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, USA) and recombinant human BMP7 (30 ng/ml, a gift from K. Sampath, Sanofi-Genzyme Research Center

. Kowanetz, Cells were transfected with siRNAs targeting mRNAs at a final concentration of 20 nM, in a single transfection, or at a final concentration of 2 3 20 nM, in two sequential transfections, performed on successive days. Transient transfections with siRNAs targeting lncRNAs were performed using Dharmafect 1 reagent (Dharmacon/GE Healthcare, Uppsala, Sweden), according to the manufacturer's instructions, at a final concentration of 25 nM, The periods of the treatments with growth factors are indicated in the figures. The GW6604 TbRI kinase inhibitor was synthesized by the Ludwig Cancer Research Ltd, vol.3000, 2002.

. Qiagen-ab, . Sollentuna, and ). Sweden, ) for 1 h at 4 C, with rotation. Protein lysates were generated from HEK293T cells expressing endogenous proteins or transiently transfected with the pCMV-HA-EED, pCMV-HA-EZH2 or pCMV-HA-SUZ12 plasmids that express full-length wild-type human EED, EZH2 or SUZ12 proteins, respectively. Protein lysates from parental HaCaT cells expressing endogenous PRC2 proteins were also analyzed using the same protocol. Next, the protein-RNA-beads complexes were washed in 1 3 Wash Buffer and proteins were eluted in Elution Buffer, according to the protocol by the manufacturer, and were purified using the RNA cleanup protocol from the RNeasy kit

, Briefly, the beads were covered with enough volume (50 ml) of 25 mM ammonium bicarbonate, followed by reduction with dithiothreitol (DTT) and alkylation with iodoacetamide (IAA) at end concentrations of 1 mM and 5 mM, respectively. Then, 5 mM DTT was added to quench the alkylation reaction after IAA incubation. To each sample, 0.1 mg LysC was added and digestion was performed at 37 C overnight. Then, the samples were further digested by 0, On-beads digestion: the 9 protein samples (protein-RNA complexes) bound with magnetic beads were digested with lysC and trypsin in 25 mM ammonium bicarbonate, vol.1

, Sp3 peptide enrichment and clean-up: the peptide mixtures generated from the on-beads digestion were subjected to Single-Pot Solid-Phase-enhanced Sample Preparation (SP3) procedure 1. In brief, Sera-Mag SP3 bead mix (5.5 ml) was transferred into the approx. 55 ml sample together with > 96% acetonitrile (ACN). The mix was incubated at room temperature for 8 min. Subsequently, the peptide mixtures were immobilized and rinsed by 100% ACN on the surface of the paramagnetic beads

. Lc-ms/ms-analysis, 2 cm, nanoViper, C18, 5 mm, 100 Å ), and separated on a C18 column (Easy spray PepMap RSLC, C18, 2 mm, 100 Å , 75 mm 3 50 cm). The nano capillary solvent A was 95% water, 5% DMSO, 0.1% formic acid; and solvent B was 5% water, 5% DMSO, 95% acetonitrile, 0.1% formic acid. At a constant flow of 0.25 ml$min À1 , the curved gradient went from 2% B up to 40% B in 180 min, followed by a steep increase to 100% B in 5 min. FTMS master scans with 70,000 resolution (and mass range 300-1700 m/z) were followed by data-dependent MS/MS (35,000 resolution) on the top 5 ions using higher energy collision dissociation (HCD) at 30%-40% normalized collision energy. Precursors were isolated with a 2 m/z window. Automatic gain control (AGC) targets were 1e6 for MS1 and 1e5 for MS2. Maximum injection times were 100 ms for MS1 and 150-200 ms for MS2. The entire duty cycle lasted $2.5 s. Dynamic exclusion was used with 60 s duration, online LC-MS was performed using a DionexUltiMate 3000 RSLCnano System coupled to a Q-Exactive(QEx)mass spectrometer, vol.100

, ) against Human Uniprot database (24-07-2017) and filtered to a 1% FDR cut off. We used a precursor ion mass tolerance of 10 ppm, and product ion mass tolerances of 0.02 Da for HCD-FTMS. The algorithm considered tryptic peptides with maximum 2 missed cleavages, Peptide and protein identification: the MS raw files were searched using Sequest-Percolator (06-10-2017) under the software platform Proteome Discoverer, vol.1

, The list of TGFB2-AS1-interacting proteins and associated peptide coverage with statistics are shown in Table S6. cDNA synthesis and real-time qPCR

A. H. Macherey-nagel and . Diagnostics, The expression levels of target genes were normalized to the expression levels of the reference genes GAPDH, HPRT1 or 18S rRNA and relative normalized expression was calculated based on the DDC t method. The results were plotted in graphs as average values of relative normalized expression, ) using the qPCRBIO SyGreen 2 3 Master Mix (PCR Biosystems, 2000.

, Cell Reports, vol.28, pp.3182-3198, 2019.

