A . Ribbon diagram of yeastMdm12 dimer . crystal structure of full - Mdm12 was determined by SAD refined with native data to 3 . 1 Å resolution . Lipids bound to Mdm12 are drawn with black stick models .

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 B-MEASURED_VAR O O O O O O O

A. Ribbon diagram of the yeastMdm12 dimer. The crystal structure of full-length Mdm12 was determined by SAD and refined with native data to 3.1 Å resolution. Lipids bound to Mdm12 are drawn with black stick models.


B . Schematic diagram indicating secondary structure elements their organization in Mdm12 . Three structural elements of Mdm12 are highlighted in different colored boxes .

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

B. Schematic diagram indicating the secondary structure elements and their organization in Mdm12. Three structural elements of Mdm12 are highlighted in different colored boxes.


C , D . Surface representations of Mdm12 dimer are shown in different orientations .

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

C, D. Surface representations of the Mdm12 dimer are shown in different orientations.


A . Ribbon diagram showing 2 - fold dimerization interface of Mdm12 . Oxygen nitrogen atoms are shown in red blue , respectively . orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12 . alignment of yeastMdm12 orthologs is shown to highlight conservation in N - terminus β1 - strand . Ten orthologs are aligned from residues 1 to 11 . Absolute highly conserved residues are indicated in red orange , respectively .

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

A. Ribbon diagram showing the 2-fold dimerization interface of Mdm12. Oxygen and nitrogen atoms are shown in red and blue, respectively. The orange dotted lines indicate intermolecular hydrogen bonds between two protomers of Mdm12. The sequence alignment of yeastMdm12 orthologs is shown to highlight the sequence conservation in the N-terminus β1-strand. Ten orthologs are aligned from residues 1 to 11. Absolute and highly conserved residues are indicated in red and orange, respectively.


B . molecular of Mdm12 ( I5P ) mutant was measured by - exclusion chromatography ( below ) ultracentrifugation ( top ) as in Figure 1B C .

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

B. The molecular weight of the Mdm12 (I5P) mutant was measured by size-exclusion chromatography (below) and ultracentrifugation (top) as in Figure 1B and C.


C . Ribbon diagram showing structures of SMP domain in E - SYT2 , CETP , BPI for comparison of dimeric interfaces among SMP domains . Note that CETP BPI are not dimers but monomers containing two tandem SMP domains .

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

C. Ribbon diagram showing the structures of the SMP domain in E-SYT2, CETP, and BPI for the comparison of dimeric interfaces among SMP domains. Note that CETP and BPI are not dimers but monomers containing two tandem SMP domains.


A . Simulated annealing omit map ( Fo - Fc , contoured at 1 . 5σ ) showing molecule bound to Mdm12 ( left ) . final model for bound PE is shown as in stick representation . electron density ( 2Fo - Fc ) calculated in final model is shown with stick model of PE in right ( 3 . 1 Å resolution , contoured at 0 . 8σ ) .

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 O O O O O O O O O O O O

A. Simulated annealing omit map (Fo-Fc, contoured at 1.5σ) showing the molecule bound to Mdm12 (left). The final model for the bound PE is shown as in stick representation. The electron density (2Fo-Fc) calculated in the final model is shown with the stick model of PE in the right (3.1 Å resolution, contoured at 0.8σ).


B . Surface representation of Mdm12 dimer . Hydrophobic lining Mdm12 channel are indicated by a blue mesh . Lipids built in Mdm12 are in space - filling representation .

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

B. Surface representation of the Mdm12 dimer. Hydrophobic amino acids lining the Mdm12 channel are indicated by a blue mesh. Lipids built in Mdm12 are in space-filling representation.


C . Ribbon diagram showing lipid coordination by Mdm12 . Mdm12 residues lipid fatty are colored in green yellow , respectively .

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

C. Ribbon diagram showing lipid coordination by Mdm12. Mdm12 residues and lipid fatty acids are colored in green and yellow, respectively.


D . Mdm12 binds NBD - PE . Wild - type monomeric ( I5P mutant ) Mdm12 were incubated with NBD - PE separated from free NBD - PE in native PAGE . Coomassie ( left ) ( right ) detection indicates that Mdm12 directly interacts with NBD - PEin vitro . E . Quantitative data showing binding affinities for NBD - PE by Mdm12 . binding affinities of Mdm12 ( monomer / dimer shown in Native PAGE I5P mutant ) for NBD - PE was measured with a NBD - PE - dependent manner . experiments were carried out three times means ± SD are given .

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

D. Mdm12 binds NBD-PE. Wild-type and monomeric (I5P mutant) Mdm12 were incubated with NBD-PE and separated from free NBD-PE in native PAGE. Coomassie staining (left) and fluorescent (right) detection indicates that Mdm12 directly interacts with NBD-PEin vitro.E. Quantitative data showing binding affinities for NBD-PE by Mdm12. The binding affinities of Mdm12 (monomer/dimer shown in Native PAGE and I5P mutant) for NBD-PE was measured with a NBD-PE concentration-dependent manner. All experiments were carried out three times and the means ± SD are given.


F . Mdm12 mutants ( L256W , I262W , L256W / I262W double mutant ) were incubated with NBD - PE , subjected to native PAGE . Because wild - type Mdm12 separates as both monomer dimer on native PAGE , purely monomeric form ( I5P ) of Mdm12 was used as wild type for clarity . graph in right indicates quantities measured in experiments . bar shows relative amounts of band ( / Coomassie ) . Values represent means SD from three independent experiments .

O O B-CONTROLLED_VAR 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 B-MEASURED_VAR 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

F. Mdm12 mutants (L256W, I262W, and L256W/I262W double mutant) were incubated with NBD-PE, and subjected to native PAGE. Because wild-type Mdm12 separates as both monomer and dimer on native PAGE, the purely monomeric form (I5P) of Mdm12 was used as the wild type for clarity. The graph in the right indicates the quantities measured in the experiments. The bar shows the relative amounts of the band ratio (fluorescence/Coomassie). Values represent the means and SD from three independent experiments.


A . Structural views suggesting that Mdm12 might have a preference for certain phospholipids . surface representation of Mdm12 positioned around binding site for head group of PE is shown with charge distribution ( left ) conservation ( right ) in same orientation . Surface electrostatics conservation were calculated using an APBS program [ 31 ] with nonlinear Poisson - Boltzmann equation contoured at ± 3kT / e , ConSurf website ( consurf . tau . ac . il ) [ 32 ] with 34 different yeast orthologs , respectively . ribbon diagram shown in middle indicates overall orientation of Mdm12 .

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

A. Structural views suggesting that Mdm12 might have a preference for certain phospholipids. The surface representation of Mdm12 positioned around the binding site for the head group of PE is shown with charge distribution (left) and the sequence conservation (right) in the same orientation. Surface electrostatics and sequence conservation were calculated using an APBS program [31] with the nonlinear Poisson-Boltzmann equation and contoured at ±3kT/e, and ConSurf website (consurf.tau.ac.il) [32] with 34 different yeast orthologs, respectively. The ribbon diagram shown in the middle indicates the overall orientation of Mdm12.


B . Lipid displacement experiments to identity natural ligands of Mdm12in vitro ( see material methods section for details ) . NBD - PE - preloaded Mdm12 - GST was mixed with a series of glycerophospholipids at different concentrations , displacement of NBD - PE by non - labeled ligand was estimated from decrease in . Means ± SD are shown ( n = 3 independently performed experiments ) .

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 B-MEASURED_VAR 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 O O O O O O O

B. Lipid displacement experiments to identity the natural ligands of Mdm12in vitro (see material and methods section for details). NBD-PE-preloaded Mdm12-GST was mixed with a series of glycerophospholipids at different concentrations, and displacement of NBD-PE by non-labeled ligand was estimated from the decrease in fluorescence. Means ± SD are shown (n=3 independently performed experiments).


A . Overall structure ( left ) electron density ( right ) of Mdm12 in asymmetric unit . Four molecules ( two Mdm12 dimers ) are organized with 2 - fold rotation symmetry . 2 - fold axes are indicated with a black dotted . B . Overall structure ( left ) electron density ( right ) of ΔMdm12 in asymmetric unit . Six ΔMdm12 molecules ( three Mdm12 dimers ) are arranged with 2 - fold rotation symmetry as shown above .

O O O O O O O O O O O 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 O O O O 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

A. Overall structure (left) and electron density (right) of Mdm12 in the asymmetric unit. Four molecules (two Mdm12 dimers) are organized with 2-fold rotation symmetry. The 2-fold axes are indicated with a black dotted line.B. Overall structure (left) and electron density (right) of ΔMdm12 in the asymmetric unit. Six ΔMdm12 molecules (three Mdm12 dimers) are arranged with 2-fold rotation symmetry as shown above.


C . Ribbon diagram showing a 2 - fold interface ( tail - to - tail ) in two crystal structures ( See text for details ) .

O O O O 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. Ribbon diagram showing a 2-fold interface (tail-to-tail) in two crystal structures (See text for details).


D . - exclusion chromatography revealing that N - terminus - truncated version of Mdm12 ( residues 11 - 271 ) retains ability to interact with Mmm1 . experiment was performed as in Figure 1B . Eluted fractions indicated by shading were subjected to SDS - PAGE followed by Coomassie Blue .

