D . relative fold - change of transwell migration for indicated knockdown over those of control . * * P < 0 . 01 , Student ' s t - , mean ± SD .

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D. The relative fold-change of the transwell migration for indicated knockdown cells over those of control cells. **P < 0.01, Student's t-test, mean ± SD.


E - F . Representative hematoxylin eosin ( H & E ) statistical results of micro - metastatic nodules in liver from mice injected with indicated into spleen for 45 days . N = 8 per group . * * P < 0 . 01 , Student ' s t - , mean ± SD .

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E-F. Representative hematoxylin and eosin (H&E) staining and statistical results of the micro-metastatic nodules in the liver from mice injected with the indicated cells into the spleen for 45 days. N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


G - H . Representative H & E statistical results of metastatic lung nodules from mice injected with indicated via tail vein for 60 days . ( Five sections evaluated per lung ) . N = 8 per group . * * P < 0 . 01 , Student ' s t - , mean ± SD .

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G-H. Representative H&E staining and statistical results of metastatic lung nodules from mice injected with the indicated cells via the tail vein for 60 days. (Five sections evaluated per lung). N=8 per group. **P < 0.01, Student's t-test, mean ± SD.


B . Still of GFP ( syncytia ) Propidium Iodide ( PI ) ( ) at different time - points . Scale bar : 100 µm .

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B. Still images of GFP (syncytia) and Propidium Iodide (PI) (cell death) at different time-points. Scale bar: 100 µm.


C . of U2OS - ACE2 fusion by time - lapse microscopy . Results are mean ± sd from 3 fields per condition .

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C. Quantification of U2OS-ACE2 fusion and death by time-lapse microscopy. Results are mean±sd from 3 fields per condition.


D . S of infected U2OS - ACE2 analyzed by immunofluorescence . Hoechst dye stains nuclei . Scale bar : 40 µm .

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D. S staining of infected U2OS-ACE2 cells analyzed by immunofluorescence. The Hoechst dye stains the nuclei. Scale bar: 40 µm.


E . Surface S of infected U2OS - ACE2 analyzed by flow cytometry . Results are representative of at least three independent experiments .

E. Surface S staining of infected U2OS-ACE2 cells analyzed by flow cytometry. Results are representative of at least three independent experiments.


were infected at indicated multiplicity of infection ( MOI ) analyzed after 20h . A . TMPRSS2 increases fusion mortality . Right panel : Areas of GFP + PI +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. A. TMPRSS2 increases fusion and cell mortality. Right panel: Areas of GFP+ cells and PI+ cells


were infected at indicated multiplicity of infection ( MOI ) analyzed after 20h . B . IFITM1 , but not IFITM2 3 , inhibits SARS - CoV - 2 - induced syncytia formation . Right panels : of GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. B. IFITM1, but not IFITM2 and 3, inhibits SARS-CoV-2-induced syncytia formation. Right panels: Area of GFP+ cells


were infected at indicated multiplicity of infection ( MOI ) analyzed after 20h . C . fusion of IFITM1 + with U2OS - ACE2 syncytia is drastically reduced . Right panel : of IFITM + GFP +

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. C. The fusion of IFITM1+ cells with U2OS-ACE2 syncytia is drastically reduced. Right panel: Area of IFITM+ GFP+ cells


were infected at indicated multiplicity of infection ( MOI ) analyzed after 20h . D . IFITM1 decreases number of infected . Right panel : Fraction of positive for S , normalized to control ( transduced with pQCXIP - empty vector ) .

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Cells were infected at the indicated multiplicity of infection (MOI) and analyzed after 20h. D. IFITM1 decreases the number of infected cells. Right panel: Fraction of cells positive for S, normalized to control cells (transduced with pQCXIP-empty vector ).


B . Representative of - fusion . Scale bar : 100 µm .

B. Representative images of cell-cell fusion. Scale bar: 100 µm.


C . of GFP + areas . Results are mean ± sd from 5 independent experiments .

C. Quantification of GFP+ areas. Results are mean±sd from 5 independent experiments.


D . TMPRSS2 accelerates fusion . were monitored by video microscopy GFP was quantified over time . Left panel : one representative experiment . Results are mean ± sd from 3 fields per condition . Right panel : Mean ± sd from 7 independent experiments ( at 12h post transfection ) .

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D. TMPRSS2 accelerates fusion. Cells were monitored by video microscopy and the GFP area was quantified over time. Left panel: one representative experiment. Results are mean±sd from 3 fields per condition. Right panel: Mean±sd from 7 independent experiments (at 12h post transfection).


E . Impact of TMPRSS2 IFITMs on kinetics of fusion by S - expressing 293T . One representative out of three independent experiment is shown .

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E. Impact of TMPRSS2 and IFITMs on the kinetics of fusion by S-expressing 293T cells. One representative out of three independent experiment is shown.


A . S - expressing ( Donor ) , ACE2 - expressing ( Acceptor ) were co - transfected with TMPRSS2 , IFITM or control plasmids . fusion was quantified by measuring GFP + after 18h . indicated combinations were tested . impact of IFITMs was measured in absence of TMPRSS2 ( left panel ) , in presence of TMPRSS2 in donor ( middle panel ) or acceptor ( right panel ) . Data are mean ± sd of 4 independent experiments . Statistical : One - Way ANOVA , ns : non - significant , ∗ ∗ p < 0 . 01 , ∗ ∗ ∗ ∗ p < 0 . 0001 .

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A. S-expressing cells (Donor cells), ACE2-expressing cells (Acceptor cells) were co-transfected with TMPRSS2, IFITM or control plasmids. Cell fusion was quantified by measuring the GFP+ area after 18h. The indicated combinations were tested. The impact of IFITMs was measured in absence of TMPRSS2 (left panel), in presence of TMPRSS2 in donor (middle panel) or acceptor cells (right panel). Data are mean±sd of 4 independent experiments. Statistical analysis: One-Way ANOVA, ns: non-significant, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.


Impact of TMPRSS2 on IFITMs . B . TMPRSS2 does not decrease IFITM amounts measured by flow cytometry . 293T were transfected with indicated IFITM plasmids , with or without TMPRSS2 , analyzed 18h later .

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Impact of TMPRSS2 on IFITMs levels. B. TMPRSS2 does not decrease IFITM amounts measured by flow cytometry. 293T cells were transfected with the indicated IFITM plasmids, with or without TMPRSS2, and analyzed 18h later.


C . Impact of TMPRSS2 on S ACE2 , measured by Western Blot . 293T were transfected with or without IFITM1 , TMPRSS2 , S or ACE2 plasmids , analyzed 18h later .

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C. Impact of TMPRSS2 on S and ACE2, measured by Western Blot. 293T cells were transfected with or without IFITM1, TMPRSS2, S or ACE2 plasmids, and analyzed 18h later.


Impact of TMPRSS2 on S . D . TMPRSS2 decreases S surface , measured by flow cytometry . 293T were transfected with S plasmid , with or without TMPRSS2 , analyzed 18h later . Murine polyclonal anti - S antibodies , one serum from a convalescent COVID - 19 ( sera n ° 111 ) , two monoclonal antibodies ( anti - S1 anti - S2 ) were tested . Data are representative of three independent experiments .

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Impact of TMPRSS2 on S levels. D. TMPRSS2 decreases S surface levels, measured by flow cytometry. 293T cells were transfected with S plasmid, with or without TMPRSS2, and analyzed 18h later. Murine polyclonal anti-S antibodies, one serum from a convalescent COVID-19 patient (sera n°111), and two monoclonal antibodies (anti-S1 and anti-S2) were tested. Data are representative of three independent experiments.


A Deletion of siaA , siaB , siaC , or siaD regulates biofilm formation . biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down )

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A Deletion of siaA, siaB, siaC, or siaD regulates biofilm formation. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down)


B intracellular of c - di - GMPin PAO1 , ΔsiaC , ΔsiaB corresponding complemented strains were detected by LC - MS / MS

B The intracellular levels of c-di-GMPin PAO1, ΔsiaC, ΔsiaB and the corresponding complemented strains were detected by LC-MS/MS


E two - hybrid reveals an interaction between SiaC SiaD . recombinant strains harboring different vectors were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . NA represents empty vector pBT or pTRG . expressing LGF2 Gal11P was used as positive controls

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E Bacterial two-hybrid assay reveals an interaction between SiaC and SiaD. The recombinant strains harboring different vectors were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P was used as positive controls


F Pull down assays confirmed interaction between SiaC SiaD . lysates of P . aeruginosa containing pMM67EH - siaD - Flag were incubated with GST or GST - SiaC individually , complexes were captured by glutathione beads

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F Pull down assays confirmed the interaction between SiaC and SiaD. Cell lysates of P. aeruginosa containing pMM67EH-siaD-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads


G SPR measurements of SiaC ( left ) or SiaCP ( right ) binding at varying concentrations to SiaD . SiaC specifically interacted with SiaD with a KD of 24 . 7nM , whereas SiaCP failed to interact with SiaB . Shown are measured binding responses ( black ) curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant

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G SPR measurements of SiaC (left) or SiaCP (right) binding at varying concentrations to SiaD. SiaC specifically interacted with SiaD with a KD of 24.7nM, whereas SiaCP failed to interact with SiaB. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant


H Production of c - di - GMP by either SiaD or SiaD with SiaC at indicated time - point was determined by HPLC

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H Production of c-di-GMP by either SiaD or SiaD with SiaC at the indicated time-point was determined by HPLC


A two - hybrid reveals an interaction between SiaB SiaC . recombinant strains harboring different were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . NA represents empty vector pBT or pTRG . expressing LGF2 Gal11P were used as positive controls .

