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1 DOI: 1.138/ncb3623 In the format provided by the authors and unedited. a Relative cell numbers to day WB e Mel Moderate KD shctr sh shctr Days sh M r (K) 51Mel b Relative cell numbers to day WB shctr 5 1 ME142 Moderate KD shctr sh Days sh M r (K) c Cell density relative to CTR d mrna level (2- Ct) CTR KD MM57 MM165 Mel624 CTR KD N.D. MM57 MM165 Mel624 shctr sh shctr sh shctr sh - 1 kda - 7 kda α-pep1 α-ab23 α-g17 SKMel28 shctr sh shctr sh shctr sh - 1 kda - 7 kda α-pep1 α-ab23 α-g17 Supplementary Figure 1 Moderate mrna knockdown reduces melanoma cell proliferation. (a-b) Proliferation rate of shctr and sh (moderate KD) 51Mel (a) or ME142 cells (b). Cells were counted every two days during eight or ten days (n = 2 biologically independent experiments in triplicate). protein was detected by Western blot experiments; pictures are representative of three independent experiments. serves as loading control. (c-d) knockdown in two melanoma short-term cultures (MM57 & MM165) and in Mel624 melanoma cell line. Cell density (c) and mrna levels (d) have been evaluated 4 or 7 days after infection (sh) or transfection (si). N.D. for not detected. Each histogram represents the mean of 2 or 3 biologically independent experiments (n=2 for cell density experiments; n=2 for MM57 & Mel624 and n=3 for MM165 for mrna level quantification). (e) knockdown in two melanoma cell lines expressing mrna. Three different antibodies (PEP1, AB23 & G17) were used to confirm the absence of the protein in SKMel28-luc cells. Pictures are representative of three independent experiments. Source data are available in Supplementary Table 8 and unprocessed original blots are shown in Supplementary Fig
2 Supplementary Figure 2 mir-16 reduces mrna decay induced by mir-155. (a) Cartoon illustrating the 3 UTR of human with the position of the SNPs rs683 and rs91, the three MRE-155 sites (blue) and the three putative MRE-16 sites (orange). (b) MRE-155 sequences on 3 UTR. -C corresponds to the NM_55.2 and -A to NM_55.1. Alignments have been performed using RNAhybrid 34 only a few targets are known. In contrast to plant mirnas, which usually bind nearly perfectly to their targets, animal mirnas bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of mirna/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of mirnas in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between mirna nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila mirna targets in 3 UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server ( bibiserv.techfak.uni-bielefeld.de/rnahybrid/. Underlined sequences on MRE- 155#2C and MRE-155#3A are detailed in boxes on the right to show the position of the SNPs in the two alleles of. Arrows indicate the SNP rs683 and rs91 positions. (c) Effects of synthetic mir-155 on the identified regions of MRE-155 in 51Mel. Luciferase activity was evaluated 48h after transfection. Each histogram represents the mean ± s.d. of n=3 biologically independent experiments; two-sided unpaired t-test with Welch s correction; p < 5. (d) Northern blot quantification of mir-16 in 51Mel cells. The signal (from 51Mel cells) was fit to the standard curve from synthetic titration signals to give final copy number per cell. RNU6 served as a loading control. Pictures are representative of three experiments. (e) Northern blot quantification of mrna in 51Mel cells. The signal (from 51Mel cells) was fit to the standard curve from the 3 UTR s synthetic titration signal to give final copy number per cell. GAPDH serves as a loading control. Picture presents three biological replicates of 51Mel. Source data are available in Supplementary Table
3 Supplementary Figure 3 MRE-16 on human and mouse mrna and biological consequences. (a) MRE-16s sequences on mouse 3 UTR (NM_3122.3). Alignments have been performed using RNAhybrid 34 only a few targets are known. In contrast to plant mirnas, which usually bind nearly perfectly to their targets, animal mirnas bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of mirna/target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of mirnas in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between mirna nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila mirna targets in 3 UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server ( de/rnahybrid/. (b) Schematic representation of the interaction (purple base paring) of mir-16 (orange) and mir-155 (blue) with human and mouse MRE-16#3 and MRE-155#3 respectively. (c-d) knockdown in mouse B16-F1 melanoma cells using three different sirnas. mrna levels (c) and cell density (d) have been respectively evaluated at 5 and 3 days after sirna transfection; one-way ANOVA with Holm-Sidak s multiple comparisons test. (e) Effect on cell density of synthetic mir-16 transfected in mouse B16-F1 melanoma cells 3 days after mirna transfection; two-sided unpaired t-test with Welch s correction, p < 5. Each histogram represents the mean ± s.d. (n = 3 biologically independent experiments). Source data are available in Supplementary Table
