( 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 ) 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-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 and / or rapamycin treatment in U2OS cells . Scale bar , 20 µm .

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

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


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 % ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

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

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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , 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 .

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

B. 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 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 .

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

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 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 .

O O O O O O 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

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 nuclear 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 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

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 cytoplasmic 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 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

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 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 .

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

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 . 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). 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 . The inset highlights a double helix - like structure resulting from juxtaposition of two individual filaments . Scale bars : 50 nm and 10 nm ( inset ) .

O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 protein peaks , F1 and 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 weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE .

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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 weight markers . The different SEC elution volumes of F1 and F2 ( A ) reflect different levels of egg coat filament digestion by HCE / LCE .

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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 ) 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 ) .

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

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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 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 ) .

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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 ) . 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 ) .

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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 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 .

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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 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 .

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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.


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 .

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

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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.


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 .

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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 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 .

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 the 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 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 .

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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 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 .

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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 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 ) .

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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 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 .

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

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 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 .

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

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 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 − ) .

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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 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 .

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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 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 .

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 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 .

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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 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 .

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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.


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

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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 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 ) .

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 . Staining for β - catenin and SOX9 confirmed activation of Wnt signalling in the observed lesions . Scale bar , 100 μm .

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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.


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

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


Western blot of purified crypts from the small intestine and 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 and colon . Expression of mRNA ( 2 ( − dCt ) ) calculated relative to GAPDH ( N = 3 mice ) . Statistics : one - sided Mann - Whitney U - test .

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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 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 .

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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 ) 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 .

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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 and day 8 . Graphs show average of experiments ( N = 3 mice for each genotype and time point ) ; error bars represent s . e . m .

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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 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 ) .

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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 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 ) .

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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 . 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 .

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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 ) / + 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 .

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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 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 .

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 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 .

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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 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 .

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 .

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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 the same cell as shown in a and b .

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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 - 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 .

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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 the presence of 30 and 105 mM Fe2 + ( pH 4 . 6 ) .

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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 cell .

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 and ML4 mutant TRPML1Va channels . Asterisk indicates statistical difference ( P < 0 . 01 ) compared to TRPML1Va .

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 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 .

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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 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 .

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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 cells ( typically n = 20 - 40 cells ) to 1 or 10 mM Fe2 + ( pH 4 . 6 ) .

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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 cells .

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


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

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 cells .

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


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 ) .

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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 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 .

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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 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 + .

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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 . 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 ) .

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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 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 ×

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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 , 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 .

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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 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 .

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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.


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 .

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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.


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 .

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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.


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 .

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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 + / + 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 .

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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 . 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 .

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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 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 .

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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 + / + 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 ) .

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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 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 .

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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 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 .

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 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

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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 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 .

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 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 .

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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 + / + 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 .

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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 + / + 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 .

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 + / + 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 ) .

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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 + / + 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

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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 + / + 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

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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


F - H . Asynchronous Rev3l - / - MEFs complemented with empty vector ( EV ) or REV3L were processed as in ( C ) . Quantification of 53BP1 foci in EdU positive cells at different time points is shown ( F ) . Quantification of the intensity of chromatin - bound RPA2 ( G ) and RAD51 foci ( H ) 24 hours after UV - irradiation was performed . 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 .

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F-H. Asynchronous Rev3l-/- MEFs complemented with empty vector (EV) or REV3L were processed as in (C). Quantification of 53BP1 foci in EdU positive cells at different time points is shown (F). Quantification of the intensity of chromatin-bound RPA2 (G) and RAD51 foci (H) 24 hours after UV-irradiation was performed. 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.


A RNF123 suppresses the SeV - and EMCV - induced but not the cGAS and STING - mediated activation of the IFN - βpromoter . HEK293T cells ( 3 × 105 ) were transfected with the IFN - β reporter plasmid ( 100 ng ) , the pRL - TK Renilla luciferase plasmid ( 100 ng ) , the expression plasmid for cGAS ( 50 ng ) and STING ( 50 ng ) or empty vector ( 100 ng ) , and 0 , 20 , 50 , or 100 ng ( wedges ) of RNF123 for 12 h , and then were infected with SeV or EMCV or left uninfected . Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells .

