Additional file 8: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S6. The repression of MMP13 reduces the anchorage-independent growth capacity of MMT-ETV4-overexpressing cells. a Relative MMP13 mRNA expression in the transiently transfected MMT-siCtrl and MMT-siMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-siCtrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. b Anchorage-independent growth. MMT-ETV4-siCtrl and MMT-ETV4-siMMP13 cells were cultured for 10 days in soft agar. This histogram represents the number of clones counted for experimental time points. Soft agar assays were conducted three times in triplicate. Magnification × 5. Error bars indicate SD. ****P ≤ 0.0001. (PDF 45 kb

Additional file 7: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S5. Expression of ETV4 in MMT-shMMP13-repressing cells. a Relative ETV4 mRNA expression in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl + shCtrl cells was arbitrarily = 1. Error bars indicate SD. The results were not statistically significant. b Western blot analysis of ETV4 protein expression (61 kDa) in the MMT-ETV4 + shCtrl and MMT-ETV4 + shMMP13 cells. GAPDH expression served as the loading control. (PDF 71 kb

Additional file 10: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S8. Metastasis-free survival analysis from the publicly available NKI datasets of breast tumors. a Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 12), High-ETV4 and Low-MMP13 (n = 243), or High-ETV4 and High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. b Metastasis-free survival (MFS) curves for breast tumor patients according to Low-MMP13 (n = 255) or High-MMP13 (n = 9) mRNA levels. ****P ≤ 0.0001. c Metastasis-free survival (MFS) curves for patients with breast tumors according to Low-ETV4 (n = 13) and High-ETV4 (n = 251) mRNA levels. ****P ≤ 0.0001. (PDF 19 kb

Additional file 6: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Figure S4. Expression of MMP13 in MMT cells overexpressing or repressing MMP13. a and b Relative MMP13 mRNA expression in the MMT-Ctrl and MMT-MMP13 (a) or MMT-shCtrl and MMT-shMMP13 cells (b) determined by real-time PCR and normalized to cyclophilin A levels. mRNA expression in MMT-Ctrl cells was arbitrarily = 1. Error bars indicate SD. ****P ≤ 0.0001. c Western blot analysis of MMP13 protein expression (60 kDa) in the MMT-Ctrl and MMT-MMP13 cells. GAPDH expression served as the loading control. d Zymographic analysis of MMP13 protein activity (55 kDa) from the supernatant of MMT-Ctrl and MMT-MMP13 cells. (PDF 72 kb

Additional file 2: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Table S1. Pathological and clinical characteristics of patients in relation to metastasis-free survival (MFS). (PDF 33 kb

Additional file 11: of ETV4 transcription factor and MMP13 metalloprotease are interplaying actors of breast tumorigenesis

Table S2. Multivariate Cox proportional hazards analysis of MFS for MMP13 and ETV4 expression levels in the series of 456 breast tumors. (PDF 42 kb

H7 restores expression of the <i>TP53</i> gene harboring a UGA or UAA nonsense mutation but not of the <i>TP53</i> gene harboring a UAG mutation.

<p>(A) Calu-6 (UGA nonsense mutation at codon 196 of the <i>TP53</i> gene), (B) Caov-3 (UAA nonsense mutation at codon 136 of the <i>TP53</i> gene), or (C) Caco-2 (UAG nonsense mutation at the codon 204 of <i>TP53</i> gene) cells were incubated for 24 h with DMSO as control or with H7 in increasing amounts (from 0.2 to 125 ng/μl) or G418 (from 25 to 1000 ng/μl for (A) and (B) or from 25 to 2000 ng/μl for (C)) before protein extraction and analysis. Western blotting with anti-p53 antibody raised against the N-terminal part of the protein or with anti-CBP80 antibody as a loading control. Truncated (p53 TR) and full-length p53 (p53 FL) are indicated on the right side of each gel and the molecular weight (MW) is shown on the left side of each gel. The three leftmost lanes show twofold serial dilutions of untreated HeLa cell extract. The results presented in Figure are representative of three independent experiments.</p

Validation of the reporter genes used in the screen.

<p>(A) The Fluc-int-WT reporter RNA is spliced. PCR amplification was performed on the Fluc-WT or Fluc-int-WT construct (lanes 1 and 3, respectively), or on the reverse-transcription reaction (RT) performed with extracted RNAs from HeLa cells transfected with the Fluc-int-WT construct (lane 2) in the presence of radioactive dCTP(α³³P). The amplified fragments were electrophoresed through an acrylamide gel to demonstrate that the intron introduced in the firefly luciferase cDNA is efficiently spliced out. The position of each species is indicated on the right side of the gel and a quantification of the relative proportion of pre-mRNA (black box) and mRNA (spotted box) is shown on the right panel. (B) Luciferase expression associated with the constructs used in this study. Firefly luciferase activity normalized by renilla luciferase measured in wells of a 96-well plate containing untransfected HeLa cells, or HeLa cells transfected with pRluc and pFluc-WT, pFluc-int-WT, pFluc-int-UGA, pFluc-int-UAG, or pFluc-int-UAA constructs. (C) Measure of the firefly luciferase (Fluc) and renilla luciferase (Rluc) mRNAs by RT-PCR from HeLa cells transfected with pRluc and pFluc-WT, Fluc-int-WT, Fluc-int-UGA, Fluc-int-UAG, or Fluc-int-UAA constructs. (D) The extent of NMD is shown on a bar plot depicting levels of Fluc-int-PTC RNAs measured by quantitative RT-PCR in the absence and in the presence of cycloheximide (+CHX). The values shown are from two independent experiments. Error bar = S.D., Student t-test: **P<0.01; ***P<0.001.</p

H7 is not an NMD inhibitor.

<p>Calu-6 (A), Caov-3 (B) cells and HeLa cells transfected with pFluc-int-PTC and pIE-MUP (C) were incubated with DMSO, H7 extract at 25 ng/μl, or G418 at 1000 ng/μl for 24 h before RNA extraction and RT-qPCR. Quantification based on two independent experiments is shown. Error bar = S.D., Student t-test: *P<0.05.</p

Cell toxicity in the presence of DMSO, H7, or G418.

<p>Cell proliferation was measured in the presence of DMSO, H7 extract, or G418. Calu-6 cells were counted and treated with DMSO (dashed line), H7 extract (H7) at 25 ng/μl (black line) or G418 at 1000 ng/μl (dotted line) every two days from day 0 (D0) to day 10 (D10). Error bars represent standard deviations. The results presented in the figure are based on two independent experiments.</p