4 a b sictr si #1 si #3 si #2 n=3 si n=5 n=4 #1 #2 #3 vs sictr n=3 n=3 #1 #2 Significance Analysis of Microarrays FDR=, (GEO GSE7561) Relative mrna levels to sictr MAFF NRTN RAB17 RasGRP si #2 #3 #1 MYH7B A2M MUC7 PLB1 BAIAP2L2 PPY NRTN COL7A1 GALNTL4 TNFSF9 MAFF ST3GAL4 PLA2G6 PRSS56 RAB17 SDC3 RASGRP3 PLTP HIF1A FBLN1 SEPT9 CELF2 MOXD1 HLCS ITGB8 MRPL36 C14orf169 EIF4EBP2 B3GALT6 FBXL7 FAM12C LINC34 C9orf13 ERAP1 CREM FAM198B PTP4A1 LOC IFIT1 PMP2 IFI6 FLJ4696 BFSP1 ISG15 IFI44 CHMP1B MAP2 CLDN2 proliferation of cells migration of cells quantity of cells formation of cellular protrusions generation of cells quantity of neurons invasion of cells invasion of tumor cell lines vasculogenesis proliferation of lymphocytes growth of epithelial tissue differentiation of cells Activation z-score DN UP DN UP c Relative mrna levels to shctr d Cell density relative to sictr e Relative mrna levels to mir-ctr f 2 1 MAFF NRTN RAB17 RasGRP3 CTR MAFF#1 mir-ctr mir-16 MAFF NRTN shctr RASGRP3 sh MAFF#2 NRTN#1 NRTN#2 RAB17#1 RAB17#2 RasGRP3#1 RasGRP3#2 Supplementary Figure 4 silencing decreases expression level of several mrnas. (a) Workflow to identify deregulated RNAs in KD cells. Gene expression profile of cells transfected with three different sirnas targeting or sirna CTR. Significance analysis of microarrays was done as described in methods. sirna efficacy for KD is #2>#3>#1. Heatmap focused on deregulated RNAs in function of si efficacy (top) and a z-score has been calculated using Ingenuity Pathway Analysis (IPA) (bottom). (b) mrna expression levels of, MAFF, NRTN, RAB17 and RasGRP3 in response to KD using three different sirnas (#1-3) targeting the ORF of in 51Mel cells (n = 4 biologically independent experiments except for RASGRP3 expression measurement which results from n = 3 biologically independent experiments). (c) mrna expression levels of, MAFF, NRTN, RAB17 and RasGRP3 in response to KD using sh targeting the ORF of in SKMel28-luc cells (n = 3 biologically independent experiments). (d) Cell density of 51Mel cells in response to sirnas targeting MAFF, NRTN, RAB17 or RasGRP3 (n = 3 biologically independent experiments; one-way ANOVA with Holm-Sidak s multiple comparisons test, p < 5). Two sirnas were used by target. (e) mrna expression levels in response to synthetic mir-16. Each histogram represents the mean ± s.d. (n = 3 biologically independent experiments). and RAB17 mrna expression in response to synthetic mir-16 are reported in Fig. 2d and 4b, respectively. (f) The MRE-16s sequence on human MAFF and NRTN mrnas have been identified using RNAhybrid 34 only a few targets are known. In contrast to plant mirnas, which usually bind nearly perfectly to their targets, animal mirnas bind less tightly, with a few nucleotides being unbound, thus producing more complex secondary structures of mirna/ target duplexes. Here, we present a program, RNA-hybrid, that predicts multiple potential binding sites of mirnas in large target RNAs. In general, the program finds the energetically most favorable hybridization sites of a small RNA in a large RNA. Intramolecular hybridizations, that is, base pairings between target nucleotides or between mirna nucleotides are not allowed. For large targets, the time complexity of the algorithm is linear in the target length, allowing many long targets to be searched in a short time. Statistical significance of predicted targets is assessed with an extreme value statistics of length normalized minimum free energies, a Poisson approximation of multiple binding sites, and the calculation of effective numbers of orthologous targets in comparative studies of multiple organisms. We applied our method to the prediction of Drosophila mirna targets in 3 UTRs and coding sequence. RNAhybrid, with its accompanying programs RNAcalibrate and RNAeffective, is available for download and as a Web tool on the Bielefeld Bioinformatics Server ( For b-c and e, two-sided unpaired t-tests with Welch s correction were done (p<5). For b-e, values correspond to the mean ± s.d. Source data are available in Supplementary Table