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A RNF123 suppresses the SeV- and EMCV-induced but not the cGAS and STING-mediated activation of the IFN-βpromoter. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmid for cGAS (50 ng) and STING (50 ng) or empty vector (100 ng), and 0, 20, 50, or 100 ng (wedges) of RNF123 for 12 h, and then were infected with SeV or EMCV or left uninfected. Luciferase assays were performed after 18 h and results are presented relative to the luciferase activity in control cells.


B RNF123 inhibits the SeV - , but not the cGAS and STING - induced activation of the NF - κB promoter . HEK293T cells ( 3 × 105 ) were transfected with the NF - κB reporter plasmid ( 100 ng ) , the pRL - TK Renilla luciferase plasmid ( 100 ng ) , the expression plasmids for cGAS ( 50 ng ) and STING ( 50 ng ) , or empty vector ( 100 ng ) , and 0 , 20 , 50 or 100 ng ( wedges ) of RNF123 for 12 h , and were then infected for 24 h with SeV or left uninfected , followed by analysis as described in A .

B RNF123 inhibits the SeV-, but not the cGAS and STING-induced activation of the NF-κB promoter. HEK293T cells (3×105) were transfected with the NF-κB reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), the expression plasmids for cGAS (50 ng) and STING (50 ng), or empty vector (100 ng), and 0, 20, 50 or 100 ng (wedges) of RNF123 for 12 h, and were then infected for 24 h with SeV or left uninfected, followed by analysis as described in A.


C , D RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated RANTES and IFN - β transcription . The transfection and treatment were as in A . Total RNA was isolated and RT - PCR ( C ) was performed using the indicated primers .

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C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and RT-PCR (C) was performed using the indicated primers.


C , D RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated RANTES and IFN - β transcription . The transfection and treatment were as in A . Total RNA was isolated and Q - PCR ( D ) was performed using the indicated primers .

C, D RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated RANTES and IFN-β transcription. The transfection and treatment were as in A. Total RNA was isolated and Q-PCR (D) was performed using the indicated primers.


E RNF123 inhibits the SeV - induced , but not the cGAS and STING - mediated IFN - β secretion . The transfection and treatment were as in A . Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells .

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E RNF123 inhibits the SeV-induced, but not the cGAS and STING-mediated IFN-β secretion. The transfection and treatment were as in A. Bioassays for type I interferon in the supernatants of HEK293T cells were performed and the results are presented relative to untreated control cells.


F Overexpression of RNF123 enhances NDV - eGFP replication . HEK293T cells ( 1 × 105 ) were transfected with the expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , cells were infected with NDV - eGFP . At 30 h after infection , viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot .

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F Overexpression of RNF123 enhances NDV-eGFP replication. HEK293T cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with NDV-eGFP. At 30 h after infection, viral replication was determined by GFP expression visualized by fluorescence microscopy and analyzed by western blot.


G RNF123 enhances EMCV replication . HeLa cells ( 1 × 105 ) were transfected with the expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , cells were infected with EMCV or given DMEM with no virus . At 18 h after infection , viral replication was determined by VP1 expression , which was analyzed by western blot .

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G RNF123 enhances EMCV replication. HeLa cells (1×105) were transfected with the expression plasmids for Flag-RNF123 or empty vector (2 µg). At 24 h after transfection, cells were infected with EMCV or given DMEM with no virus. At 18 h after infection, viral replication was determined by VP1 expression, which was analyzed by western blot.


A HEK293T cells with stable knockdown of RNF123 were generated using shRNA . Western blot was performed with anti - RNF123 , anti - VISA ( as a control ) , and anti - GAPDH antibodies after infection with five lentiviruses encoding a shRNA control ( GFPsh ) and four shRNAs targeting RNF123 ( RNF123 sh # 1 , # 2 , # 3 , and # 4 ) in HEK293T cells .

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A HEK293T cells with stable knockdown of RNF123 were generated using shRNA. Western blot was performed with anti-RNF123, anti-VISA (as a control), and anti-GAPDH antibodies after infection with five lentiviruses encoding a shRNA control (GFPsh) and four shRNAs targeting RNF123 (RNF123 sh #1, #2, #3, and #4) in HEK293T cells.