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 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

D. Size-exclusion chromatography revealing that the N-terminus-truncated version of Mdm12 (residues 11-271) retains the ability to interact with Mmm1. The experiment was performed as in Figure 1B. Eluted fractions indicated by shading were subjected to SDS-PAGE followed by Coomassie Blue staining.


( C ) BN - PAGE SDS - PAGE assays with inactive activated Rx - NRC2 . C - terminally 6xHA tagged Rx C - terminally 4xMyc - tagged NRC2EEE were co - expressed with either free GFP or C - terminally GFP - tagged CP . Free mCherry - 4xMyc mCherry - 6xHA fusions were used as controls in treatments without NRC2 Rx , respectively . Total was extracted with a Tris - HCl - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . Red asterisks indicate bands corresponding to activated NRC2 . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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 B-CONTROLLED_VAR 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 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 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

(C) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2. C-terminally 6xHA tagged Rx and C-terminally 4xMyc-tagged NRC2EEE were co-expressed with either free GFP or C-terminally GFP-tagged CP. Free mCherry-4xMyc and mCherry-6xHA fusions were used as controls in the treatments without NRC2 and Rx, respectively. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE SDS - PAGE assays with inactive activated Rx - NRC2 using different heavy sensor - helper combinations . Total was extracted with a Tris - HCl - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . Red asterisk indicates bands corresponding to activated NRC2 . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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 O O O O O O O O O O O O O O O O O O O O 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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different heavy and light sensor-helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complex. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE SDS - PAGE assays with inactive activated Rx - NRC2 using different " heavy " " " helper combinations . Total was extracted with a Tris - HCl - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . A non - specific band was observed at ~ 110 kDa for anti - FLAG antibody . Red asterisks indicate bands corresponding to activated " heavy " NRC2 complexes . Blue asterisks indicate bands corresponding to " " NRC2 complexes . Purple bands indicate intermediate molecular complexes combining " heavy " " " NRC2 . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated Rx-NRC2 using different "heavy" and "light" helper combinations. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. A non-specific band was observed at ~110 kDa for the anti-FLAG antibody. Red asterisks indicate bands corresponding to the activated "heavy" NRC2 complexes. Blue asterisks indicate bands corresponding to "light" NRC2 complexes. Purple bands indicate intermediate molecular weight complexes combining "heavy" and "light" NRC2. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( B ) BN - PAGE SDS - PAGE assays with inactive activated NRC - dependent sensors NRC2 . Total was extracted with a Tris - HCl - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . Red asterisk indicates bands corresponding to activated NRC2 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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 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

(B) BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


BN - PAGE SDS - PAGE assays with inactive activated NRC - dependent sensors NRC4AAA . Total was extracted with a HEPES - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . Red asterisk indicates bands corresponding to activated NRC4 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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 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

BN-PAGE and SDS-PAGE assays with inactive and activated NRC-dependent sensors and NRC4AAA. Total protein was extracted with a HEPES-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisk indicates bands corresponding to the activated NRC4 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


C - terminally GFP - tagged NRC2EEE C - terminally RFP - tagged Rx were co - expressed either with an EV - 4xMyc construct or a CP - 4xMyc construct in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines . ( A - B ) Single - plane confocal show localization of both components of inactive active Rx - NRC2 system , together with PM marker RPW8 . 2 - BFP . Scale bars represent 10 µm . ( A ) NRC2EEE - GFP Rx - RFP co - localize in cytoplasm prior to activation . ( B ) Upon co - expression of CP activation of system , NRC2EEE forms associated with PM while Rx remains in cytoplasm .

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

C-terminally GFP-tagged NRC2EEE and C-terminally RFP-tagged Rx were co-expressed either with an EV-4xMyc construct or a CP-4xMyc construct in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines. (A-B) Single-plane confocal micrographs show the localization of both components of the inactive and active Rx-NRC2 system, together with PM marker RPW8.2-BFP. Scale bars represent 10 µm. (A) NRC2EEE-GFP and Rx-RFP co-localize in the cytoplasm prior to activation. (B) Upon co-expression of CP and activation of the system, NRC2EEE forms puncta associated with the PM while Rx remains in the cytoplasm.


( C ) enrichment assays are consistent with microscopy , showing that inactive NRC2EEE - GFP is mostly present in soluble ( ) fraction , whereas activated NRC2EEE - GFP exhibits equal distribution across soluble fractions . Rx is mostly present in soluble fraction exhibits no change upon activation of system with CP . T = total , S = soluble , M = . ATPase was used as a marker . Rubisco was used as a marker for total soluble fractions visualized by Ponceau ( PS ) . Red asterisks indicate bands matching expected MW for each . experiment was repeated two times .

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 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 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

(C) Membrane enrichment assays are consistent with microscopy, showing that inactive NRC2EEE-GFP is mostly present in the soluble (cytoplasmic) fraction, whereas activated NRC2EEE-GFP exhibits equal distribution across soluble and membrane fractions. Rx is mostly present in the soluble fraction and exhibits no change upon activation of the system with CP. T = total, S = soluble, M = membrane. ATPase was used as a membrane marker. Rubisco was used as a marker for total and soluble fractions and visualized by Ponceau staining (PS). Red asterisks indicate bands matching the expected MW for each protein. The experiment was repeated two times.


( B ) BN - PAGE SDS - PAGE assays with infected uninfected leaves expressing Rx NRC2 . Total was extracted with a Tris - HCl - buffer , as described in materials methods . Extracts were run on native denaturing PAGE assays in parallel immunoblotted with appropriate antisera labelled on left . Approximate molecular weights ( kDa ) of are shown on right . Red asterisks indicate bands corresponding to activated NRC2 complexes . Rubisco loading control was carried out using Ponceau stain ( PS ) . experiment was repeated three times .

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 O O O O O O O O O O O O O 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

(B) BN-PAGE and SDS-PAGE assays with infected and uninfected leaves expressing Rx and NRC2. Total protein was extracted with a Tris-HCl-based buffer, as described in materials and methods. Extracts were run on native and denaturing PAGE assays in parallel and immunoblotted with the appropriate antisera labelled on the left. Approximate molecular weights (kDa) of the proteins are shown on the right. Red asterisks indicate bands corresponding to the activated NRC2 complexes. Rubisco loading control was carried out using Ponceau stain (PS). The experiment was repeated three times.


( A - B ) Single - plane confocal show localization of NRC2 together with Rx or free RFP during PVX infection . C - terminally GFP - tagged NRC2EEE was co - infiltrated with either EV - RFP or Rx - RFP in leaves of nrc2 / 3 / 4 N . benthamiana CRISPR mutant lines infected with PVX by agroinfection . Scale bars represent 10 µm . ( A ) In absence of Rx , NRC2EEE - GFP is localized to cytoplasm during PVX infection . ( B ) When Rx is present , NRC2EEE forms associated with PM during PVX infection .

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-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 B-MEASURED_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 O O

(A-B) Single-plane confocal micrographs show the localization of NRC2 together with Rx or free RFP during PVX infection. C-terminally GFP-tagged NRC2EEE was co-infiltrated with either EV-RFP or Rx-RFP in leaves of nrc2/3/4 N. benthamiana CRISPR mutant lines and infected with PVX by agroinfection. Scale bars represent 10 µm. (A) In the absence of Rx, NRC2EEE-GFP is localized to the cytoplasm during PVX infection. (B) When Rx is present, NRC2EEE forms puncta associated with the PM during PVX infection.


a , GFP - MAP1LC3B foci after RNAi / or rapamycin treatment in U2OS . Scale bar , 20 µm .

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

a, GFP-MAP1LC3B foci after RNAi and/or rapamycin treatment in U2OS cells. Scale bar, 20 µm.


B . Violin plots of 3 ' READS signal on TNFα induced genes ( n = 307 ) or set of TNFα induced genes longer than 40 kb ( n = 111 ) . * * * * p < 0 . 0001 . Statistical : Friedman with Dunn ' s multiple correction . Violin plot : minimal to maximal value , dashed horizontal : median , dotted horizontal lines : interquartile ( 25 % 75 % ) .

O O O O O O O O O O B-CONTROLLED_VAR 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 O O O O O O O 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. Violin plots of the 3'READS signal on all the TNFα induced genes (n=307) or the set of TNFα induced genes longer than 40 kb (n=111). **** p < 0.0001. Statistical test: Friedman test with Dunn's multiple test correction. Violin plot: minimal to maximal value, dashed horizontal line: median, dotted horizontal lines: interquartile (25% and 75%).


C . Western blot of total pol II , Ser2P , Xrn2 , CPSF2 , CPSF73 , histone H3 as a loading control , on fraction . D . Western blot of Xrn2 , CPSF2 , Nucleolin as a loading control , on nucleoplasm fraction ( CPSF73 antibody does not provide reliable results on nucleoplasm fraction ) . E . of western blots shown in C D . n = 2 biological replicates , mean .

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

C. Western blot of total pol II, Ser2P, Xrn2, CPSF2, CPSF73, and histone H3 as a loading control, on the chromatin fraction. D. Western blot of Xrn2, CPSF2, and Nucleolin as a loading control, on the nucleoplasm fraction (the CPSF73 antibody does not provide reliable results on the nucleoplasm fraction). E. Quantification of the western blots shown in C and D. n=2 biological replicates, mean.