5 a RAB17 (IJB cohort) b + RAB17 (IJB cohort) c + RAB17 (TCGA cohort) High (n=92) p=52 HR=1.4 95% CI=-1.97 Low (n=92) Time (years) p<1.8 HR= % CI= High (n=13) Low (n=54) Time (years) High (n=157) p < 1 HR=2. 95 % Cl= Low (n=157) Time (years) d f NRTN (TCGA cohort) low NRTN (n = 46) high NRTN (n = 46) p =.1172 HR = % Cl = Time (Days) mir-16 (IJB cohort) e g + NRTN (TCGA cohort) mir-16 (TCGA cohort) low NRTN+ (n = 46) high NRTN + (n=46) p = 19 HR = % Cl = Time (Days) p=.7142 HR=96 95 % Cl= low mir-16 (n=43) high mir-16 (n=42) Time (years) p=.8336 HR=3 95 % Cl= low mir-16 (n=175) high mir-16 (n=174) Time (years) Supplementary Figure 5 of patients with metastatic melanoma according to, RAB17, NRTN mrnas or mir-16 expression levels. (a-b) Determination of overall survival (OS) curves by Kaplan-Meier analysis, according to the expression levels of RAB17 (a) or and RAB17 (b). Based on 184 skin and lymph node metastases from melanoma patients (IJB cohort). High and low expression of and RAB17 (a) alone were defined based on their median values and scored as 1 and, respectively. For combination (b), scores of (1 or ) and RAB17 (1 or ) are added and the resulting scores 1 and 2 are combined as the high score (n = 13 patients), which was significantly different from the low score (n = 54 patients) regarding to OS values (two-sided Mann-Whitney test, p = 4). Cox regression was used to calculate P values, hazard ratios (HR) and 95% confidence intervals (CI). (c) Association of and RAB17 expression with patient survival. and RAB17 expression levels were assessed in the TCGA SKCM melanoma cohort. Patients were ranked decreasingly according to and RAB17 expression, resulting in almost three equal groups. The Kaplan-Meier curve representing the highest third of and RAB17 expressers shows a significantly lower OS as compared to the lowest third (log-rank test). (d-e) Determination of OS curves by Kaplan-Meier analysis, according to the expression levels of NRTN (d) or and NRTN (e). Patients were ranked decreasingly according to NRTN (d) or and NRTN expression (e), resulting in almost four equal groups. The Kaplan-Meier curve represents the highest and the lower quarters of expressers (log-rank test). (f-g) Determination of OS curves by Kaplan-Meier analysis, according to the expression levels of mir-16 from n = 85 patients of the IJB cohort (f) or from n=349 patients from the TCGA cohort (g). High and low expression groups of mir-16 were defined based on its median value (log-rank test). 5
6 a b 2,5 TSB-C1 TSB-T3 TSB: C1 T3 mouse 1 - liver mouse 3 - liver 2, mouse 2 - liver mouse 4 - liver Tumor volume (mm 3 ) 1,5 1, 5 TSB-C1 mouse 1 - kidney TSB-T3 mouse 3 - kidney c Days after treatment 2 mouse 2 - kidney mouse 4 - kidney LAMP2 mrna level (2- Ct) 1 n.s. TSB-C1 TSB-T3 Supplementary Figure 6 Histological analyses of liver and kidney from PDX mice and long-term tumor growth. (a) Representative micrographs of liver and kidney slices stained with hematoxylin from PDX-mice exposed to TSB- C1 or TSB-T3. Toxicity evaluation was blindly examined by two independent pathologists. Two mice have been showed per group among five mice analysed given similar results. Scale bar : 1µm. (b) Tumor volume for individual PDX mice treated with TSB-C1 or TSB-T3 as described in Fig. 6b. (c) Quantification of LAMP2 mrna in melanoma tumors treated with TSB-C1 or TSB-T3 (n = 5 mice per group). LAMP2 is not a mir-16 target. For c, lines represent the mean ± s.d.; values represent fold change relative to the mean of TSB-C1 condition; two-sided unpaired t-tests with Welch s correction; n.s., non-significant. Source data are available in Supplementary Table
7 Figure 1b Figure 1e sh shctr Figure 3b GAPDH GAPDH 15 kda - Figure 4a aspecific signal RAB17 Figure 4b RAB17 15 kda - RAB17 Figure 4f RAB17 4 kda - 15 kda - 15 kda - 15 kda - aspecific signal Sup. Figure 1a Sup. Figure 1b Sup. Figure 1e : PEP1 51Mel AB93 G17 Sup. Figure 1e SKMel28 : PEP1 AB93 G17 Supplementary Figure 7 Unprocessed original scans of blots. 7
