of Histone H3.3, E-cadherin and Vimentin mRNAs reported as ratio of P over NP (2−[ΔCt(P)–ΔCt(NP)]) (n = 3, p < 0.01).

Table S1.A TMAs used to detect FMRP expression

B FMRP expression on the TMA

Table S2. Clinical and pathological informations of the consecutive cohort of breast cancer patients (IFOM-Milan).

Table S3. Clinical and pathological informations of the breast cancer samples (UZLeuven).

Table S4.A Cancer occurring in the Cohort of 241 women with FMR1 pre-mutation and full-mutation.

B Cancer incidence in the Cohort of 199 women with FMR1 pre-mutation and full-mutation

Table S5. FMRP target mRNAs in breast cancer cells.

Table S6. Clinical and pathological informations of the breast cancer samples (San Giovanni Rotondo Hospital).

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