F . of different CPA termination factors to total pol II . n = 3 biological replicates , mean ± , p - value : * p < 0 . 05 , * * p < 0 . 01 . Statistical : two - unpaired t .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F. Ratio of the different CPA and termination factors to total pol II. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01. Statistical test: two-tailed unpaired t test.


A . Western blot of total pol II , Ser2P ( Abcam or Signaling ) , Ser5P ( Abcam or Signaling ) , histone H3 as a loading control , on fraction of CDK9as or wild - type HEK293 treated with DMSO or 15 µM 1 - NA - PP1 for 15 or 30 minutes . B . of western blots shown in A . n = 2 biological replicates , except for Ser2P / pol II ( Abcam ) CDK9as NA 30 min , Ser5P / pol II ( Abcam ) CDK9as NA 15 min NA 30 min , Ser5P / pol II ( Signaling ) CDK9as NA 30 min , where n = 3 biological replicates .

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

A. Western blot of total pol II, Ser2P (Abcam or Cell Signaling), Ser5P (Abcam or Cell Signaling), and histone H3 as a loading control, on the chromatin fraction of CDK9as or wild-type HEK293 cells treated with DMSO or 15 µM 1-NA-PP1 for 15 or 30 minutes. B. Quantification of the western blots shown in A. n=2 biological replicates, except for Ser2P / pol II (Abcam) CDK9as NA 30 min, Ser5P / pol II (Abcam) CDK9as NA 15 min and NA 30 min, and Ser5P / pol II (Cell Signaling) CDK9as NA 30 min, where n=3 biological replicates.


C . ChIP - qPCR of total pol II of Ser2P ( Abcam or Signaling ) , Ser5P ( Abcam or Signaling ) ratioed to total pol II on KPNB1 . n = 3 biological replicates , mean ± , p - value : * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . Statistical : two - unpaired t .

O O O O O 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 O O O O O O O

C. ChIP-qPCR of total pol II and of Ser2P (Abcam or Cell Signaling), and Ser5P (Abcam or Cell Signaling) ratioed to total pol II on KPNB1. n=3 biological replicates, mean ± SEM, p-value: * p < 0.05, ** p < 0.01, *** p < 0.001. Statistical test: two-tailed unpaired t test.


B . Western blot of total SF3B1 , SF3B1 T142P , histone H3 as a loading control , on fraction of CDK9as treated for 30 minutes with DMSO , NA , tautomycetin ( TT ) , calyculin A ( CA ) , NA + TT , or NA + CA . histone H3 loading control ( CA samples ) is same as loading control shown in Appendix Figure S3B as same western blot experiment is shown in two different figures .

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

B. Western blot of total SF3B1, SF3B1 T142P, and histone H3 as a loading control, on the chromatin fraction of CDK9as cells treated for 30 minutes with DMSO, NA, tautomycetin (TT), calyculin A (CA), NA+TT, or NA+CA. The histone H3 loading control (CA samples) is the same as the loading control shown in Appendix Figure S3B as the same western blot experiment is shown in two different figures.


C . Co - immunoprecipitation of CPSF2 in CDK9as treated for 30 minutes with DMSO or NA followed by western blot with total pol II , CPSF2 , CPSF3 , SF3B1 , SF3B3 β - actin ( negative control ) antibodies .

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

C. Co-immunoprecipitation of CPSF2 in the CDK9as cell treated for 30 minutes with DMSO or NA followed by western blot with total pol II, CPSF2, CPSF3, SF3B1, SF3B3 and β-actin (negative control) antibodies.


D . Co - immunoprecipitation of SF3B1 in CDK9as treated for 30 minutes with DMSO , NA , or DRB followed by western blot with total pol II , SF3B1 , SF3B2 , GAPDH ( negative control ) antibodies .

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

D. Co-immunoprecipitation of SF3B1 in the CDK9as cell treated for 30 minutes with DMSO, NA, or DRB followed by western blot with total pol II, SF3B1, SF3B2, and GAPDH (negative control) antibodies.


B . qRT - PCR of polyadenylated mRNAs of several TNFα induced or non - induced genes with a 30 minutes DMSO , DRB , CA , or DRB + CA treatment .

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

B. qRT-PCR of nuclear polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.


C . qRT - PCR of polyadenylated mRNAs of several TNFα induced or non - induced genes with a 30 minutes DMSO , DRB , CA , or DRB + CA treatment .

C. qRT-PCR of cytoplasmic polyadenylated mRNAs of several TNFα induced or non-induced genes with a 30 minutes DMSO, DRB, CA, or DRB+CA treatment.


D . ChIP - qPCR of Ser2P , CPSF73 , or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO , DRB , CA , or DRB + CA on KPNB1 .

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

D. ChIP-qPCR of Ser2P, CPSF73, or CPSF2 ratioed to total pol II after 30 minutes treatment with DMSO, DRB, CA, or DRB+CA on KPNB1.


E . Co - immunoprecipitation of total pol II from HeLa treated for 30 minutes with DMSO , DRB , CA , or DRB + CA followed by western blot with total pol II , Ser2P , SF3B1 , CPSF2 , Xrn2 , GAPDH antibodies .

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

E. Co-immunoprecipitation of total pol II from HeLa cells treated for 30 minutes with DMSO, DRB, CA, or DRB+CA followed by western blot with total pol II, Ser2P, SF3B1, CPSF2, Xrn2, and GAPDH antibodies.


A Electron micrograph of unstained UMOD filament sheets in human urine . inset highlights a double helix - like structure resulting from juxtaposition of two filaments . Scale bars : 50 nm 10 nm ( inset ) . B , C " Tree " front view " zig - zag " side view of purified native UMOD filaments , imaged using a Volta phase plate . Scale bars : 10 nm . D , E Front side views of UMODe filaments , showing absence of branches . Scale bars : 10 nm .

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

A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset). B, C "Tree" front view and "zig-zag" side view of purified native UMOD filaments, imaged using a Volta phase plate. Scale bars: 10 nm. D, E Front and side views of UMODe filaments, showing absence of branches. Scale bars: 10 nm.


A Electron micrograph of unstained UMOD filament sheets in human urine . inset highlights a double helix - like structure resulting from juxtaposition of two filaments . Scale bars : 50 nm 10 nm ( inset ) .

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

A Electron micrograph of unstained UMOD filament sheets in human urine. The inset highlights a double helix-like structure resulting from juxtaposition of two individual filaments. Scale bars: 50 nm and 10 nm (inset).


A Immunofluorescence of unpermeabilized stably transfected MDCK shows that , unlike wt full - UMOD , mutants ZP - N ( R415A ) or ZP - C ( ΔFA ) do not assemble into filaments . Scale bar : 50 µm .

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 O

A Immunofluorescence of unpermeabilized stably transfected MDCK cells shows that, unlike wt full-length UMOD, mutants ZP-N (R415A) or ZP-C (ΔFA) do not assemble into filaments. Scale bar: 50 µm.


B - G Immunofluorescence of unpermeabilized co - expressing FLAG - tagged wt UMOD ( green ) indicated HA - tagged isoforms ( red ) . UMOD R415A does not incorporate into polymers that only contain wt ( C ) . UMOD ΔFA has a dominant negative effect on wt polymerization ( D ) that is rescued in a double mutant carrying both ZP - N ZP - C mutations ( F ) . Similarly , dominant negative effect of a CCS mutation ( 4A ) that prevents EHP dissociation ( E ) is suppressed by introducing R415A mutation in 4A isoform ( G ) . Scale bar : 50 µm .

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 B-MEASURED_VAR 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 O 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-G Immunofluorescence of unpermeabilized cells co-expressing FLAG-tagged wt UMOD (green) and the indicated HA-tagged isoforms (red). UMOD R415A does not incorporate into polymers that only contain wt protein (C). UMOD ΔFA has a dominant negative effect on wt protein polymerization (D) that is rescued in a double mutant carrying both ZP-N and ZP-C mutations (F). Similarly, the dominant negative effect of a CCS mutation (4A) that prevents EHP dissociation (E) is suppressed by the introducing the R415A mutation in the 4A isoform (G). Scale bar: 50 µm.


H Immunofluorescence of permeabilized expressing soluble isoforms of UMOD truncated before EHP ( UMOD - CCS ) shows that wt forms intracellular polymers whereas polymerization interface mutants do not . intracellular polymers are localized in endoplasmic reticulum ( ER ) , as shown by co - with KDEL used as ER marker . Scale bar : 10 µm .

O O O O O O O O B-CONTROLLED_VAR 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

H Immunofluorescence of permeabilized cells expressing soluble isoforms of UMOD truncated before the EHP (UMOD-CCS) shows that wt forms intracellular polymers whereas polymerization interface mutants do not. The intracellular polymers are localized in the endoplasmic reticulum (ER), as shown by co-staining with the KDEL sequence used as ER marker. Scale bar: 10 µm.


A Analytical SEC of UFE HCE / LCE - digests produces two peaks , F1 F2 .

A Analytical SEC of UFE HCE/LCE-digests produces two protein peaks, F1 and F2.