A Bacterial two-hybrid assay reveals an interaction between SiaB and SiaC. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). NA represents the empty vector pBT or pTRG. The strain expressing LGF2 and Gal11P were used as positive controls.


B Pull - down assays confirmed interaction between SiaB SiaC . lysates of P . aeruginosa containing pMM67EH - siaC - Flag were incubated with GST or GST - SiaB individually , complexes were captured by glutathione beads .

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B Pull-down assays confirmed the interaction between SiaB and SiaC. Cell lysates of P. aeruginosa containing pMM67EH-siaC-Flag were incubated with GST or GST-SiaB individually, and protein complexes were captured by glutathione beads.


C SPR measurements of SiaC binding at varying concentrations to SiaB . SiaC specifically interacted with SiaB with a KD of 40 . 1 nM . Shown are measured binding responses ( black ) curve fits to a 1 : 1 interaction ( red ) . Plots are representative from two experiments with similar results . RU , response units ; KD , dissociation constant .

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C SPR measurements of SiaC binding at varying concentrations to SiaB. SiaC specifically interacted with SiaB with a KD of 40.1 nM. Shown are measured binding responses (black) and curve fits to a 1:1 interaction (red). Plots are representative from two experiments with similar results. RU, response units; KD, dissociation constant.


D SiaB - mediated regulation of biofilm formation is dependent on SiaC . biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) .

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D SiaB-mediated regulation of biofilm formation is dependent on SiaC. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E two - hybrid assayreveals an interaction between SiaC SiaA PP2C - like domain . NA represents empty vector pBT or pTRG . recombinant strains harboring different were separately streaked on nonselective dual - selective media ( 3 - - 1 , 2 , 4 - triazole + streptomycin ) . expressing LGF2 Gal11P were used as positive controls .

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E Bacterial two-hybrid assayreveals an interaction between SiaC and SiaA PP2C-like domain. NA represents the empty vector pBT or pTRG. The recombinant strains harboring different proteins were separately streaked on nonselective and dual-selective media (3-amino-1, 2, 4- triazole + streptomycin). The strain expressing LGF2 and Gal11P were used as positive controls.


F Pull - down assays confirmed interaction between SiaC SiaA . lysates of P . aeruginosa containing pMM67EH - siaA - Flag were incubated with GST or GST - SiaC individually , complexes were captured by glutathione beads .

F Pull-down assays confirmed the interaction between SiaC and SiaA. Cell lysates of P. aeruginosa containing pMM67EH-siaA-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


G Pull - down assays confirmed interaction between SiaC SiaA PP2C - like domain . lysates of P . aeruginosa containing pMMB67EH - siaA386 - Flag were incubated with GST or GST - SiaC individually , complexes were captured by glutathione beads .

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G Pull-down assays confirmed the interaction between SiaC and SiaA PP2C-like domain. Cell lysates of P. aeruginosa containing pMMB67EH-siaA386-Flag were incubated with GST or GST-SiaC individually, and protein complexes were captured by glutathione beads.


H biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) .

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H The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate . Extracted ion chromatograms of doubly protonated peptide are shown with peak intensities indicating relative amount of either modified or unmodified peptides .

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B MS detection of SiaB peptide L63LYLNTSSIK72 that was covalently modified with one molecule of phosphate. Extracted ion chromatograms of the doubly protonated peptide are shown with peak intensities indicating the relative amount of either the modified or unmodified peptides.


C Determination of modification sites by collision induced dissociation ( CID ) . MS / MS spectrum of modified L63LYLNTSSIK72 is shown . fragment ions b6 y6 to y9 have a mass increase of 80 corresponding to phosphorylation , suggesting phosphorylation of Thr68 .

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C Determination of modification sites by collision induced dissociation (CID) analysis. The MS/MS spectrum of modified L63LYLNTSSIK72 is shown. The fragment ions b6 and y6 to y9 have a mass increase of 80 corresponding to phosphorylation, suggesting phosphorylation of Thr68.


D SiaB phosphorylates SiaA dephosphorylates SiaC in vitro . SiaA or SiaAT68A were added to reactions containing SiaB or SiaA386 γ32P ATP . Phosphorylated products were separated by SDS - PAGE .

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D SiaB phosphorylates and SiaA dephosphorylates SiaC in vitro. SiaA or SiaAT68A were added to the reactions containing SiaB or SiaA386 and γ32P ATP. Phosphorylated products were separated by SDS-PAGE.


E Over - expression of SiaCT68A restored ability of ΔsiaA to form biofilm . biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) . F Deletion of ΔsiaB in ΔsiaA background restored ability of ΔsiaA to form biofilm . biofilm formation of indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) .

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E Over-expression of SiaCT68A restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). F Deletion of ΔsiaB in the ΔsiaA background strain restored the ability of ΔsiaA to form biofilm. The biofilm formation of the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


G Production of c - di - GMP at indicated time - point by SiaD with SiaC or SiaCT68A was determined by HPLC . To evaluate effect of SiaB - mediated phosphorylation on SiaD DGC activity , SiaC or SiaCT68A was pretreated by SiaB in presence of 1 mM ATP before reaction initiation .

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G Production of c-di-GMP at the indicated time-point by SiaD with SiaC or SiaCT68A was determined by HPLC. To evaluate the effect of SiaB-mediated phosphorylation on SiaD DGC activity, SiaC or SiaCT68A was pretreated by SiaB in the presence of 1 mM ATP before reaction initiation.


C SiaCE33A partially restored biofilm formation of ΔsiaA . biofilm formation by indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) .

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C SiaCE33A partially restored the biofilm formation of ΔsiaA. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


D Triple mutation of E61A - Q64A - R67A is important for function of SiaB . biofilm formation by indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) .

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D Triple mutation of E61A-Q64A-R67A is important for the function of SiaB. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down).


E Leu110 Phe174 residues are important for SiaB function . biofilm formation by indicated strains was displayed with crystal violet ( up ) quantified with optical density measurement ( down ) . Data represent means SDs of three biological replicates . * * , P < 0 . 01 on one - way ANOVA .

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E Leu110 and Phe174 residues are important for SiaB function. The biofilm formation by the indicated strains was displayed with crystal violet staining (up) and quantified with optical density measurement (down). Data represent the means and SDs of three biological replicates. **, P<0.01 based on one-way ANOVA test.


A - C ARR1 / 10 / 12 were examined by western blot when five - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - F of of ARR1 / 10 / 12 in ( A - C ) .

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A-C ARR1/10/12 protein levels were examined by western blot when five-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP were treated with or without 200 mM NaCl for the indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-F Quantification analysis of the protein levels of ARR1/10/12 in (A-C).


C In vivo Coimmunoprecipitation of MPK3 / 4 / 6 ARR1 / 10 / 12 . MPK3 / 4 / 6 - YFP ARR1 / 10 / 12 - MYC were co - expressed in Arabidopsis protoplast . extracts ( Input ) were immunoprecipitated with GFP Trap magnetic agarose beads . Immunoblots were performed with anti - MYC antibody to detect ARR1 / 10 / 12 with anti - GFP antibody to detect MPK3 / 4 / 6 .

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C In vivo Coimmunoprecipitation assay of MPK3/4/6 and ARR1/10/12. MPK3/4/6-YFP and ARR1/10/12-MYC were co-expressed in Arabidopsis protoplast cells. Protein extracts (Input) were immunoprecipitated with GFP Trap magnetic agarose beads. Immunoblots were performed with anti-MYC antibody to detect ARR1/10/12 and with anti-GFP antibody to detect MPK3/4/6.


A - C Thiophosphorylation assays of wild type mutated forms ARR1 / 10 / 12 by MPK3 / 6 in vitro . Potential phosphorylated Ser residues or Thr residues of ARR1 , ARR10 , ARR12 were mutated to Ala ( ARR1m / ARR10m / ARR12m ) . Recombinant ARR1 / 10 / 12 - His ARR1m / 10m / 12m - His were incubated with MPK3 MPK6 which were activated by MKK5DD . Phosphorylated ARR1 / 10 / 12 , ARR1m / 10m / 12m , MPK3 / 6 were visualized by an anti - thiophosphate ester - specific antibody ( α - TPE ) ( upper panel ) . Asterisk in ( B ) indicates non - specific bands . Recombinant MKK5DD , MPK3 , MPK6 , ARR1 / 10 / 12 , ARR1m / 10m / 12m were detected by comassie brilliant blue ( CBB ) as loading controls ( bottom panel ) .