8 Supplementary Table Legends Supplementary Table 1 Metastatic melanoma cell lines and short-term cultures. Supplementary Table 2 mirna quantification in 51Mel cells. Supplementary Table 3 Clinical characteristics of the patients and tissues from the Institute Jules Bordet cohort. Supplementary Table 4 Correlation between and RAB17, NRTN or MAFF in the TCGA cutaneous skin cancer cohort. Supplementary Table 5 TSB-T3 efficacy, mutation state and mrnas expression in 51Mel and short-term cultures. Supplementary Table 6 sirna, mirna, TSB, inserts and probes sequences. Supplementary Table 7 Antibodies. Supplementary Table 8 Statistics Source Data. 8
9 Life Sciences Reporting Summary Corresponding author(s): Galibert M.-D. & Gilot D. Initial submission Revised version Final submission Nature Research wishes to improve the reproducibility of the work that we publish. This form is intended for publication with all accepted life science papers and provides structure for consistency and transparency in reporting. Every life science submission will use this form; some list items might not apply to an individual manuscript, but all fields must be completed for clarity. For further information on the points included in this form, see Reporting Life Sciences Research. For further information on Nature Research policies, including our data availability policy, see Authors & Referees and the Editorial Policy Checklist. Experimental design 1. Sample size Describe how sample size was determined. 2. Data exclusions Describe any data exclusions. 3. Replication Describe whether the experimental findings were reliably reproduced. 4. Randomization Describe how samples/organisms/participants were allocated into experimental groups. 5. Blinding Describe whether the investigators were blinded to group allocation during data collection and/or analysis. No sample size calculation was performed. Three to more independent results were used to perform statistical analyses. If less, no statistics were performed from these samples. All raw data required for statistical tests are in indicated in supplementary Table 1 (Statistics data source) Data were excluded only for PDX mice treatments. PDX mice were chosen by the range of their initial tumor volume: 18 mm3 (median, mm3). All attempts at replication were successful. Samples were allocated to groups randomly but no specific methods were used. Investigators were blinded to group allocation during data collection only for the PDX mice treatments. For other experiments, investigators who performed experiments were the same who took care of cell lines, did extractions, etc., so blinding was not possible. Note: all studies involving animals and/or human research participants must disclose whether blinding and randomization were used. 6. Statistical parameters n/a For all figures and tables that use statistical methods, confirm that the following items are present in relevant figure legends (or in the Methods section if additional space is needed). Confirmed The exact sample size (n) for each experimental group/condition, given as a discrete number and unit of measurement (animals, litters, cultures, etc.) A description of how samples were collected, noting whether measurements were taken from distinct samples or whether the same sample was measured repeatedly A statement indicating how many times each experiment was replicated The statistical test(s) used and whether they are one- or two-sided (note: only common tests should be described solely by name; more complex techniques should be described in the Methods section) A description of any assumptions or corrections, such as an adjustment for multiple comparisons The test results (e.g. P values) given as exact values whenever possible and with confidence intervals noted A clear description of statistics including central tendency (e.g. median, mean) and variation (e.g. standard deviation, interquartile range) Clearly defined error bars nature research life sciences reporting summary June 217 See the web collection on statistics for biologists for further resources and guidance. 1