Reducing SDS - PAGE of SEC purified UFE digests with indicated molecular markers . different SEC elution volumes of F1 F2 ( A ) reflect different of egg coat filament digestion by HCE / LCE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Reducing SDS-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE.


native - PAGE of SEC purified UFE digests with indicated molecular markers . different SEC elution volumes of F1 F2 ( A ) reflect different of egg coat filament digestion by HCE / LCE .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

native-PAGE of SEC purified UFE digests with indicated molecular weight markers. The different SEC elution volumes of F1 and F2 (A) reflect different levels of egg coat filament digestion by HCE/LCE.


D , E TOF - MS spectrograms of purified F2 ( D ) F1 ( E ) products . Round - shaped symbols near peaks indicate two moieties of intact ZP module of ZI - 3 ( green ) or separated ZP - N ZP - C domains of LCE - ZI - 1 , 2 ( purple ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

D, E TOF-MS spectrograms of purified F2 (D) and F1 (E) products. Round-shaped symbols near the peaks indicate the two moieties of the intact ZP module of ZI-3 (green) or the separated ZP-N and ZP-C domains of LCE-cleaved ZI-1,2 (purple).


F , G TOF - MS spectrograms of cross - linked products of F2 ( F ) F1 ( G ) , with domain symbols indicating deduced subunit composition of cross - linked products .

F, G TOF-MS spectrograms of cross-linked products of F2 (F) and F1 (G), with domain symbols indicating the deduced subunit composition of cross-linked products.


I Electron micrograph of negatively - stained fish egg coat filaments . On right are representative 2D classes , indicating a separation of ~ 65 Å between repeating units within polymers ( arrows ) . Scale bars : 100 nm ( left panel ) 10 nm ( bottom right panel ) .

O O O O O O 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

I Electron micrograph of negatively-stained fish egg coat filaments. On the right are representative 2D classes, indicating a separation of ~65 Å between repeating units within the polymers (white arrows). Scale bars: 100 nm (left panel) and 10 nm (bottom right panel).


A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl ( top ) . of this material by correlation averaging reveals a periodic pattern ( bottom ) with a two - fold symmetry axis perpendicular to plane of sheet ( symbol ) . Scale bars : 250 Å ( top ) 125 Å ( bottom ) .

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 O O O O O O O

A Negative stain micrograph of a sheet formed by purified UMOD filaments in 100 mM NaCl (top). Analysis of this material by correlation averaging reveals a periodic pattern (bottom) with a two-fold symmetry axis perpendicular to the plane of the sheet (white symbol). Scale bars: 250 Å (top) and 125 Å (bottom).


Aci2 Pa were both grown in monoculture in two - resource environment . same data is shown in both plots . left plot shows average of eight technical replicates on a log scale , while right plot shows each of replicates on a linear scale . Overlain on left plot are growth rate fits of gAci2 = 0 . 88hr - 1 gPa = 0 . 67hr - 1 . Data Information : Data in A were collected at 400nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aci2 and Pa were both grown in monoculture in the two-resource environment. The same data is shown in both plots. The left plot shows the average of eight technical replicates on a log scale, while the right plot shows each of the replicates on a linear scale. Overlain on the left plot are growth rate fits of gAci2 = 0.88hr-1 and gPa = 0.67hr-1. Data Information: Data in A were collected at 400nm.


Aci2 was grown with alanine glutamate supplied at 25 different ratios from 1 : 16 to 16 : 1 with total supply kept constant at 0 . 1 % w / Data Information : Data were collected at 600nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Aci2 was grown with alanine and glutamate supplied at 25 different ratios from 1:16 to 16:1 with the total supply kept constant at 0.1%w/ Data Information: Data were collected at 600nm.


population at which Aci2 ' s diauxic shift occurred is linearly correlated to alanine supply , indicating that Aci2 initially consumes almost entirely alanine . Data Information : Data were collected at 600nm .

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

The population size at which Aci2's diauxic shift occurred is linearly correlated to the alanine supply, indicating that Aci2 initially consumes almost entirely alanine. Data Information: Data were collected at 600nm.


Instantaneous growth rates were extracted from Aci2 monoculture data A variation in Aci2 ' s lag time is clearly visible in this representation . Data Information : Data were collected at 600nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Instantaneous growth rates were extracted from the Aci2 monoculture data A variation in Aci2's lag time is clearly visible in this representation. Data Information: Data were collected at 600nm.


same monoculture experiments were performed for Pa . appearance of Pa having a long initial lag is primarily due to growth that it needs to accomplish before its population becomes significant on a linear scale as well as a small optical density artifact Data Information : Data were collected at 600nm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The same monoculture experiments were performed for Pa. The appearance of Pa having a long initial lag is primarily due to the growth that it needs to accomplish before its population size becomes significant on a linear scale as well as a small optical density artifact Data Information: Data were collected at 600nm.


Pa ' s population at onset of its diauxic shift correlates to supply of whichever resource is supplied in a more limiting amount , indicating coutilization . Data Information : Data were collected at 600nm .

Pa's population size at the onset of its diauxic shift correlates to the supply concentration of whichever resource is supplied in a more limiting amount, indicating coutilization. Data Information: Data were collected at 600nm.


Instantaneous growth rates extracted from Pa monoculture data . Data Information : Data were collected at 600nm .

Instantaneous growth rates extracted from the Pa monoculture data. Data Information: Data were collected at 600nm.


Diauxic lag times were fit from monoculture data are plotted as circles . Lag times could not be fit for conditions with too little growth on remaining glutamate . lag times used in modeling are shown as lines through data .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Diauxic lag times were fit from the monoculture data and are plotted as circles. Lag times could not be fit for conditions with too little growth on the remaining glutamate. The lag times used in the modeling are shown as lines through the data.


In order to validate modeling its predictive power , we repeated competition experiments between Aci2 Pa across a 54 - condition grid of resource supply ratios dilution factors Shown is fraction Pa on Days 1 , 3 , 5 , 7 for eight total replicates of each condition ( two biological replicates , each with four technical replicates started at different initial species fractions We used model to predict steady - state population fractions for each condition . We calculated mean final population fractions from experimental data compared these fractions to model predictions .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In order to validate the modeling and test its predictive power, we repeated the competition experiments between Aci2 and Pa across a 54-condition grid of resource supply ratios and dilution factors Shown is the fraction Pa on Days 1, 3, 5, and 7 for eight total replicates of each condition (two biological replicates, each with four technical replicates started at different initial species fractions We used the model to predict steady-state population fractions for each condition. We calculated mean final population fractions from the experimental data and compared these fractions to the model predictions.


Monoculture experiments were performed to determine species ' growth rates lag times for each two - resource environment . For each pair of species , coculture competition experiments were performed in each two - resource environment , qualitative competitive outcomes were determined There were 44 competitions in which both species could be assigned growth rates lag times Each point represents one competition . Pie charts summarize competitive outcomes for cases in which slow - grower was fast - switcher ( above Δtlag = 0 ) in which slow - grower was not slow - switcher ( on below Δtlag = 0 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Monoculture experiments were performed to determine species' growth rates and lag times for each two-resource environment. For each pair of species, coculture competition experiments were performed in each two-resource environment, and qualitative competitive outcomes were determined There were 44 competitions in which both species could be assigned growth rates and lag times Each point represents one competition. Pie charts summarize the competitive outcomes for cases in which the slow-grower was the fast-switcher (above the Δtlag = 0 line) and in which the slow-grower was not the slow-switcher (on and below Δtlag = 0).


Cross - sectional longitudinal of association between amyloid - PET ( in centiloid ) , CSF p - tau181 , CSF sTREM2 in early Aβ - accumulators ( i . e . Aβ CSF + / PET − ; n = 70 ) late Aβ - accumulators ( i . e . Aβ CSF + / PET + ; n = 201 ) . Cross - sectional linear regressions between A ) centiloid p - tau181 B ) centiloid sTREM2 . Standardized beta - estimates ( β ) , T - values , p - values were derived from linear regressions controlling for , sex , education , clinical status .

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

Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Cross-sectional linear regressions between A) centiloid and p-tau181 and B) centiloid and sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.


Cross - sectional longitudinal of association between amyloid - PET ( in centiloid ) , CSF p - tau181 , CSF sTREM2 in early Aβ - accumulators ( i . e . Aβ CSF + / PET − ; n = 70 ) late Aβ - accumulators ( i . e . Aβ CSF + / PET + ; n = 201 ) . Longitudinal linear regressions between C ) centiloid change in p - tau181 D ) centiloid change in sTREM2 . Standardized beta - estimates ( β ) , T - values , p - values were derived from linear regressions controlling for , sex , education , clinical status .

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

Cross-sectional and longitudinal analysis of the association between amyloid-PET (in centiloid), CSF p-tau181, and CSF sTREM2 in early Aβ-accumulators (i.e. Aβ CSF+/PET−; n=70) and late Aβ-accumulators (i.e. Aβ CSF+/PET+; n=201). Longitudinal linear regressions between C) centiloid and change in p-tau181 and D) centiloid and change in sTREM2. Standardized beta-estimates (β), T-values, and p-values were derived from linear regressions controlling for age, sex, education, and clinical status.


B ) T - value projection of association between sTREM2 on FDG - PET , stratified by group . FDG - PET z - scores were derived by referencing FDG - PET SUVRs to cognitively normal controls ( i . e . n = 131 ; Aβ CSF − / PET − ) .