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A-C Thiophosphorylation assays of wild type and mutated forms ARR1/10/12 by MPK3/6 in vitro. Potential phosphorylated Ser residues or Thr residues of ARR1, ARR10, and ARR12 were mutated to Ala (ARR1m/ARR10m/ARR12m). Recombinant ARR1/10/12-His and ARR1m/10m/12m-His proteins were incubated with MPK3 and MPK6 which were activated by MKK5DD. Phosphorylated ARR1/10/12, ARR1m/10m/12m, and MPK3/6 were visualized by an anti-thiophosphate ester-specific antibody (α-TPE) (upper panel). Asterisk in (B) indicates non-specific bands. Recombinant MKK5DD, MPK3, MPK6, ARR1/10/12, and ARR1m/10m/12m were detected by comassie brilliant blue (CBB) staining as loading controls (bottom panel).


A - C Immunoblot with anti - MYC antibody or anti - GFP antibody showing upshift of ARR1 , ARR10 , ARR12 in a phos - tag gel . phosphorylated ARR1 , ARR10 , ARR12 were abolished after treatment with calf intestinal alkaline phosphatase ( CIAP ) . Seven - day - old seedlings of 35S : ARR1 : MYC ( A ) , 35S : ARR10 : YFP ( B ) , 35S : ARR12 : YFP ( C ) in Col MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Total were extracted subjected to immunoblot . ARR1 , ARR10 , ARR12 separated on SDS - PAGE gel without phos - tag were detected as loading controls .

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A-C Immunoblot analysis with anti-MYC antibody or anti-GFP antibody showing the upshift of ARR1, ARR10, and ARR12 in a phos-tag gel. The phosphorylated ARR1, ARR10, and ARR12 were abolished after treatment with calf intestinal alkaline phosphatase (CIAP). Seven-day-old seedlings of 35S:ARR1:MYC (A), 35S:ARR10:YFP (B), and 35S:ARR12:YFP (C) in Col and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Total proteins were extracted and subjected to immunoblot analysis. ARR1, ARR10, and ARR12 proteins separated on SDS-PAGE gel without phos-tag were detected as loading controls.


A - C Immunoblot effects of MPK3 / 6 on salt stress - induced ARR1 / 10 / 12 degradation . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP in Col - 0 MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I of MPK3 / 6 effect on salt stress - induced ARR1 / 10 / 12 degradation in ( A - C ) .

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A-C Immunoblot analysis the effects of MPK3/6 on salt stress-induced ARR1/10/12 protein degradation. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, and 35S:ARR12:YFP in Col-0 and MPK3SR backgrounds were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of MPK3/6 effect on salt stress-induced ARR1/10/12 protein degradation in (A-C).


A - C Immunoblot stability of ARR1 / 10 / 12 ARR1T553A / 10S383A / 12S323A under salt stress . Seven - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR10 : YFP , 35S : ARR12 : YFP , 35S : ARR1T553A : MYC , 35S : ARR10S383A : YFP , 35S : ARR12S323A : YFP were treated with or without 200 mM NaCl for indicated time points . Actin was used as internal reference . relative of band detected by anti - MYC or anti - GFP antibody to that by anti - Actin antibody without treatment was set to 1 . 0 . D - I of stability of ARR1 / 10 / 12 ARR1T553A / 10S383A / 12S323A under salt stress in ( A - C ) .

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A-C Immunoblot analysis stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress. Seven-day-old seedlings of 35S:ARR1:MYC, 35S:ARR10:YFP, 35S:ARR12:YFP, 35S:ARR1T553A:MYC, 35S:ARR10S383A:YFP, and 35S:ARR12S323A:YFP were treated with or without 200 mM NaCl for indicated time points. Actin was used as the internal reference. The relative intensity of band detected by anti-MYC or anti-GFP antibody to that by anti-Actin antibody without treatment was set to 1.0. D-I Quantification analysis of stability of ARR1/10/12 and ARR1T553A/10S383A/12S323A proteins under salt stress in (A-C).


Four - day - old seedlings of 35S : ARR1 : MYC , 35S : ARR1T553A : MYC , 35S : ARR1T553D : MYC were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , ( A ) were determined .

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Four-day-old seedlings of 35S:ARR1:MYC, 35S:ARR1T553A:MYC, and 35S:ARR1T553D:MYC were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Four - day - old seedlings of 35S : ARR10 : YFP , 35S : ARR10S383A : YFP , 35S : ARR10S383D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , ( A ) were determined .

Four-day-old seedlings of 35S:ARR10:YFP, 35S:ARR10S383A:YFP, and 35S:ARR10S383D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes (A) were determined.


Four - day - old seedlings of 35S : ARR12 : YFP , 35S : ARR12S323A : YFP , 35S : ARR12S323D : YFP were transferred to 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . After three days of salt treatment , were determined .

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Four-day-old seedlings of 35S:ARR12:YFP, 35S:ARR12S323A:YFP, and 35S:ARR12S323D:YFP were transferred to 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. After three days of salt treatment, plant phenotypes were determined.


Three - day - old seedlings of Col - 0 , MPK3SR , MPK3SRarr1 / 12 , arr1 / 12 were transferred to 0 . 5 μM NA - PP1 - containing 1 / 2 MS medium for 2 d to inhibit MPK3TG in MPK3SR MPK3SRarr1 / 12 then were transferred to 0 . 2 μM NA - PP1 - containing 1 / 2 MS medium supplemented with or without 50 mM or 75 mM NaCl . Pictures were taken elongated root was determined 3 d

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Three-day-old seedlings of Col-0, MPK3SR, MPK3SRarr1/12, and arr1/12 were transferred to 0.5 μM NA-PP1-containing 1/2 MS medium for 2 d to inhibit MPK3TG in MPK3SR and MPK3SRarr1/12 and then the plants were transferred to 0.2 μM NA-PP1-containing 1/2 MS medium supplemented with or without 50 mM or 75 mM NaCl. Pictures were taken and the elongated root length was determined 3 d later


A - C . Positioning of Upf1 , Upf2 Upf3 in abundance distribution of yeast , on data compiled by Ho et al . , 2018 ( A )

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A-C. Positioning of Upf1, Upf2 and Upf3 in the abundance distribution of all yeast proteins, based on the data compiled by Ho et al., 2018 (A)


distribution of abundance for identified by mass spectrometry in input ( B ) show expected bias for present in high copy

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The distribution of abundance for the proteins identified by mass spectrometry in input (B) show the expected bias for proteins present in high copy numbers


distribution of abundance for identified by mass spectrometry i purified samples ( C ) , show expected bias for present in high copy numbers . grey colour highlights positions of that were only identified in Upf1 - TAP samples ( 6 replicates ) not in total extract ( 3 replicates )

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The distribution of abundance for the proteins identified by mass spectrometry i purified samples (C), show the expected bias for proteins present in high copy numbers. Light grey colour highlights the positions of proteins that were only identified in the Upf1-TAP samples (6 replicates) and not in the total extract (3 replicates)


D . Correlation of LTOP2 - level estimates in a total yeast extract with published abundance data ( Ho et al , 2018 )

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D. Correlation of LTOP2-based protein level estimates in a total yeast protein extract with published protein abundance data (Ho et al, 2018)


E . Distribution of for quantified in association with Upf1 ( LTOP2 score , log2 scale ) . Positioning of Upf1 is highlighted as a rectangle while Upf2 Upf3 are highlighted in red . grey indicates region of intensities corresponding to ribosomal F . Distribution of enrichment values for Upf1 associated on LTOP2 scores known abundance in a total yeast extract . Upf1 , Upf2 Upf3 positions are also highlighted as rectangles G . A combination of data presented in ( E ) ( F ) as a scatter plot , to see both amount enrichment of in Upf1 - TAP purified samples . horizontal axis represents LTOP2 score , with vertical axis showing enrichment over input values . Both axes use log2 transformed values

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E. Distribution of the intensity for the proteins quantified in association with Upf1 (LTOP2 score, log2 scale). Positioning of Upf1 is highlighted as a white rectangle while Upf2 and Upf3 are highlighted in red. The dark grey line indicates the region of intensities corresponding to ribosomal proteins F. Distribution of the protein enrichment values for Upf1 associated proteins based on LTOP2 scores and known protein abundance in a total yeast extract. Upf1, Upf2 and Upf3 positions are also highlighted as white rectangles G. A combination of the data presented in (E) and (F) as a scatter plot, to see both the amount and enrichment of proteins in Upf1-TAP purified samples. The horizontal axis represents the LTOP2 score, with the vertical axis showing enrichment over input values. Both axes use log2 transformed values