, Nucleo-cytoplasmic fractionation

, ) and split into two fractions: from the major fraction, bound RNA, together with total RNA from input samples was purified using the RNA cleanup protocol from the RNeasy kit (QIAGEN AB, Sollentuna, Sweden); from the minor fraction, washed lysate was loaded on polyacrylamide gels for immunoblotting using the same antibody with which the RNA immunoprecipitation was performed and described above or a different antibody against the same protein in the case of EZH2 (Active Motif Europe, PBS. Then, cells were resuspended in Cell Fractionation Buffer (PARIS kit) on ice and incubated at 4 C for 10 min. Lysates were centrifuged at 500 3 g at 4 C for 5 min and the supernatant (cytoplasmic fraction) was transferred to new tubes. Then, the nuclear pellet was washed once in PBS and lysed in ice-cold Cell Disruption Buffer (PARIS kit), with vigorous vortexing, p.2

, ), mounted in Fluoromount-G (SouthernBiotech, AH Diagnostics, Solna, Sweden) and examined on a Zeiss Axioplan 2 fluorescence microscope with the Zeiss 40 3 objective lens (Carl Zeiss AB, Then, hybridization step was performed, using hybridization buffer, containing RNA probes specific for TGFB2-AS1, vol.1

, The cells were permeabilized with 0.1% Triton X-100 for 10 min at room temperature and blocked in 5% FBS/PBS for 1 h at room temperature. Then, the samples were incubated with primary antibodies in 5% FBS/PBS overnight at 4 C. The next day, the coverslips were incubated with Alexa Fluor-488-labeled secondary antibody (Invitrogen, Thermo Fisher Scientific, Immunofluorescence HaCaT or A549 cells were fixed in 3.7% (w/v) formaldehyde stabilized with 10% (v/v) methanol for 10 min at room temperature, vol.1

, Cell Signaling Technology, vol.9523

, The lysates were cleared by centrifugation at maximum speed for 10 min. The supernatant was transferred to new tubes and protein concentration was measured with the bicinchoninic acid (BCA) assay. Next, 2 3 sample buffer (0.12 M Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 0.01% bromophenol blue, 100 mM DTT) was added to the lysates, which were then boiled at 95 C for 5 min and subjected to SDS-PAGE. Equal amount of protein (40 mg) was loaded to each polyacrylamide gel. The resolved proteins were transferred to a nitrocellulose filter using a Bio-Rad wet or semidry transfer unit, Immunoblotting Total proteins were extracted using lysis buffer (20 mM Tris-HCl, pH = 8.0, 1% Nonidet P-40, 150 mM NaCl, 2 mM EDTA, and complete protease inhibitor mixture from Roche Diagnostics Scandinavia AB, Bromma, Sweden)

, A complete list of the oligonucleotides used for ChIP-qPCR assays is shown in Table S3. Luciferase assays HaCaT, HepG2 or A549 cells transiently transfected with the TGFb/Smad-responsive CAGA 12 promoter reporter construct (CAGA 12 -luc) were used for luciferase assays. The HaCaT CAGA 12 -Luc/TK-Renilla cells, which stably express the CAGA 12 -luc construct and the TK-Renilla luciferase reporter plasmid, were also used for luciferase assays. The BMP/Smad responsive promoter reporter (BRE 2 -luc) construct was transiently transfected to shControl or shTGFB2-AS1 HaCaT cells. The pCMV-b-gal construct, which encodes the b-galactosidase, was co-transfected with the transiently transfected promoter reporter plasmids for normalization of the firefly luciferase measurements. In the case of the HaCaT CAGA 12 -Luc/TK-Renilla cells, the firefly luciferase values were normalized to the Renilla-luc values. Luciferase reporter assays were performed using the Firefly and Renilla Dual Luciferase Assay kit from Biotium, Chromatin immunoprecipitation HaCaT cells expressing pcDNA3 (empty vector) or pcDNA3-TGFB2-AS1 were grown to 80% confluency in 15-cm dishes and crosslinked in 1% formaldehyde for 10 min at room temperature. The crosslinking was quenched by addition of 125 mM glycine for 5 min at room temperature. The cells were then washed and scraped in ice-cold PBS and centrifuged. The cell pellets were stored at À80 C until further use. The cell pellets were lysed in ChIP lysis buffer (50 mM Tris-HCl, pH 8, 10 mM EDTA, 1% SDS), supplemented with protease inhibitors (Roche Diagnostics Scandinavia AB, vol.8

N. Y. Corning, ) and incubated at 37 C for 1 h. After trypsinization, 4 3 10 4 cells were seeded in DMEM/0.1% FBS in the upper chamber with or without TGFb1 (5 ng/ml), and DMEM/6% FBS was placed in the lower chamber, Cell Reports, vol.28, pp.3182-3198, 2019.

, For quantification, 10-15 pictures of each insert were taken at 20 3 magnification, and the nuclei were counted using the ImageJ software (National Institutes of Health, methanol and stained with DAPI

, HaCaT cell invasion was analyzed as described above for A549 cells with the following alterations: cells were first treated with GSK343 in DMEM/0.1% FBS for 24 h, then media were changed to DMEM/0.1% FBS containing GSK343 (5 mM) with or without TGFb1 (5 ng/ml) for 24 h. Cells on the upper chamber received DMEM/0.1% FBS with or without TGFb1 (5 ng/ml) in the presence or absence of GSK343 (5 mM

, For immunoblotting experiments of plain lysates or after RNA pull-down or RIP, one representative result is presented. The exact number of repeated experiments is indicated in the Figure legends. For immunofluorescence and FISH experiments, only single (or two, FISH) representative photomicrographs are shown. Each independent immunofluorescence or FISH experiment was repeated 2 to 4 times (n = 2-4 biological repeats) and included single technical repeats; out of each technical repeat 4-5 independent photomicrographs were collected and compared subjectively for assessment of high reproducibility. Comparisons of quantitative measurements were performed using two-tailed paired Student's t test in Excel. In all experimental conditions statistical significance was accepted based on a p value smaller than 0.05. The exact degree of significance for each assay is, QUANTIFICATION AND STATISTICAL ANALYSIS The results of RT-qPCRs, luciferase assays and thymidine incorporation assays represent the mean from at least three independent experiments (n = 3