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

B) T-value projection of the association between sTREM2 on FDG-PET, stratified by group. FDG-PET z-scores were derived by referencing FDG-PET SUVRs to cognitively normal controls (i.e. n=131; Aβ CSF−/PET−).


Wild - type mice have small defined crypts in small intestine with little nuclearβ - catenin at bottom of crypt . small intestine of AhCreERAPCfl / fl , AhCreERCatnblox ( ex3 ) / lox ( ex3 ) ( day 5 ) or AhCreERGsk3alphafl / fl betafl / fl ( day 6 ) show crypt - progenitor ( CPC ) phenotype with increased crypt ( red bar ) nuclearβ - catenin ( arrows ) along crypt - villus axis . Scale bar , 100 μm .

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

Wild-type mice have small defined crypts in the small intestine with little nuclearβ-catenin at the bottom of the crypt. The small intestine of AhCreERAPCfl/fl, AhCreERCatnblox(ex3)/lox(ex3) (day 5) or AhCreERGsk3alphafl/fl betafl/fl (day 6) show the crypt-progenitor cell (CPC) phenotype with increased crypt size (red bar) and nuclearβ-catenin (arrows) along the crypt-villus axis. Scale bar, 100 μm.


A heterozygous activation of β - catenin ( AhCreER Catnblox ( ex3 ) / + ) shows no increase in crypt or β - catenin at days 5 - 10 . At day 15 post - induction , accumulation of β - catenin ( arrows ) becomes evident with a dramatic CPC phenotype at about day 25 . Scale bar , 100 μm .

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

A heterozygous activation of β-catenin (AhCreER Catnblox(ex3)/+) shows no increase in crypt size or nuclear β-catenin at days 5-10. At day 15 post-induction, accumulation of nuclear β-catenin (arrows) becomes evident with a dramatic CPC phenotype at about day 25. Scale bar, 100 μm.


Culture of small intestinal crypts of WT VilCreER Apcfl / fl ( or AhCreER Apcfl / fl ) mice with / without R - spo1 shows dependence of Wnt agonist in WT organoids but not in Apc - deficient spheres . Representative photos were taken at day 5 in culture . Black scale bar , 50 μm .

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

Culture of small intestinal crypts of WT and VilCreER Apcfl/fl (or AhCreER Apcfl/fl) mice with/without R-spo1 shows the dependence of the Wnt agonist in WT organoids but not in Apc-deficient spheres. Representative photos were taken at day 5 in culture. Black scale bar, 50 μm.


At day 5 post - induction , only crypts from AhCreERCatnblox ( ex3 ) / lox ( ex3 ) but not AhCreERCatnblox ( ex3 ) / + survive in culture without addition of R - spo1 . At day 10 post - induction , we observed a mixed phenotype of more organoid - like structures in AhCreERCatnblox ( ex3 ) / + compared to spheres from Catnblox ( ex3 ) / lox ( ex3 ) crypts in first week of culture . Black scale bar , 50 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O

At day 5 post-induction, only crypts from AhCreERCatnblox(ex3)/lox(ex3) but not AhCreERCatnblox(ex3)/+ survive in culture without the addition of R-spo1. At day 10 post-induction, we observed a mixed phenotype of more organoid-like structures in AhCreERCatnblox(ex3)/+ compared to spheres from Catnblox(ex3)/lox(ex3)crypts in the first week of culture. Black scale bar, 50 μm.


of organoids / spheres at day 10 post - induction ( N = 2 or 3 mice per genotype , mean of 6 technical replicates per mouse ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Quantification of organoids/spheres at day 10 post-induction (N = 2 or 3 mice per genotype, mean of 6 technical replicates per mouse).


Table shows that only homozygous activation of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / lox ( ex3 ) ) or loss of Apc ( Lgr5CreER Apcfl / fl ) in Lgr5 - positive results in colonic lesions in cohorts , sampled when signs of sickness were apparent . There were no lesions observed when only one allele of β - catenin ( Lgr5CreER Catnblox ( ex3 ) / + ) was mutated ( chi - squared , P < 0 . 001 ) .

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

Table shows that only homozygous activation of β-catenin (Lgr5CreER Catnblox(ex3)/lox(ex3)) or loss of Apc (Lgr5CreER Apcfl/fl) in Lgr5-positive stem cells results in colonic lesions in cohorts, sampled when signs of sickness were apparent. There were no lesions observed when only one allele of β-catenin (Lgr5CreER Catnblox(ex3)/+) was mutated (chi-squared test, P < 0.001).


Colonic lesions were scored from H & amp ; amp ; E by identification of disorganised epithelium . for β - catenin SOX9 confirmed activation of Wnt signalling in observed lesions . Scale bar , 100 μm .

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

Colonic lesions were scored from H&amp;E by identification of disorganised epithelium. Staining for β-catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions. Scale bar, 100 μm.


of small intestinal colonic crypts for E - cadherin . Scale bar , 100 μm .

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

Staining of small intestinal and colonic crypts for E-cadherin. Scale bar, 100 μm.


Western blot of purified crypts from small intestine colon ( N = 4 mice ) .

Western blot of purified crypts from the small intestine and colon (N = 4 mice).


qRT - PCR of whole tissue from small intestine colon . Expression of mRNA ( 2 ( − dCt ) ) calculated relative to GAPDH ( N = 3 mice ) . Statistics : one - sided Mann - Whitney U - .

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

qRT-PCR of whole tissue from small intestine and colon. Expression of mRNA (2(−dCt)) calculated relative to GAPDH (N = 3 mice). Statistics: one-sided Mann-Whitney U-test.


Proximity ligation for E - cadherin β - catenin . For each mouse ( N = 6 ) , at least 10 crypts per tissue were analysed to calculate mean . Statistics : one - sided Mann - Whitney U - ; scale bar , 50 μm .

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

Proximity ligation assay for E-cadherin and β-catenin. For each mouse (N = 6), at least 10 crypts per tissue were analysed to calculate the mean. Statistics: one-sided Mann-Whitney U-test; white scale bar, 50 μm.


Immunoprecipitation ( IP ) of E - cadherin from VilCreER Catnblox ( ex3 ) / lox ( ex3 ) mice , 24 h ( day 1 , top ) 48 h ( day 2 , bottom ) after induction . of wild - type ( WT , top lane ) mutant exon 3 β - catenin ( Δex3 , bottom lane ) bound to E - cadherin ( IP : Ecad ) was calculated for each tissue . Graphs show average of experiments ( N = 3 mice for each genotype time point ) ; error bars represent s . e . m .

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

Immunoprecipitation (IP) of E-cadherin from VilCreER Catnblox(ex3)/lox(ex3) mice, 24 h (day 1, top) and 48 h (day 2, bottom) after induction. The ratio of wild-type (WT, top lane) and mutant exon 3 β-catenin (Δex3, bottom lane) bound to E-cadherin (IP:Ecad) was calculated for each tissue. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.


IP of E - cadherin from VilCreER Catnblox ( ex3 ) / + mice at day 4 day 8 . Graphs show average of experiments ( N = 3 mice for each genotype time point ) ; error bars represent s . e . m .

O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR 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

IP of E-cadherin from VilCreER Catnblox(ex3)/+ mice at day 4 and day 8. Graphs show average of experiments (N = 3 mice for each genotype and time point); error bars represent s.e.m.


AhCreERCatnblox ( ex3 ) / + compared to AhCreER Catnblox ( ex3 ) / + Cdh1fl / + at day 10 post - induction . Note presence of colonic lesions in colon of AhCreER Catnblox ( ex3 ) / + Cdh1fl / + mice ( arrows ) . Scale bar , 100 μm ; red bar indicates proliferative zone ( BrdU ) .

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 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

AhCreERCatnblox(ex3)/+ compared to AhCreER Catnblox(ex3)/+ Cdh1fl/+ at day 10 post-induction. Note the presence of colonic lesions in the colon of AhCreER Catnblox(ex3)/+ Cdh1fl/+ mice (arrows). Scale bar, 100 μm; red bar indicates proliferative zone (BrdU).


Scoring of BrdU - positive per half - crypt in small intestine of wild - type , AhCreERCdh1fl / + , AhCreERCatnblox ( ex3 ) / + AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice at day 10 post - induction . N ≥ 3 , at least 25 crypts per mouse were scored . There was significantly higher proliferation in AhCreERCatnblox ( ex3 ) / + Cdh1fl / + mice ( P = 0 . 028 , one - sided Mann - Whitney U - ) .

O O O O O O O O O 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 O O O O O O O O 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 O O O O O O O O O O O O O O O O O

Scoring of BrdU-positive cells per half-crypt in the small intestine of wild-type, AhCreERCdh1fl/+, AhCreERCatnblox(ex3)/+ and AhCreERCatnblox(ex3)/+Cdh1fl/+mice at day 10 post-induction. N ≥ 3, at least 25 crypts per mouse were scored. There was significantly higher proliferation in the AhCreERCatnblox(ex3)/+Cdh1fl/+mice (P = 0.028, one-sided Mann-Whitney U-test).


In vitro growth of crypts ( small intestine ) from mice as indicated at day 5 post - induction without R - spo1 . of sphere - forming efficiency of AhCreERCatnblox ( ex3 ) / + AhCreERCatnblox ( ex3 ) / + Cdh1fl / + , day 5 post - induction . N = 3 mice per genotype , mean of 6 technical replicates per mouse , P = 0 . 04 one - sided Mann - Whitney U - .