A - F . Enrichment values for identified in tagged Upf1 ( A ) , Upf2 ( B ) , Upf3 ( C ) , Nmd4 ( D ) , Ebs1 ( E ) , Dcp1 ( F ) purifications . Tagged are indicated in bold for each experiment . Black bars correspond to average enrichment values obtained in purifications done without RNase orange bars with an RNase A RNase T1 treatment . Error bars represent SD . Dots represent enrichment values for each in 6 replicates for Upf1 - TAP , 3 for Upf1 - TAP RNase , 6 for Upf2 - TAP , 3 for Upf2 - TAP RNase , 5 for Upf3 - TAP , 3 for Upf3 - TAP RNase , 4 for Nmd4 - TAP , 3 for Nmd4 - TAP RNase , 3 for Ebs1 - TAP , with without RNase , 3 for Dcp1 - TAP with or without RNase . Only enriched by a factor of 16 or more in one of purifications presented here are shown . For clarity , groups of related were combined ( group of Lsm1 to Lsm7 is marked as " Lsm1 - 7 " values correspond to mean of values found in purifications ; CK2 group corresponds to Cka1 , Cka2 , Ckb1 Ckb2 )

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A-F. Enrichment values for proteins identified in tagged Upf1 (A), Upf2 (B), Upf3 (C), Nmd4 (D), Ebs1 (E), and Dcp1 (F) purifications. Tagged proteins are indicated in bold for each experiment. Black bars correspond to average enrichment values obtained in purifications done without RNase and orange bars with an RNase A and RNase T1 treatment. Error bars represent SD. Dots represent individual enrichment values for each protein in the 6 replicates for Upf1-TAP, 3 for Upf1-TAP and RNase, 6 for Upf2-TAP, 3 for Upf2-TAP and RNase, 5 for Upf3-TAP, 3 for Upf3-TAP and RNase, 4 for Nmd4-TAP, 3 for Nmd4-TAP and RNase, 3 for Ebs1-TAP, with and without RNase, and 3 for Dcp1-TAP with or without RNase. Only proteins enriched by a factor of 16 or more in one of the purifications presented here are shown. For clarity, groups of related proteins were combined (the group of Lsm1 to Lsm7 is marked as "Lsm1-7" and the values correspond to the mean of all the values found in purifications; CK2 group corresponds to Cka1, Cka2, Ckb1 and Ckb2)


G . Immunoblot validation of Upf1 - TAP interactions with HA tagged Nmd4 Ebs1 . Control strains did not express Upf1 -

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G. Immunoblot validation of Upf1-TAP interactions with HA tagged Nmd4 and Ebs1. Control strains did not express Upf1-TAP


H . Immunoblot validation of Upf1 - TAP interaction with Pat1 - HA , Lsm1 - HA Edc3 - HA . purification was performed with a mix of three strains , expressing Upf1 - TAP or not ( control )

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H. Immunoblot validation of Upf1-TAP interaction with Pat1-HA, Lsm1-HA and Edc3-HA. The purification was performed with a mix of three strains, expressing Upf1-TAP or not (control)


B - D . Results of purifications using Upf1 - FL ( B ) , Upf1 - CH ( C as bait with x - axis showing average enrichment value ( log2 scale ) for Upf1 - decapping Upf1 - 23

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B-D. Results of purifications using Upf1-FL (B), Upf1-CH (C as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components


B - D . Results of purifications usin Upf1 - Cter ( D ) as bait with x - axis showing average enrichment value ( log2 scale ) for Upf1 - decapping Upf1 - 23 components . colour of bars illustrates treatment used during purification , black without RNase , orange with an RNase treatment . Error bars represent SD . dots correspond to enrichment values obtained in

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B-D. Results of purifications usin Upf1-Cter (D) as bait with the x-axis showing the average enrichment value (log2 scale) for Upf1-decapping and Upf1-23 components. The colour of the bars illustrates the treatment used during the purification, black without RNase, and orange with an RNase treatment. Error bars represent SD. White dots correspond to enrichment values obtained in individual experiments


E . Comparison of enrichment of Upf1 , Nmd4 Ebs1 in purification of Upf1 fragments . Upf1 C - terminal region ( 854 - 971 ) affected association with Nmd4 or Ebs1 ( Student t - with a one - sided alternative hypothesis ) . We compared enrichment of each of Ebs1 Nmd4 between purifications of Upf1 fragments having this region ( Upf1 - FL Upf1 - HD - Cter , 6 experiments ) purifications of Upf1 fragments lacking extension ( Upf1 - CH - HD Upf1 - HD , 4 experiments ) . C - terminal region had no effect on Nmd4 enrichment ( p - value ≈ 0 . 98 ) , whereas there was significantly less associated Ebs1 on this small region ( p - value ≈ 0 . 0013 )

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E. Comparison of the enrichment of Upf1, Nmd4 and Ebs1 in the purification of the Upf1 fragments. The Upf1 C-terminal region (854-971) affected the association with Nmd4 or Ebs1 (Student t-test with a one-sided alternative hypothesis). We compared the enrichment of each of Ebs1 and Nmd4 between purifications of Upf1 fragments having this region (Upf1-FL and Upf1-HD-Cter, 6 experiments) and purifications of Upf1 fragments lacking the extension (Upf1-CH-HD and Upf1-HD, 4 experiments). The C-terminal region had no effect on Nmd4 enrichment (p-value ≈ 0.98), whereas there was significantly less associated Ebs1 on this small region (p-value ≈ 0.0013)


F . Western blot of input eluates of Upf1 domains purification in a Nmd4 - HA . band with # might corresponds to a dimer of Upf1 - CH , bands marked with a star correspond to residual signal with anti - HA antibodies ( Nmd4 ) . Fragments in eluate have a smaller because A part of tag was removed by digestion with TEV protease . G6PDH served as a loading control in input

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F. Western blot of input and eluates of Upf1 domains purification in a Nmd4-HA strain. The band with the # might corresponds to a dimer of Upf1-CH, bands marked with a star correspond to residual signal with the anti-HA antibodies (Nmd4). Fragments in the eluate have a smaller size because the protein A part of the tag was removed by digestion with the TEV protease. G6PDH served as a loading control in the input samples


A . Average enrichment of Upf1 - 23 Upf1 - decapping components in purified Upf1 - TAP in presence ( black bars ) absence ( blue bars ) of NMD4 . Pink blue vertical lines highlight of Upf1 - 23 Upf1 - decapping complexes respectively . dots correspond to enrichment values obtained in replicate experiments , 6 for Upf1 - TAP 3 for nmd4Δ condition . Error bars correspond to SD

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A. Average enrichment of Upf1-23 and Upf1-decapping components in purified Upf1-TAP in the presence (black bars) and absence (blue bars) of NMD4. Pink and dark blue vertical lines highlight proteins of the Upf1-23 and Upf1-decapping complexes respectively. White dots correspond to individual enrichment values obtained in replicate experiments, 6 for Upf1-TAP and 3 for the nmd4Δ condition. Error bars correspond to SD


B . Average enrichment of Upf1 - decapping components in Nmd4 - TAP in presence ( black bars ) absence ( blue bars ) of UPF1 . dots correspond to enrichment values obtained in replicate experiments , 4 for Nmd4 - TAP 2 for upf1Δ condition . Error bars correspond to SD

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B. Average enrichment of Upf1-decapping components in Nmd4-TAP in the presence (black bars) and absence (blue bars) of UPF1. White dots correspond to individual enrichment values obtained in replicate experiments, 4 for Nmd4-TAP and 2 for the upf1Δ condition. Error bars correspond to SD


D . CBP - Nmd4 was mixed with hUPF1 - HD or yUPF1 - HD ( overexpressed in E . coli purified ) . mixtures before ( input , 20 % of total ) or after purification on calmodulin affinity beads were separated on 10 % SDS - PAGE ( w / v ) acrylamide gels

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D. CBP-Nmd4 was mixed with hUPF1-HD or yUPF1-HD (all the proteins overexpressed in E. coli and purified). Protein mixtures before (input, 20% of total) or after purification on calmodulin affinity beads were separated on 10% SDS-PAGE (w/v) acrylamide gels


E . Upf1 interaction with a 30 - mer 3 ' biotinylated fragment was tested by mixing purified Upf1 helicase domain with Nmd4 testing fraction of recovered on streptavidin beads after washes with 150 mM 300 mM NaCl containing buffer solution

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E. Upf1 interaction with a 30-mer 3' biotinylated RNA fragment was tested by mixing purified Upf1 helicase domain with Nmd4 and testing the fraction of recovered protein on streptavidin beads after washes with 150 mM and 300 mM NaCl containing buffer solution