10 Software Policy information about availability of computer code 7. Software Describe the software used to analyze the data in this study. 4peaks, NDP.view (Nanozoomer digital pathology), serial cloner 3., IGV (Integrative Genomics Viewer), R, microsoft excel, Image J, SDS v2. (Applied Biosystems), Bowtie, bioconductor LIMMA, SVA, MultiExperiment Viewer (MeV v4.8), IPA (Ingenuity Pathway Analysis), Prism 6 (GraphPad), SPSS Statistics 15., nsolver Analysis Software (Nanostring) and ArrayScan VTI High-Content Systems (ThermoFisher Scientific) For manuscripts utilizing custom algorithms or software that are central to the paper but not yet described in the published literature, software must be made available to editors and reviewers upon request. We strongly encourage code deposition in a community repository (e.g. GitHub). Nature Methods guidance for providing algorithms and software for publication provides further information on this topic. Materials and reagents Policy information about availability of materials 8. Materials availability Indicate whether there are restrictions on availability of unique materials or if these materials are only available for distribution by a for-profit company. 9. Antibodies Describe the antibodies used and how they were validated for use in the system under study (i.e. assay and species). Target site blockers are only available for distribution by a for-profit company (Exiqon). Anti- (PEP1) antibody was kindly provided by Prof. V.J. Hearing (National Institute of Health, Bethesda, Maryland). Full information about the anitbodies used in this study is provided in Supplementary Table 9. All primary antibodies anti- and anti-rab17 were also tested and validated in our lab using human shrna cell lines against and RAB17 gene by Western- Blot. Anti- detects a weaker band in sh cell extracts compared to shcontrol cell extracts, validating the antibody. Anti-RAB17 detects a weaker band in shrab17 cell extracts compared to shcontrol cell extracts, validating the antibody. 1. Eukaryotic cell lines a. State the source of each eukaryotic cell line used. Metastatic melanoma (MM) cell lines were derived from tumors by the Laboratory of Oncology and Experimental Surgery (Prof. G Ghanem) at the Institute Jules Bordet, Brussels. 51Mel, ME142 and SKMel28 cell lines were obtained from ATCC. SKMel28-luc was obtained from Prof. T. Montier (INSERM U178) and Mel624 from Dr. G. Lizée (Department of Melanoma Medical Oncology and Department of Immunology, Houston, TX, USA). Mouse melanoma B16-F1-luc-G5 cells were obtained from PerkinElmer (Waltham, MA). Huh7.5 Drosha knockout cells were obtained from Prof. R.B. Darnell (Rockefeller University, New York, USA). b. Describe the method of cell line authentication used. Sources cited above authenticated their cell lines. c. Report whether the cell lines were tested for mycoplasma contamination. d. If any of the cell lines used are listed in the database of commonly misidentified cell lines maintained by ICLAC, provide a scientific rationale for their use. Animals and human research participants All cell lines were tested negative for mycoplasma contamination. No commonly misidentified cell lines were used. Policy information about studies involving animals; when reporting animal research, follow the ARRIVE guidelines 11. Description of research animals Provide details on animals and/or animal-derived materials used in the study. ~ 6-week-old females NMRI nude mice were used. nature research life sciences reporting summary June 217 2
11 Policy information about studies involving human research participants 12. Description of human research participants Describe the covariate-relevant population characteristics of the human research participants. Cutaneous and lymph node metastases (n = 191) were collected from patients with stages III and IV melanoma undergoing surgery as a part of the diagnostic work-up or therapeutic strategy at the Institut Jules Bordet (Brussels, Belgium) between 1998 to 29. But no information about treatment categories were given. This cohort were composed of 8 males and 111 females from 2 to 87 years old (median: 55 years old). The majority of the melanomas were of superficial spreading or nodular histological subtypes with Breslow s thicknesses > 1 mm. nature research life sciences reporting summary June 217 3
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Supplemental Table S. Tumorigenicity and metastatic potential of 44SQ cell subpopulations a Tumorigenicity b Average tumor volume (mm ) c Lung metastasis d CD high /4 8. 8/ CD low /4 6./ a Mice were injected
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Corresponding Author: Manuscript Number: Manuscript Type: Bernhard Staresina A51406B Article Reporting Checklist for Nature Neuroscience # Main Figures: 5 # Supplementary Figures: 10 # Supplementary Tables:
More informationReporting Checklist for Nature Neuroscience
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