O O O O O O O O O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

In vitro growth of crypts (small intestine) from mice as indicated at day 5 post-induction without R-spo1. Quantification of sphere-forming efficiency of AhCreERCatnblox(ex3)/+AhCreERCatnblox(ex3)/+Cdh1fl/+, day 5 post-induction. N = 3 mice per genotype, mean of 6 technical replicates per mouse, P = 0.04 one-sided Mann-Whitney U-test.


Survival of Lgr5CreERCatnblox ( ex3 ) / + Lgr5CreERCatnblox ( ex3 ) / + Cdh1fl / + shows significant acceleration ( P = 0 . 000123 , log - rank ) after E - cadherin reduction . About 85 % ( 6 / 7 mice ) had colonic lesions , as identified with β - cateninIHC in contrast to Lgr5CreERCatnblox ( ex3 ) / + mice .

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

Survival of Lgr5CreERCatnblox(ex3)/+ and Lgr5CreERCatnblox(ex3)/+Cdh1fl/+ shows significant acceleration (P = 0.000123, log-rank test) after E-cadherin reduction. About 85% (6/7 mice) had colonic lesions, as identified with β-cateninIHC in contrast to Lgr5CreERCatnblox(ex3)/+mice.


Proximity ligation for E - cadherin : β - catenin on a tissue microarray of SPT . PLA dots / in normal tumour tissue were counted . Each dot in boxplot represents average for a single . for β - catenin confirms accumulation of β - catenin in tumour tissue . Representative pictures are shown . N = 18 , statistics : Mann - Whitney U - , P = 0 . 0009051 . scale bar , 50 μm .

O 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 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 O

Proximity ligation assay for E-cadherin:β-catenin on a tissue microarray of SPT patients. PLA dots/cells in normal and tumour tissue were counted. Each dot in the boxplot represents the average for a single patient. Staining for β-catenin confirms accumulation of nuclear β-catenin in tumour tissue. Representative pictures are shown. N = 18, statistics: Mann-Whitney U-test, P = 0.0009051. White scale bar, 50 μm.


Correlation of several Wnt target genes with expression of E - cadherin ( CDH1 ) was analysed in 269 HCC ( TCGA provisional ) , linear regression ( blue ) confidence region ( shaded ) were added .

O O 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 O O O O O O O O O O O O O O O O O

Correlation of several Wnt target genes with the expression of E-cadherin (CDH1) was analysed in 269 HCC patients (TCGA provisional), and linear regression line (blue) and confidence region (shaded) were added.


a , A TRPML1Va - expressing HEK293T showed a large inwardly rectifying whole - current elicited by voltage steps ( from - 140 mV to + 80 mV in increments of 20 mV ) in standard extracellular ( Tyrode ' s ) bath solution . Step duration , 90 ms ; holding potential , 0 mV ; Vm , potential .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, A TRPML1Va-expressing HEK293T cell showed a large inwardly rectifying whole-cell current elicited by voltage steps (from -140 mV to +80 mV in increments of 20 mV) in the standard extracellular (Tyrode's) bath solution. Step duration, 90 ms; holding potential, 0 mV; Vm, membrane potential.


b , No significant inward current was detected in NMDG + ( Na + - free , Ca2 + - free , pH 4 . 6 ) solution .

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

b, No significant inward current was detected in NMDG+ (Na+-free, Ca2+-free, pH 4.6) solution.


c , Inwardly rectifying step currents were evoked by 30 mM Fe2 + solution ( pH 4 . 6 ) in same as shown in a b .

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

c, Inwardly rectifying step currents were evoked by 30 mM Fe2+ solution (pH 4.6) in the same cell as shown in a and b.


d , pH - dependence of IFe / TRPML1Va . Whole - currents were elicited by repeated voltage ramps ( - 100 to + 100 mV ; 400 ms ) with a 4 - s interval between ramps . Only a portion of voltage protocol is shown . Holding potential , 0 mV .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

d, pH-dependence of IFe/TRPML1Va. Whole-cell currents were elicited by repeated voltage ramps (-100 to +100 mV; 400 ms) with a 4-s interval between ramps. Only a portion of the voltage protocol is shown. Holding potential, 0 mV.


e , Large IFe / TRPML2Va was seen in presence of 30 105 mM Fe2 + ( pH 4 . 6 ) .

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

e, Large IFe/TRPML2Va was seen in the presence of 30 and 105 mM Fe2+ (pH 4.6).


f , Little or no IFe was seen in a TRPML3Va - expressing .

f, Little or no IFe was seen in a TRPML3Va-expressing cell.


a , Current densities ( mean ± s . e . m . , n = 4 - 10 ) of IFe ( 30 mM Fe2 + , pH 4 . 6 ) for TRPML1Va ML4 mutant TRPML1Va channels . Asterisk indicates statistical difference ( P < 0 . 01 ) compared to TRPML1Va .

O O O O O O O O O O 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 O O O O O O O O O O O O O O O O O

a, Current densities (mean ± s.e.m., n = 4-10) of IFe (30 mM Fe2+, pH 4.6) for TRPML1Va and ML4 mutant TRPML1Va channels. Asterisk indicates statistical difference (P < 0.01) compared to TRPML1Va.


b , 55Fe2 + uptake ( normalized ) in HEK293T transfected with vector control , with TRPML1Va , with F408D - TRPML1Va with T232P - TRPML1Va constructs . Error bars indicate standard deviation on basis of two independent triplicate experiments .

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

b, 55Fe2+uptake (normalized) in HEK293T cells transfected with vector control, with TRPML1Va, with F408D-TRPML1Va and with T232P-TRPML1Va constructs. Error bars indicate the standard deviation on the basis of two independent triplicate experiments.


c , [ Fe2 + ] o - dependent quenching of Fura - 2 in TRPML1Va - transfected ( arrows ) , but not in non - transfected control ( arrowheads ) or T232P - TRPML1Va - transfected ( bottom row ) . was measured at an excitation wavelength of 360 nm ( F360 ) . original magnification used for was × 200 .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 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

c, [Fe2+]o-dependent quenching of Fura-2 fluorescence in TRPML1Va-transfected cells (arrows), but not in non-transfected control cells (arrowheads) or T232P-TRPML1Va-transfected cells (bottom row). The fluorescence intensity was measured at an excitation wavelength of 360 nm (F360). The original magnification used for all micrographs was ×200.


d , Average normalized responses of EGFP - positive TRPML1Va - transfected ( typically n = 20 - 40 ) to 1 or 10 mM Fe2 + ( pH 4 . 6 ) .

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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O

d, Average normalized responses of EGFP-positive TRPML1Va-transfected cells (typically n = 20-40 cells) to 1 or 10 mM Fe2+ (pH 4.6).


e , No significant quenching was seen in T232P - TRPML1Va - transfected .

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

e, No significant quenching was seen in T232P-TRPML1Va-transfected cells.


f , Slightly less quenching was observed for F408Dgr ; - TRPML1Va - expressing .

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

f, Slightly less quenching was observed for the F408Dgr;-TRPML1Va-expressing cells.


g , A small but significant quenching reaction was detected ( with 10 mM Fe2 + ) in R403C - TRPML1Va - expressing .

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

g, A small but significant quenching reaction was detected (with 10 mM Fe2+) in R403C-TRPML1Va-expressing cells.


a , Co - localization of mCherry - TRPML1 EGFP - LAMP1 at of an isolated enlarged LEL ( see Methods ) . patch pipette was filled with red rhodamine B dye ( shown in blue for purpose of illustration ) .

O O O O O O O O B-MEASURED_VAR 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

a, Co-localization of mCherry-TRPML1 and EGFP-LAMP1 at the membrane of an isolated enlarged LEL (see Methods). The patch pipette was filled with red rhodamine B dye (shown in blue for the purpose of illustration).


c , Lysosomal ITRPML1Va . Switching from lysosome - attached to ( lysosome ) luminal - side - out configuration significantly reduced amplitude of current . luminal - side - out patch was exposed to Tyrode ' s solution . A Cs + - solution ( 147 mM Cs - methanesulphonate ( Cs - MSA ) ) was used as a pipette solution for both configurations .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, Lysosomal ITRPML1Va. Switching from lysosome-attached to (lysosome) luminal-side-out configuration significantly reduced the amplitude of the current. The luminal-side-out patch was exposed to the Tyrode's solution. A Cs+-based solution (147 mM Cs-methanesulphonate (Cs-MSA)) was used as a pipette solution for both configurations.


d , NMDG + - impermeable lysosomal ITRPML1Va was much larger in absence of divalent cations ( nominal divalent - free ) . e , Lowering pH potentiated lysosomal ITRPML1Va . f , IFe / TRPML1Va induced by 30 mM 105 mM Fe2 + .

O O O O O O O O O O O O O O O O O O O O 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 I-CONTROLLED_VAR O

d, NMDG+-impermeable lysosomal ITRPML1Va was much larger in the absence of divalent cations (nominal divalent-free). e, Lowering pH potentiated lysosomal ITRPML1Va. f, IFe/TRPML1Va induced by 30 mM and 105 mM Fe2+.


g , Whole - lysosome current in an enlarged lysosome expressing wild - type TRPML1 . pipette ( lumen ) solution contained nominal divalent - free Tyrode solution . A Cs + - bath solution ( 147 mM Cs - MSA ) was used . h , Whole - lysosome ITRPML1Va . i , Whole - lysosome IFe / TRPML1 . pipette ( lumen ) solution contained 105 mM Fe2 + ( pH 4 . 6 ) .