B . Scatter plot of mean fold change of transcripts in nmd4 ∆ RNAseq experiment against transcript mean fold change in ebs1 ∆ ( Pearson correlation , r = 0 . 66 , p - value < 2 . 10 - 16 ) . Black dots correspond to NMD substrate examples for which traces are shown in Fig . EV3 . Dashed lines represent boundaries of five bins of equal numbers of transcripts ( 2046 transcripts or notable features per bin ) , with bin 5 containing transcripts that were most increased in mutant condition compared with wild

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B. Scatter plot of the mean fold change of transcripts in nmd4∆ RNAseq experiment against transcript mean fold change in ebs1∆ (Pearson correlation, r = 0.66, p-value < 2.10-16). Black dots correspond to NMD substrate examples for which individual traces are shown in Fig. EV3. Dashed lines represent boundaries of five bins of equal numbers of transcripts (2046 transcripts or notable features per bin), with bin 5 containing the transcripts that were most increased in the mutant condition compared with wild type


C . of transcripts affected by UPF1 deletion ( increase by at least 1 . 4 fold ) among nmd4 ∆ bins , as defined in B . Differences between percentages of upf1 ∆ affected transcripts in bin 4 5 bin 3 4 were significant ( binomial , p < 10 - 24 ) . D . Same as in C , for ebs1 ∆ . Differences between distribution of transcripts in bin n bin n + 1 were significant ( binomial , p < 10 - 9 )

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C. Percentage of transcripts affected by UPF1 deletion (increase by at least 1.4 fold) among nmd4∆ bins, as defined in B. Differences between percentages of upf1∆ affected transcripts in bin 4 and 5 and bin 3 and 4 were significant (binomial test, p<10-24). D. Same as in C, for ebs1∆. Differences between distribution of transcripts in bin n and bin n+1 were significant (binomial test, p<10-9)


E . NMD efficiency of WT , upf1 ∆ or double mutant strains complemented with Upf1 - FL , Upf1 - HD - Cter or an empty plasmid . NMD efficiency for each is on reverse - transcription followed by quantitative PCR for RPL28 pre - mRNA . A wild - type has 100 % NMD efficiency a upf1 ∆ has 0 % NMD efficiency , by definition . Each value is an average of replicate experiments , 3 for WT condition , 5 for upf1Δ , 3 for ufp1Δ / upf2Δ , upf1Δ / ufp3Δ upf1Δ / ebs1Δ 4 for upf1Δ / nmd4Δ condition . Error bars represent SD . Dots represent values obtained in various replicate

O O O O O O O B-CONTROLLED_VAR O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O

E. NMD efficiency of WT, upf1∆ or double mutant strains complemented with Upf1-FL, Upf1-HD-Cter or an empty plasmid. The NMD efficiency for each strain is based on reverse-transcription followed by quantitative PCR for RPL28 pre-mRNA. A wild-type strain has 100% NMD efficiency and a upf1∆ strain has 0% NMD efficiency, by definition. Each value is an average of replicate experiments, 3 for the WT condition, 5 for upf1Δ, 3 for ufp1Δ/upf2Δ, upf1Δ/ufp3Δ and upf1Δ/ebs1Δ and 4 for the upf1Δ/nmd4Δ condition. Error bars represent SD. Dots represent values obtained in the various replicate experiments


G . Changes in steady - state level of reporter encoded HA - tagged in upf1Δ , nmd4Δ , ebs1Δ nmd4Δ / ebs1Δ strains were estimated by immunoblot from three independent experiments , with one example

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G. Changes in the steady-state level of the reporter encoded HA-tagged protein in upf1Δ, nmd4Δ, ebs1Δ and the nmd4Δ/ebs1Δ strains were estimated by immunoblot from three independent experiments, with one example shown


H . decay in a wild - type nmd4Δ / ebs1Δ strains was tested by reverse - transcription quantitative PCR specific to 5 " end of reporter at different times after transcription shut - off . quantifications represent mean amounts standard deviation of average ( three independent

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H. RNA decay in a wild-type and nmd4Δ/ebs1Δ strains was tested by reverse-transcription quantitative PCR specific to the 5" end of the reporter RNA at different times after transcription shut-off. The quantifications represent mean RNA amounts and standard deviation of the average (three independent experim


A - B . Comparison between average enrichment values for Upf1 - decapping Upf1 - 23 components , Lsm CK subunits in Upf1 - TAP purified samples in presence ( grey bars ) or absence ( blue bars ) of UPF2 ( AError bars represent SD . dots represent enrichment values for replicates , 6 for Upf1 - TAP , 3 for upf2Δ an

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O B-CONTROLLED_VAR O

A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1 -TAP purified samples in the presence (grey bars) or absence (blue bars) of UPF2 (AError bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for the upf2Δ an conditions


A - B . Comparison between average enrichment values for Upf1 - decapping Upf1 - 23 components , Lsm CK subunits in Upf1 - TAP purified samples in presence ( grey bars ) or absence ( blue bars ) o UPF3 ( B ) . Error bars represent SD . dots represent enrichment values for replicates , 6 for Upf1 - TAP , 3 for th upf3Δ

O O O O O O O O O O B-MEASURED_VAR O O B-MEASURED_VAR O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O B-CONTROLLED_VAR

A-B. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf1-TAP purified samples in the presence (grey bars) or absence (blue bars) o UPF3 (B). Error bars represent SD. White dots represent enrichment values for individual replicates, 6 for Upf1-TAP, 3 for th upf3Δ conditions


C - D . Evaluation of effects of UPF2 deletion on Upf3 - TAP ( C in total extracts , in comparison with a loading control ( G6PDH ) was done by

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C-D. Evaluation of the effects of UPF2 deletion on Upf3-TAP levels (C in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot


C - D . Evaluation of effect of UPF3 deletion on Upf2 - TAP ( D ) in total extracts , in comparison with a loading control ( G6PDH ) was done by

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C-D. Evaluation of the effect of UPF3 deletion on Upf2-TAP levels (D) in total extracts, in comparison with a loading control (G6PDH) was done by immunoblot


E . Comparison between average enrichment values for Upf1 - decapping Upf1 - 23 components , Lsm CK subunits in Upf2 - TAP purified samples in presence ( grey bars , 6 experiments ) or absence ( blue bars , 2 experiments ) of UPF1 . Error bars represent SD . of expression of Upf2 - TAP Upf3 - TAP have been verified in this condition ( Appendix Fig . S2 )

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E. Comparison between average enrichment values for Upf1-decapping and Upf1-23 components, Lsm proteins and CK subunits in Upf2-TAP purified samples in the presence (grey bars, 6 experiments) or absence (blue bars, 2 experiments) of UPF1. Error bars represent SD. The levels of expression of Upf2-TAP and Upf3-TAP proteins have been verified in this condition (Appendix Fig. S2)


A . Unspliced RPL28 , an NMD substrate , was enriched in Nmd4 - TAP purification in comparison with a control untagged , as measured by reverse transcription followed by quantitative PCR . Bars correspond to mean of pre - RPL28 enrichment for 3 independent experiments , as compared with RIM1 , a non - NMD mRNA . Error bars correspond to SD . indicated p - value was computed using Welch t - , single - sided comparison

O O O B-MEASURED_VAR O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. Unspliced RPL28, an NMD substrate, was enriched in Nmd4-TAP purification in comparison with a control untagged strain, as measured by reverse transcription followed by quantitative PCR. Bars correspond to the mean of pre-RPL28 enrichment for 3 independent experiments, as compared with RIM1, a non-NMD mRNA. Error bars correspond to SD. The indicated p-value was computed using the Welch t-test, single-sided comparison


B - D . Distribution of Nmd4 - TAP , Nog1 ( marker of free 60S subunits ) Rps8 ( marker of 40S , 80S polysome fractions ) in wild type ( B ) , upf1 ∆ ( C ) upf2 ∆ ( D ) strains , tested by immunoblot . Fractions 1 , 2 , 3 are fractions , fractions 4 , 5 6 correspond respectively to ribosomal 40S , 60S 80S positions , fractions 7 to 12 correspond to polysomes . of total signal for Nmd4 - TAP in three independent replicates along with standard deviation of values are indicated for each

O O O O O O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

B-D. Distribution of Nmd4-TAP, Nog1 (marker of free 60S subunits) and Rps8 (marker of 40S, 80S and polysome fractions) in wild type (B), upf1∆ (C) and upf2∆ (D) strains, tested by immunoblot. Fractions 1, 2, 3 are light fractions, fractions 4, 5 and 6 correspond respectively to ribosomal 40S, 60S and 80S positions, fractions 7 to 12 correspond to polysomes. The percent of total signal for Nmd4-TAP in three independent replicates along with standard deviation of the values are indicated for each fraction


E . Quantified relative changes in Nmd4 - TAP average in monosome / polysome fractions in upf1 ∆ upf2 ∆ strains ( 100 % correspond to in wild type ) . Indicated p - values correspond to Welch t - , single - sided comparison of 4 replicate experiments ( values are indicated as dots ) . Error bars correspond to SD

O O O O O O B-MEASURED_VAR O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Quantified relative changes in Nmd4-TAP average levels in light and monosome/polysome fractions in upf1∆ and upf2∆ strains (100% correspond to levels in the wild type strain). Indicated p-values correspond to the Welch t-test, single-sided comparison of 4 replicate experiments (individual values are indicated as dots). Error bars correspond to SD


a ) number of somatic mutations discovered from each paired NPC case subjected to WES key clinical parameters ( Supplementary Table 2a ) clustered according to clinical stage . Indel , insertion or deletion . ( b ) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC . font of nucleotides at 3 ′ 5 ′ positions is proportional to their frequencies ( Supplementary Fig . 3b ) .