B , C Knockdown of RNF123 facilitated the activation of IFN - β and NF - κB promoters induced by SeV and EMCV but not by cGAS and STING . Stable HEK293T cells with GFPsh or RNF123 shRNAs ( 2 × 105 ) were transfected with the indicated reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , cGAS ( 50 ng ) , and STING ( 50 ng ) , or empty vector ( 100 ng ) . At 12 h after transfection , they were infected for 12 h with SeV or EMCV or left uninfected , followed by analysis as described in the legend of Fig 1A .

B, C Knockdown of RNF123 facilitated the activation of IFN-β and NF-κB promoters induced by SeV and EMCV but not by cGAS and STING. Stable HEK293T cells with GFPsh or RNF123 shRNAs (2×105) were transfected with the indicated reporter plasmid (100 ng), pRL-TK Renilla luciferase plasmid (100 ng), cGAS (50 ng), and STING (50 ng), or empty vector (100 ng). At 12 h after transfection, they were infected for 12 h with SeV or EMCV or left uninfected, followed by analysis as described in the legend of Fig 1A.


D , E Knockdown of RNF123 enhances the IFN - β and RANTES transcription induced by SeV and EMCV , but not that by cGAS and the STING . HEK293T cells were transfected with cGAS ( 1 μg ) and STING ( 1 μg ) or empty vector for 12 h , and then infected with SeV or EMCV for 12 h or left uninfected . Total RNA was isolated and RT - PCR ( D ) were performed using the indicated primers .

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D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and RT-PCR (D) were performed using the indicated primers.


D , E Knockdown of RNF123 enhances the IFN - β and RANTES transcription induced by SeV and EMCV , but not that by cGAS and the STING . HEK293T cells were transfected with cGAS ( 1 μg ) and STING ( 1 μg ) or empty vector for 12 h , and then infected with SeV or EMCV for 12 h or left uninfected . Total RNA was isolated and Q - PCR ( E ) were performed using the indicated primers .

D, E Knockdown of RNF123 enhances the IFN-β and RANTES transcription induced by SeV and EMCV, but not that by cGAS and the STING. HEK293T cells were transfected with cGAS (1 μg) and STING (1 μg) or empty vector for 12 h, and then infected with SeV or EMCV for 12 h or left uninfected. Total RNA was isolated and Q-PCR (E) were performed using the indicated primers.


F , G Time - course of potentiation of SeV - induced IFN - β transcription ( F ) by knockdown of RNF123 . HEK293T cells were infected with SeV for 6 , 12 , 18 , or 24 h , total RNA was isolated , and RT - PCR was performed using the indicated primers ( F ) .

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F, G Time-course of potentiation of SeV-induced IFN-β transcription (F) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated, and RT-PCR was performed using the indicated primers (F).


F , G Time - course of potentiation of SeV - induced IFN - β secretion ( G ) by knockdown of RNF123 . HEK293T cells were infected with SeV for 6 , 12 , 18 , or 24 h , total RNA was isolated . Bioassays of type - I interferon in the supernatants of HEK293T cells were performed , and the results are presented relative to untreated control cells ( G ) .

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F, G Time-course of potentiation of SeV-induced IFN-β secretion (G) by knockdown of RNF123. HEK293T cells were infected with SeV for 6, 12, 18, or 24 h, total RNA was isolated. Bioassays of type-I interferon in the supernatants of HEK293T cells were performed, and the results are presented relative to untreated control cells (G).


H HeLa cells with stable knockdown of RNF123 were generated by shRNA . Western blot was performed as described in Fig 2A .

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H HeLa cells with stable knockdown of RNF123 were generated by shRNA. Western blot was performed as described in Fig 2A.


I , J Knockdown of RNF123 enhances SeV - and EMCV - induced IFN - β expression in HeLa cells . HeLa cells were infected with SeV or EMCV for 12 h , total RNA was isolated , and RT - PCR ( I ) was performed using the indicated primers .

I, J Knockdown of RNF123 enhances SeV- and EMCV- induced IFN-β expression in HeLa cells. HeLa cells were infected with SeV or EMCV for 12 h, total RNA was isolated, and RT-PCR (I) was performed using the indicated primers.