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

g, Whole-lysosome current in an enlarged lysosome expressing wild-type TRPML1. The pipette (lumen) solution contained nominal divalent-free Tyrode solution. A Cs+-based bath solution (147 mM Cs-MSA) was used. h, Whole-lysosome ITRPML1Va. i, Whole-lysosome IFe/TRPML1. The pipette (lumen) solution contained 105 mM Fe2+ (pH 4.6).


a , Cultured TRPML1 - / - ( ML1 - / - ) skin fibroblasts showed less de - quenching of iron - sensitive than ML1 + / - did . De - quenching was achieved by preloading fibroblasts with an iron - sensitive dye , Phen Green SK ( PG SK ) , then adding - permeable transition metal chelator , 2 , 2 ′ - bipyridyl ( BPD ) . 2 , 2 ′ - BPD is predicted to chelate free iron ( also referred to as chelatable or labile iron ) , which subsequently increases PG SK . original magnification used was ×

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

a, Cultured TRPML1-/- (ML1-/-) skin fibroblasts showed less de-quenching of the iron-sensitive fluorescence than ML1+/- cells did. De-quenching was achieved by preloading the fibroblasts with an iron-sensitive dye, Phen Green SK (PG SK), and then adding the membrane-permeable transition metal chelator, 2,2′-bipyridyl (BPD). 2,2′-BPD is predicted to chelate free cellular iron (also referred to as chelatable or labile iron), which subsequently increases PG SK fluorescence. The original magnification used was ×200


b , average 2 , 2 ′ - BPD induced normalized change of ( Dgr ; F / F0 ) for ML4 ( ML1 - / - ; n = 6 experiments ) is significantly ( asterisk , P < 0 . 01 ) lower than for parental ML1 + / - . Fibroblast ( n = 10 - 20 ) were analysed for each experiment . 4 , 4 ′ - BPD , a 2 , 2 ′ - BPD analogue that cannot bind Fe2 + , did not induce any significant restoration of PG SK . Error bars , s . e . m .

O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_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 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 I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O

b, The average 2,2′-BPD induced normalized change of fluorescence (Dgr;F/F0) for ML4 cells (ML1-/-; n = 6 experiments) is significantly (asterisk, P < 0.01) lower than for the parental ML1+/- cells. Fibroblast cells (n = 10-20) were analysed for each individual experiment. 4,4′-BPD, a 2,2′-BPD analogue that cannot bind Fe2+, did not induce any significant restoration of PG SK fluorescence. Error bars, s.e.m.


c , An ML1 - / - skin fibroblast showed autofluorescence in LAMP1 - positive compartments . Autofluorescence ( green ) was detected within a range of excitation wavelengths ( shown with excitation at 480 nm ) . No significant autofluorescence was observed for a ML1 + / + . Lysosomes were stained with a LAMP1 antibody ( red ) . Differential interference contrast ( DIC ) are shown for comparison .

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

c, An ML1-/- skin fibroblast cell showed autofluorescence in LAMP1-positive compartments. Autofluorescence (green) was detected within a range of excitation wavelengths (shown with excitation at 480 nm). No significant autofluorescence was observed for a ML1+/+ cell. Lysosomes were stained with a LAMP1 antibody (red). Differential interference contrast (DIC) images are shown for comparison.


central axial sections of reconstructions . Note C - terminal domain of gp104 other low - density features that do not visualize well in isosurface representation ( panels ( A - C ) ) . Letters G , H I indicate position of cross - sections shown in homonymously labeled panels below . A 69 Å - thick slice of reconstruction of extended sheath cryoEM map orthogonal to sixfold axis . proximal distal parts of one of three that run at 82 ° to sixfold axis are indicated with a semitransparent cyan magenta lines , respectively . Sections of spontaneously urea contracted sheath structures that cut through baseplate / proximal part of fiber plane , whose position in reconstructions is indicated with a red in panels ( E F ) . putative direction of distal part of fiber that is completely disordered in reconstructions is indicated with a solar cross symbol .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

The central axial sections of the reconstructions. Note the C-terminal domain of gp104 and other low-density features that do not visualize well in the isosurface representation (panels (A-C)). Letters G, H and I indicate the position of the cross-sections shown in the homonymously labeled panels below. A 69 Å-thick slice of the reconstruction of the extended sheath cryoEM map orthogonal to the sixfold axis. The proximal and distal parts of one of the three fibers that run at 82° to the sixfold axis are indicated with a semitransparent cyan and magenta lines, respectively. Sections of the spontaneously and urea contracted sheath structures that cut through the baseplate/proximal part of the fiber plane, whose position in the reconstructions is indicated with a red line in panels (E and F). The putative direction of the distal part of the fiber that is completely disordered in the reconstructions is indicated with a solar cross symbol.


of phage particles in extended , intermediate , contracted states after 5 , 20 80 minutes of incubation with L . monocytogenes walls . numbers above bars show number of particles contained in tomograms . average of tail standard deviation for each of three states is given next to color legend .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Percentage of phage particles in the extended, intermediate, and contracted states after 5, 20 and 80 minutes of incubation with L. monocytogenes cell walls. The numbers above the bars show the number of particles contained in the tomograms. The average length of the tail and the standard deviation for each of the three states is given next to the color legend.


pre - attachment state . Here elsewhere in this figure , EM image in lower panel is a slice through cryoET reconstruction . Note that in presence of Listeria wall gp108 are no longer in their stowed conformation but instead are fully or partially unraveled . Gp108 attach to host wall but sheath is not yet contracted . RBP / TF gp106 ( possibly gp107 ) pyramids reorient to point toward surface , change their conformation , bind to wall . baseplate - proximal part of sheath is in a contracted state , whereas capsid - proximal part is still in an extended state . baseplate - proximal part of sheath contains a segment where tail tube is clearly visible because it no longer interacts with sheath ( lower panel ) . post - attachment state . tail is fully contracted tail tube has pierced through wall . capsid appears full indicating that injection has not yet begun .

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

The pre-attachment state. Here and elsewhere in this figure, the EM image in the lower panel is a slice through the cryoET reconstruction. Note that in the presence of the Listeria cell wall the gp108 fibers are no longer in their stowed conformation but instead are fully or partially unraveled. Gp108 fibers attach to the host cell wall but the sheath is not yet contracted. RBP/TF gp106 (and possibly gp107) pyramids reorient to point toward the cell surface, change their conformation, and bind to the cell wall. The baseplate-proximal part of the sheath is in a contracted state, whereas the capsid-proximal part is still in an extended state. The baseplate-proximal part of the sheath contains a segment where the tail tube is clearly visible because it no longer interacts with the sheath (lower panel). The post-attachment state. The tail is fully contracted and the tail tube has pierced through the cell wall. The capsid appears full indicating that DNA injection has not yet begun.


B . Rev3l + / + Rev3l - / - MEFs were - labelled with EdU for 15min , permeabilized , fixed stained for EdU incorporation ( green ) . S phase sub - stages from I to IV were evaluated by visual inspection of cycling population ( > 300 EdU + , top panel ) . Scale bar = 5 μm . Dot plots pie charts show relative proportion ( of total S ) of each sub - stage from I to IV ( middle bottom panel , respectively ) . Each dot represents mean of two technical replicates .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Rev3l+/+ and Rev3l-/- MEFs were pulse-labelled with EdU for 15min, permeabilized, fixed and stained for EdU incorporation (green). S phase sub-stages from I to IV were evaluated by visual inspection of the cycling population (>300 EdU+ cells, top panel). Scale bar= 5 μm. Dot plots and pie charts show the relative proportion (percentage of total S) of each sub-stage from I to IV (middle and bottom panel, respectively). Each dot represents the mean of two technical replicates.


C . of active marks H3K27ac H3K4me3 facultative heterochromatin mark H3K27me3 content in early , mid , late TTR compared to disturbed replicating regions found in Rev3l - / - MEFs . Bar in boxplot represents median , red points represents mean . limit of boxes corresponds to 0 . 25 to 0 . 75 quartiles with whiskers extending to maximum value of 1 . 5 times interquartile range . Graphs show data from two biological experiments .

O O O O O B-MEASURED_VAR I-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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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. Analysis of the active marks H3K27ac and H3K4me3 and facultative heterochromatin mark H3K27me3 content in early, mid, late and TTR compared to disturbed replicating regions found in Rev3l-/- MEFs. Bar in boxplot represents the median, and red points represents the mean. The limit of the boxes corresponds to the 0.25 to 0.75 quartiles with whiskers extending to the maximum value of 1.5 times the interquartile range. Graphs show data from two biological experiments.


D . Relative mRNA of four genes involved in development ( Hoxb2 , 8 , 9 WT1 ) were validated through qRT - PCR from Rev3l + / + Rev3l - / - MEFs samples . data were normalized to amount of GAPDH mRNA . Error bars indicate standard error of mean for three independent experiments . * * * , P < 0 . 001 * * , P < 0 . 005 by Student ' s t - . E . Relative mRNA of genes involved in imprinting ( paternal expressed genes : Dlk1and Slc18a4 maternal expressed genes : Dcn1 H19 ) were validated through qRT - PCR from Rev3l + / + Rev3l - / - MEFs samples . data were normalized to amount of GAPDH mRNA . Error bars indicate standard error of mean for three independent experiments . * * * P < 0 . 001 by Student ' s t - .