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a) The number of somatic mutations discovered from each paired NPC case subjected to WES and key clinical parameters (Supplementary Table 2a) clustered according to the clinical stage. Indel, insertion or deletion. (b) Trinucleotide contexts of somatic mutations occurring at cytosine nucleotides in NPC. The font size of the nucleotides at the 3′ and 5′ positions is proportional to their frequencies (Supplementary Fig. 3b).


( c ) Rates of arm - level focal SCNVs ( Online Methods ) across different types of malignancies . Data regarding SCNVs of following types were summarized from Zack et al . : RCC , renal carcinoma ; GBM , glioblastoma multiforme ; UCEC , uterine cervix ; COAD , colorectal adenocarcinoma ; BRCA , breast ; HNSC , head neck squamous ; OV , ovary ; LUAD , lung adenocarcinoma ; BLCA , bladder ; LUSC , lung squamous .

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(c) Rates of arm-level and focal SCNVs (Online Methods) across different types of malignancies. Data regarding SCNVs of the following types were summarized from Zack et al.: RCC, renal cell carcinoma; GBM, glioblastoma multiforme; UCEC, uterine cervix; COAD, colorectal adenocarcinoma; BRCA, breast; HNSC, head and neck squamous cell; OV, ovary; LUAD, lung adenocarcinoma; BLCA, bladder; LUSC, lung squamous cell.


Data matrix showing frequent SCNVs , significantly mutated genes ( bold ; Benjamini - Hochberg FDR 0 . 2 calculated with CHASM ) their associated pathway or family mutations discovered in cases with NPC analyzed by WES , targeted deep sequencing ( TS ) or SNP array ( SNP - a ) . frequencies of alterations are plotted on right . colors shapes denoting different types of somatic events are also applied in Figure 5a . Columns , examined cases ; rows , genes ; hot spot , identical mutations have been registered in COSMIC ( see URLs ) ; LOH , loss of heterozygosity . asterisk indicates that this was also subjected to SNP array profiling . PIK3CA amplification in C666 has been previously described .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O

Data matrix showing frequent SCNVs, significantly mutated genes (bold; Benjamini-Hochberg FDR 0.2 calculated with CHASM) and their associated pathway or family mutations discovered in cases with NPC analyzed by WES, targeted deep sequencing (TS) or SNP array (SNP-a). The frequencies of the alterations are plotted on the right. The colors and shapes denoting different types of somatic events are also applied in Figure 5a. Columns, examined cases; rows, genes; hot spot, identical mutations have been registered in COSMIC (see URLs); LOH, loss of heterozygosity. The asterisk indicates that this tumor was also subjected to SNP array profiling. PIK3CA amplification in C666 cells has been previously described.


( b , c ) Significant correlations between dysregulation of modification total number of somatic mutations ( b ) overall survival probability ( c ) . Mann - Whitney ( b ) log - rank ( c ) were used to calculate statistical significance . Horizontal bars , mean value ; HR , hazard .

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(b,c) Significant correlations between dysregulation of chromatin modification and total number of somatic mutations (b) and overall survival probability (c). Mann-Whitney test (b) and log-rank test (c) were used to calculate statistical significance. Horizontal bars, mean value; HR, hazard ratio.


( a - d ) NPC stably expressing either negative control shRNA ( Scramble ) or pooled shRNAs targeting ARID1A ( shARID1A ) were subjected to anchorage - independent colony formation ( a ) , migration ( b ) , xenograft growth ( horizontal lines , mean values ) western blot ( WB ) with indicated antibodies ( d ) .

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(a-d) NPC cells stably expressing either negative control shRNA (Scramble) or pooled shRNAs targeting ARID1A (shARID1A) were subjected to anchorage-independent colony formation assay (a), migration assay (b), xenograft growth assay (horizontal lines, mean values) and western blot (WB) assay with the indicated antibodies (d).


( e - g ) NPC either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation ( e )

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(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e)


( e - g ) NPC either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation ( e ) , migration ( f )

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(e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f)


e - g ) NPC either with or without exogenously expressing ARID1A were examined by anchorage - independent colony formation ( e ) , migration ( f ) WB with indicated antibodies ( g ) .

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e-g) NPC cells either with or without exogenously expressing ARID1A were examined by anchorage-independent colony formation assay (e), migration assay (f) and WB assay with the indicated antibodies (g).


( h , i ) NPC either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation ( h )

(h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h)


h , i ) NPC either with or without exogenously expressing BAP1 were examined by anchorage - independent colony formation ( h ) WB with indicated antibodies ( i ) . β - actin was examined as a loading control . data shown in a , b , e , f h represent mean ± s . d . ; n = 3 for a , b , e f ; n = 4 for h . * P 0 . 05 , * * P 0 . 01 . Student ' s t - was used to calculate statistical significance in a - c , e , f h ; blots shown in d , g i are representative of three total blots run .

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h,i) NPC cells either with or without exogenously expressing BAP1 were examined by anchorage-independent colony formation assay (h) and WB assay with indicated antibodies (i). β-actin was examined as a loading control. The data shown in a, b, e, f and h represent the mean ± s.d.; n = 3 for a, b, e and f; n = 4 for h. *P 0.05, **P 0.01. Student's t-test was used to calculate statistical significance in a-c, e, f and h; the blots shown in d, g and i are representative of three total blots run.


( a ) Data matrix showing alterations of genes involved in ERBB - PI3K signaling pathway their related targeting molecules . Considering space limitations , numbers of targeting agents available for each are shown in parentheses . Drugs currently in phase 2 clinical trials are marked in blue , those in phase 3 are in purple , FDA - approved drugs are in brown ( see URLs ) .

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(a) Data matrix showing alterations of genes involved in the ERBB-PI3K signaling pathway and their related targeting molecules. Considering space limitations, the numbers of targeting agents available for each protein are shown in parentheses. Drugs currently in phase 2 clinical trials are marked in blue, those in phase 3 are in purple, and FDA-approved drugs are in brown (see URLs).


( b ) Kaplan - Meier survival curves of NPC subjects either with or without genetic lesions in ERBB - PI3K signaling pathway . Log - rank was used to calculate statistical significance .

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(b) Kaplan-Meier survival curves of NPC subjects either with or without genetic lesions in the ERBB-PI3K signaling pathway. Log-rank test was used to calculate statistical significance.


C Relative mRNA expression of Zeb1 , Acp5 , Ctsk during differentiation from BMDMs to osteoclasts in culture ( n = 3 ) .

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C Relative mRNA expression of Zeb1, Acp5, and Ctsk during the differentiation from BMDMs to osteoclasts in culture (n = 3).


D Western blot of Zeb1 , c - Src , Ctsk expression in BMDMs mature osteoclasts ( n = 3 ) .

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D Western blot of Zeb1, c-Src, and Ctsk expression in BMDMs and mature osteoclasts (n = 3).


F Zeb1 ( red ) TRAP ( green ) immunofluorescence of a femur section including growth plate ( GP ) metaphysis from a wild - type mouse . multinucleated associated with surface of bone marrow cavity ( BM cavity ) that are shown in red box are further enlarged in panels to right . Scale bar , 10 μm .

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F Zeb1 (red) and TRAP (green) immunofluorescence of a femur section including the growth plate (GP) and metaphysis from a wild-type mouse. The multinucleated cells associated with the surface of the bone marrow cavity (BM cavity) that are shown in the red box are further enlarged in the panels to the right. Scale bar, 10 μm.


A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of distal femur trabeculae of 3 - month - old wild - type Zeb1ΔM / ΔM mice are shown . genotype of Zeb1 + / + , Zeb1f / + , Zebf / f mice is defined as " wild - type " control . Scale bar , 500 μm . B of BV / TV , Tb . Th , Tb . N , Tb . Sp as determined by nanoCT in 3 - month - old wild - type Zeb1ΔM / ΔM mice ( n = 8 ) .

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A Representative nanoCT of sagittal sections of femurs with 3D reconstruction of the distal femur trabeculae of 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown. The genotype of Zeb1+/+, Zeb1f/+, Zebf/f mice is defined as "wild-type" control. Scale bar, 500 μm. B Quantification of BV/TV, Tb.Th, Tb.N, and Tb.Sp as determined by nanoCT in 3-month-old wild-type and Zeb1ΔM/ΔM male mice (n = 8). C


C H & E TRAP of distal femurs of 3 - month - old wild - type Zeb1ΔM / ΔM mice . Scale bar , 200 μm . D of osteoclast number per bone surface ( N . Oc / BS ) eroded surface per bone surface ( ES / BS ) , as well as serum CTX - I OCN in 3 - month - old wild - type Zeb1ΔM / ΔM mice are shown ( n = 6 ) .