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

D. Relative mRNA levels of four genes involved in development (Hoxb2, 8, 9 and WT1) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. ***, P<0.001 and **, P<0.005 by Student's t-test. E. Relative mRNA levels of genes involved in imprinting (paternal expressed genes: Dlk1and Slc18a4 and maternal expressed genes: Dcn1 and H19) were validated through qRT-PCR from Rev3l+/+ and Rev3l-/- MEFs samples. The data were normalized to the amount of GAPDH mRNA. Error bars indicate standard error of the mean for three independent experiments. *** P<0.001 by Student's t-test.


A . Total histones were acid extracted from Rev3l + / + Rev3l - / - MEFs . Samples were analyzed by western - blot using indicated antibodies specific to histone marks ( right panel ) . An anti - pan H4 or H3 was used as loading control ( left panel ) . Histograms show WT / KO Rev3l fold change relative to immunoblot shown on top panel ( using 1X sample ) .

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

A. Total histones were acid extracted from Rev3l+/+ and Rev3l-/- MEFs. Samples were analyzed by western-blot using indicated antibodies specific to histone marks (right panel). An anti-pan H4 or H3 was used as loading control (left panel). Histograms show WT/KO Rev3l fold change relative to the immunoblot shown on the top panel (using 1X sample intensity).


B . Histone mark were examined at selected genes repressed in Rev3l - / - MEFs ( Hoxb2 , Hoxb8 , WT1 ) . Histograms represent enrichment of H3K9me3 H3K27me3 at indicated loci assessed by ChIP - qPCR in Rev3l + / + Rev3l - / - MEFs . Error bars indicate standard error of mean for three independent experiments .

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

B. Histone mark levels were examined at selected genes repressed in Rev3l-/- MEFs (Hoxb2, Hoxb8, WT1). Histograms represent enrichment of H3K9me3 and H3K27me3 at indicated loci assessed by ChIP-qPCR in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.


C . H3K9me3 H3K27me3 mark were evaluated at selected genes that localized in genomic loci displaying replication timing delay downregulated in Rev3l - / - MEFs ( FoxG1 , GATA6 ) . Histograms represent enrichment of H3K9me3 H3K27me3 assessed by ChIP - qPCR at indicated loci in Rev3l + / + Rev3l - / - MEFs . Error bars indicate standard error of mean for three independent experiments .

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

C. H3K9me3 and H3K27me3 mark levels were evaluated at selected genes that localized in genomic loci displaying replication timing delay and downregulated in Rev3l-/- MEFs (FoxG1, GATA6). Histograms represent enrichment of H3K9me3 and H3K27me3 assessed by ChIP-qPCR at indicated loci in Rev3l+/+ and Rev3l-/- MEFs. Error bars indicate standard error of the mean for three independent experiments.


G . MEFs were transfected with GFP - REV3L761 - 1029 WT PSVVL or ASAVA mutant fixed with 4 % formaldehyde . distribution of GFP - REV3L constructs was detected by autofluorescence ( green ) , chromocenters were visualized by HP1α immunostaining ( red ) was counterstained with DAPI . Scale bar = 10

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 B-MEASURED_VAR 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

G. MEFs cells were transfected with GFP-REV3L761-1029 WT PSVVL or ASAVA mutant and fixed with 4% formaldehyde. The distribution of GFP-REV3L constructs was detected by autofluorescence (green), chromocenters were visualized by HP1α immunostaining (red) and DNA was counterstained with DAPI. Scale bar= 10 μm


H . Human 293T were transfected with either FH - REV3L700 - 900 , mutant V802D F - H - REV3L700 - 900 or empty vector . Forty - eight hours after transfection lysates were made used for immunoprecipitation with HP1 antibody . Western blot was processed membranes were immunoblotted with indicated antibodies .

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

H. Human 293T cells were transfected with either FH-REV3L700-900, mutant V802D F-H-REV3L700- 900 or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with HP1 antibody. Western blot was processed and membranes were immunoblotted with the indicated antibodies.


I . 293T were co - transfected with F - H - REV3L700 - 900 or empty vector V5 - HP1γ , mutant IY / EE V5 - HP1γ or empty vector . Forty - eight hours after transfection lysates were made used for immunoprecipitation with α - V5 antibody . After electrophoresis , samples were immunoblotted with anti - HA , anti - HP1γ or anti - α - Tubulin as indicated .

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

I. 293T cells were co-transfected with F-H-REV3L700-900 or empty vector and V5-HP1γ, mutant IY/EE V5-HP1γ or empty vector. Forty-eight hours after transfection cell lysates were made and used for immunoprecipitation with α-V5 antibody. After electrophoresis, samples were immunoblotted with anti-HA, anti-HP1γ or anti-α-Tubulin as indicated.


B . Asynchronous Rev3l + / + Rev3l - / - MEFs were fixed with PFA then subjected to in situ proximity ligation ( PLA ) using 53BP1 HP1α antibodies then PLA foci were counted in both lines ( more than 150 nuclei for each condition were counted ) . p - values were calculated by Mann - Whitney ( * * * * : p < 0 . 0001 ) . Red lines indicate mean values . Error bars : . Controls with a single antibody are also shown . Experiments were repeated three times . Representative are shown . Scale bar = 5 μm .

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 B-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

B. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were fixed with PFA then subjected to in situ proximity ligation assay (PLA) using 53BP1 and HP1α antibodies then PLA foci were counted in both cell lines (more than 150 nuclei for each condition were counted). p-values were calculated by Mann-Whitney test ( ****: p<0.0001). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Experiments were repeated three times. Representative images are shown. Scale bar= 5 μm.


C . Asynchronous Rev3l + / + Rev3l - / - MEFs were subjected to PLA as by using γH2AX HP1β antibodies processed as in ( B ) . p - values were calculated by Mann - Whitney ( * : p < 0 . 05 ) . Red lines indicate mean values . Error bars : . Controls with a single antibody are also shown . Three independent experiments were performed .

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

C. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were subjected to PLA as by using γH2AX and HP1β antibodies and processed as in (B). p-values were calculated by Mann-Whitney test ( *: p<0.05). Red lines indicate the mean values. Error bars: SEM. Controls with a single antibody are also shown. Three independent experiments were performed.


Rev3l + / + Rev3l - / - MEFs were incubated with or without 0 . 23 μM aphidicolin for 24h before metaphase spreading . FISH was performed using major satellite probe to quantify breaks in pericentromeric regions ( F ) . Representative of chromosomes showing abnormalities ( see arrows ) in pericentromeric regions from Rev3l - / - MEFs . Chromosomes were labelled with DAPI Error bars indicate standard error of mean for three independent experiments . Mann - Whitney ( ns : not significant , * p < 0 . 05 ; * * * p < 0 . 001 ) .

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

Rev3l+/+ and Rev3l-/- MEFs were incubated with or without 0.23 μM aphidicolin for 24h before metaphase spreading. FISH was performed using major satellite probe to quantify breaks in pericentromeric regions (F). Representative images of chromosomes showing abnormalities (see arrows) in pericentromeric regions from Rev3l-/- MEFs. Chromosomes were labelled with DAPI Error bars indicate standard error of the mean for three independent experiments. Mann-Whitney test (ns: not significant, * p<0.05; *** p< 0.001).


A . Asynchronous Rev3l + / + Rev3l - / - MEFs were UV - irradiated at indicated doses harvested 6 h or 24 h later . lysates were analyzed by immunoblotting with indicated antibodies . B . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were UV - irradiated at indicated doses harvested 6 h or 24 h later . lysates were analyzed by immunoblotting with indicated antibodies . C

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 O O O O O O O O B-CONTROLLED_VAR 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

A. Asynchronous Rev3l+/+ and Rev3l-/- MEFs were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. B. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were UV-irradiated at the indicated doses and harvested 6 h or 24 h later. Cell lysates were analyzed by immunoblotting with indicated antibodies. C


Asynchronous Rev3l + / + Rev3l - / - MEFs were labelled for 15 min using 10 μM EdU then UV - irradiated at 4 J / m2 . pre - treated with CSK buffer were fixed 53BP1 , RPA RAD51 were detected by immunofluorescence . Representative in non - irradiated ( NI ) of 53BP1 foci in EdU positive at different time points are shown Scale bar = 10 μm number of analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis ) . presented data are representative of 2 repeats ) . Bars represent median ± interquartile

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

Asynchronous Rev3l+/+ and Rev3l-/- MEFs were pulse labelled for 15 min using 10 μM EdU then UV-irradiated at 4 J/m2. Cells pre-treated with CSK buffer were fixed and 53BP1, RPA and RAD51 were detected by immunofluorescence. Representative images in non-irradiated cells (NI) and quantification of 53BP1 foci in EdU positive cells at different time points are shown Scale bar= 10 μm The number of cells analyzed is indicated in (n) on each graph. ***p < 0.001 (Kruskal-Wallis test). The presented data are representative of 2 repeats). Bars represent the median ± interquartile range