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C H&E and TRAP staining of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice. Scale bar, 200 μm. D Quantification of osteoclast number per bone surface (N.Oc/BS) and eroded surface per bone surface (ES/BS), as well as serum CTX-I and OCN levels in 3-month-old wild-type and Zeb1ΔM/ΔM male mice are shown (n = 6). E


E Golden ' s trichrome illuminating howship ' s lacunae ( arrow ) of distal femurs of 3 - month - old wild - type Zeb1ΔM / ΔM mice were performed along with double - calcein bone labeling confocal imaging to assess bone formation . Scale bar , 10 μm . F of MAR BFR as assessed in 3 - month - old wild - type Zeb1ΔM / ΔM mice ( n = 6 ) .

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E Golden's trichrome staining illuminating howship's lacunae (arrow) of the distal femurs of 3-month-old male wild-type and Zeb1ΔM/ΔM mice were performed along with double-calcein bone labeling and confocal imaging to assess bone formation. Scale bar, 10 μm. F Quantification of MAR and BFR as assessed in 3-month-old male wild-type and Zeb1ΔM/ΔM mice (n = 6). D


B Zeb1 , NFATc1 , c - Fos , c - Src , β3 integrin , Mmp9 , Mmp14 , Ctsk expression as assessed by Western blot in wild - type Zeb1ΔM / ΔM BMDMs during osteoclast differentiation ( n = 3 ) .

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B Zeb1, NFATc1, c-Fos, c-Src, β3 integrin, Mmp9, Mmp14, and Ctsk expression as assessed by Western blot in wild-type and Zeb1ΔM/ΔM BMDMs during osteoclast differentiation (n = 3).


G Supernatant CTX - I determined by ELISA ( n = 6 ) .

G Supernatant CTX-I determined by ELISA (n = 6).


H , I After a 6 - day culture atop bone slice , phalloidin ( red ) was performed in wild - type versus Zeb1ΔM / ΔM osteoclasts ( I ) , actin ring per quantified ( H ) . Scale bar , 20 μm .

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H, I After a 6-day culture atop bone slice, phalloidin staining (red) was performed in wild-type versus Zeb1ΔM/ΔM osteoclasts (I), and actin ring area per cell quantified (H). Scale bar, 20 μm.


E Relative mRNA expression of Ckmt1 in BMDMs osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice .

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E Relative mRNA expression of Ckmt1 in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice.


F MtCK1 , Ckb , VDAC , Tomm20 expression as assessed by Western blot in BMDMs osteoclasts generated from wild - type or Zeb1ΔM / ΔM mice ( n = 3 ) .

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F MtCK1, Ckb, VDAC, and Tomm20 expression as assessed by Western blot in BMDMs and osteoclasts generated from wild-type or Zeb1ΔM/ΔM mice (n = 3).


H MtCK1 ( green ) immunofluorescence of wild - type Zeb1ΔM / ΔM osteoclasts . Scale bar , 20

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H MtCK1 (green) immunofluorescence of wild-type and Zeb1ΔM/ΔM osteoclasts. Scale bar, 20 μ


A Phosphocreatine ( PCr ) creatine ( Cr ) level , as well as PCr / Cr of wild - type or Zeb1ΔM / ΔM osteoclasts cultured atop plastic substrata as determined by LC / MS ( n = 3 ) .

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A Phosphocreatine (PCr) and creatine (Cr) level, as well as PCr/Cr ratio of wild-type or Zeb1ΔM/ΔM osteoclasts cultured atop plastic substrata as determined by LC/MS analysis (n = 3).


B , C Oxygen consumption rate ( OCR ) profile plot ( B ) mitochondrial function parameters ( C ) analyzed by XF Mito Stress in osteoclasts from wild - type or Zeb1ΔM / ΔM mice after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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B, C Oxygen consumption rate (OCR) profile plot (B) and mitochondrial function parameters (C) analyzed by XF Cell Mito Stress Assay in osteoclasts from wild-type or Zeb1ΔM/ΔM mice after sequential treatment of compounds modulating mitochondrial function (n = 4).


C Wild - type BMDMs were transduced with either a mock vector , wild - type human MtCK1 , or a catalytically - inactive MtCK1C316G mutant expression vector , differentiated into osteoclasts , lysates collected for MtCK1 , Ckb , Tomm20 , VDAC , Zeb1 , c - Src , Ctsk immunoblotting ( n = 3 ) .

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C Wild-type BMDMs were transduced with either a mock vector, wild-type human MtCK1, or a catalytically-inactive MtCK1C316G mutant expression vector, differentiated into osteoclasts, and cell lysates collected for MtCK1, Ckb, Tomm20, VDAC, Zeb1, c-Src, and Ctsk immunoblotting (n = 3).


E PCr / Cr of mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts cultured on plastic substrata as determined by LC / MS ( n = 3 ) .

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E PCr/Cr ratio of mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts cultured on plastic substrata as determined by LC/MS analysis (n = 3).


OCR profile plot ( H ) analyzed by XF Mito Stress in mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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OCR profile plot (H) analyzed by XF Cell Mito Stress Assay in mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


J , K Mock vector - , MtCK1 - , or MtCK1C316G - transduced wild - type pre - osteoclasts were cultured atop bone slices for 3 days , stained with phalloidin ( red ) . Osteoclasts were removed resorption pits visualized by WGA - DAB ( J ) . actin ring per resorption pit were quantified ( K ; n = 6 ) . Scale bar , upper 20 μm , lower 100 μm .

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J, K Mock vector-, MtCK1-, or MtCK1C316G-transduced wild-type pre-osteoclasts were cultured atop bone slices for 3 days, stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (J). The actin ring area per cell and resorption pit area were quantified (K; n = 6). Scale bar, upper 20 μm, lower 100 μm.


A , B Human monocyte - derived macrophages ( hMDMs ) were transfected with either shCTRL or siRNA targeting ZEB1 , differentiated into osteoclasts , relative mRNA expression of CKMT1 ( A ) mitochondrial creatine kinase activity determined ( B ; n = 3 ) .

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A, B Human monocyte-derived macrophages (hMDMs) were transfected with either shCTRL or siRNA targeting ZEB1, differentiated into osteoclasts, and relative mRNA expression of CKMT1 (A) and mitochondrial creatine kinase activity determined (B; n = 3).


F Mitochondrial function parameters analyzed by XF Mito Stress in non - targeting siCTRL - or siZEB1 - transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function ( n = 4 ) .

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F Mitochondrial function parameters analyzed by XF Cell Mito Stress Assay in non-targeting siCTRL- or siZEB1-transfected osteoclasts after sequential treatment of compounds modulating mitochondrial function (n = 4).


G , H siCTRL - or siZEB1 - transfected human osteoclasts were cultured atop bone slices for 6 days stained with phalloidin ( red ) . Osteoclasts were removed resorption pits visualized by WGA - DAB ( G ) . Scale bar , upper 20 μm , lower 100 μm . actin ring per , resorption pit , number of TRAP + MNCs were quantified ( H ; n = 6 ) .

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G, H siCTRL- or siZEB1-transfected human osteoclasts were cultured atop bone slices for 6 days and stained with phalloidin (red). Osteoclasts were removed and resorption pits visualized by WGA-DAB staining (G). Scale bar, upper 20 μm, lower 100 μm. The actin ring area per cell, resorption pit area, and the number of TRAP+ MNCs were quantified (H; n = 6).


I - L Calvaria isolated from wild - type Zeb1ΔM / ΔM mice were cultured in presence or absence of 200 μM cyclocreatine , stained for phalloidin ( red ) Vpp3 ( green ) ( I ) . Scale bar , 10 μm . TRAP activity of whole lysates , actin ring per , supernatant CTX - I were quantified ( J - L ; n = 6 ) .

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I-L Calvaria isolated from wild-type and Zeb1ΔM/ΔM mice were cultured in the presence or absence of 200 μM cyclocreatine, and stained for phalloidin (red) and Vpp3 (green) (I). Scale bar, 10 μm. The TRAP activity of whole cell lysates, actin ring area per cell, and supernatant CTX-I were quantified (J-L; n = 6).


( B ) Density of HEAP - AGO2 peaks across lncRNAs ( n = 62 , blue ) 3 ' UTR regions of mRNAs ( n = 8798 , red ) with experimental evidence for AGO2 binding in mESCs ( > 0 AGO2 peaks ) . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution whiskers represent 5th 95th quantiles of distribution .

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(B) Density of HEAP-AGO2 peaks across cytosolic lncRNAs (n=62, blue) and the 3'UTR regions of mRNAs (n=8798, red) with experimental evidence for AGO2 binding in mESCs (>0 AGO2 peaks). Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( D ) Distribution of fold - change ( FC ) in degradation rate of mRNAs ( n = 13296 , red ) , micropeptide encoding transcripts ( n = 43 , pink ) , bona fide ( n = 759 , blue ) ( n = 4299 , grey ) lncRNAs in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) after 8 days treatment , horizontal dashed represents a KO / WT FC in degradation rate of 1 . Statistics : NS - p > 0 . 05 , * - p - value < 0 . 05 . Two - Mann - Whitney U : p - value = 0 . 044 . Central band of boxplot represents median , box depicts 25 - 75 quantiles of distribution whiskers represent 5th 95th quantiles of distribution .

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(D) Distribution of the fold-change (FC) in degradation rate of mRNAs (n=13296, red), micropeptide encoding transcripts (n=43, pink), bona fide cytosolic (n=759, blue) and nuclear (n=4299, grey) lncRNAs in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days treatment, horizontal dashed line represents a KO/WT FC in degradation rate of 1. Statistics: NS-p>0.05, *-p-value<0.05. Two-tailed Mann-Whitney U test: p-value=0.044. Central band of boxplot represents median, box depicts 25-75 quantiles of distribution and whiskers represent the 5th and 95th quantiles of the distribution.


( F ) Distribution of fold - change ( FC ) in degradation rates of mRNAs ( n = 29900 , red ) , ( n = 474 , blue ) ( n = 2348 , grey ) lncRNAs , in 4 - OHT treated ( KO ) relative to ethanol treated ( WT ) after 8 days of treatment ( estimated on 10 minutes 4sU ) , horizontal dashed represents a KO / WT FC in degradation rate of 1 Statistics : NS - p - value > 0 . 05 anband of boxplot represents median box depicts quantiles distribution whiskers represent > th 95th quantiles of distribution . ( two - Mann - Whitney U ) .

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(F) Distribution of the fold-change (FC) in degradation rates of mRNAs (n=29900, red), cytosolic (n=474, blue) and nuclear (n=2348, grey) lncRNAs, in 4-OHT treated (KO) relative to ethanol treated (WT) cells after 8 days of treatment (estimated based on the 10 minutes 4sU pulse), horizontal dashed line represents a KO/WT FC in degradation rate of 1 Statistics: NS-p-value>0.05 anband of boxplot represents median box depicts quantiles distribution and whiskers represent the>th and 95th quantiles of the distribution. (two-tailed Mann-Whitney U test).


( B ) Fraction of lncRNAs with experimental evidence for ribosomal binding with ( red ) or without ( blue ) an overlapping conserved short open reading frame .

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(B) Fraction of cytosolic lncRNAs with experimental evidence for ribosomal binding with (red) or without (blue) an overlapping conserved short open reading frame.


( B ) Expression of GFP , lncRNA - c1 , GFP - lncRNA - c1 , lncRNA - c2 GFP - lncRNA - c2 ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted ( KO ) relative to ethanol treated ( WT ) mESC ( y - axis ) 24h hours post - transfection . Four independent biological replicates were treated , transfected analyzed by RT - qPCR . Statistical significance represented on figure on comparison of KO / WT fold change in expression of GFP , with GFP - lncRNA - c1 with GFP - lncRNA - c2 , ( paired two - t - p - value = 0 . 034 0 . 032 respectively ) on comparison of KO / WT fold change in expression of GFP - lncRNAc1 with lncRNA - c1 GFP - lncRNA - c2 with lncRNA - c2 ( paired two - t - p - value = 0 . 012 0 . 018 respectively ) . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Data information : For RTqPCR analyses , transcript expression was first normalized by amount of Act - β PolII next by total amount of transfected vectors per estimated on of relative Neomycin expression . Each point corresponds to results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 * * * - p - value < 0 . 001 .

O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(B) Expression of GFP, lncRNA-c1, GFP-lncRNA-c1, lncRNA-c2 and GFP-lncRNA-c2 (x-axis) in 8 day 4-OHT treated, miRNA depleted cells (KO) relative to ethanol treated (WT) mESC (y-axis) 24h hours post-transfection. Four independent biological replicates were treated, transfected and analyzed by RT-qPCR. Statistical significance represented on the figure based on comparison of KO/WT fold change in expression of GFP, with GFP-lncRNA-c1 and with GFP-lncRNA-c2, (paired two-tailed t-test p-value= 0.034 and 0.032 respectively) and based on comparison of KO/WT fold change in expression of GFP-lncRNAc1 with lncRNA-c1 and GFP-lncRNA-c2 with lncRNA-c2 (paired two-tailed t-test p-value= 0.012 and 0.018 respectively). Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( C ) Expression of GFP - lncRNA - c1 ΔMRE relative to GFP - lncRNA - c1 ( y - axis ) in ethanol treated ( WT , circles ) or 4 - OHT treated , miRNA - depleted ( KO , triangles , x - axis ) . Four independent biological replicates were treated , transfected analyzed by RTqPCR . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Paired two - t - p - value = 0 . 0071 Data information : For RTqPCR analyses , transcript expression was first normalized by amount of Act - β PolII next by total amount of transfected vectors per estimated on of relative Neomycin expression . Each point corresponds to results of one independent biological replicate . Statistics : NS - p - value > 0 . 05 , * - p - value < 0 . 05 , * * - p - value < 0 . 01 * * * - p - value < 0 . 001 .

O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(C) Expression of GFP-lncRNA-c1 ΔMRE relative to GFP-lncRNA-c1 (y-axis) in ethanol treated (WT, circles) or 4-OHT treated, miRNA-depleted cells (KO, triangles, x-axis). Four independent biological replicates were treated, transfected and analyzed by RTqPCR. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0071 Data information: For all RTqPCR analyses, transcript expression was first normalized by the amount of Act-β and PolII and next by the total amount of transfected vectors per cell estimated based on the levels of relative Neomycin expression. Each point corresponds to the results of one independent biological replicate. Statistics: NS- p-value> 0.05, *-p-value<0.05, **-p-value<0.01 and ***-p-value<0.001.


( F ) Immunoblot of GFP ( GFP ) in extracts from mESCs transfected with mock , BoxB ( - 30 ) - GFP GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted ( G ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) BoxB ( - 30 ) - GFP - lncRNA - c1 , BoxB ( - 30 ) - GFP - lncRNA - c2 ( BoxB ( - 30 ) , triangles ) ( x - axis ) 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates were analyzed . Data is represented as mean ± SD Each point corresponds to results of one independent biological replicate . Paired two - t - p - value = 0 . 0494 for GFP - lncRNA - c1 p - value = 0 . 0355 for GPF - lncRNA - c2Data information : Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data .

O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O

(F) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(-30)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted (G) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(-30)-GFP-lncRNA-c1, BoxB (-30)-GFP-lncRNA-c2 (BoxB(-30), triangles) (x-axis) 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates were analyzed. Data is represented as mean±SD and Each point corresponds to the results of one independent biological replicate. Paired two-tailed t-test p-value= 0.0494 for GFP-lncRNA-c1 and p-value=0.0355 for GPF-lncRNA-c2Data information: Uncropped blots used for assembly of panels F are provided in Figure 4 Source Data.


( H ) Immunoblot of GFP ( GFP ) in extracts from mESCs transfected with mock , BoxB ( + 339 ) - GFP GFP expressing vectors . ACTIN - β ( ACT - β ) was used as an internal control . One representative blot is depicted . ( I ) Fold - change ( FC ) in normalized expression of GFP - lncRNA - c1 , GFP - lncRNA - c2 ( noBoxB , circles ) BoxB ( + 339 ) - GFP - lncRNA - c1 , BoxB ( + 339 ) - GFP - lncRNA - c2 ( BoxB ( + 339 ) , triangles ) ( x - axis ) in 8 day 4 - OHT treated , miRNA depleted mESCs ( KO ) relative to ethanol treated mESCs ( WT ) ( y - axis ) . Four independent biological replicates analyzed . Data information : Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data .

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(H) Immunoblot analysis of GFP (GFP) in protein extracts from mESCs transfected with mock, BoxB(+339)-GFP and GFP expressing vectors. ACTIN-β (ACT-β) was used as an internal control. One representative blot is depicted. (I) Fold-change (FC) in normalized expression of GFP-lncRNA-c1, GFP-lncRNA-c2 (noBoxB, circles) and BoxB(+339)-GFP-lncRNA-c1, BoxB (+339)-GFP-lncRNA-c2 (BoxB(+339), triangles) (x-axis) in 8 day 4-OHT treated, miRNA depleted mESCs (KO) relative to ethanol treated mESCs (WT) (y-axis). Four independent biological replicates analyzed.Data information: Uncropped blots used for assembly of panels H are provided in Figure 4 Source Data.