d , Average normalized responses of EGFP - positive TRPML1Va - transfected ( typically n = 20 - 40 ) 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 .

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


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

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 .

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

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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 amplitude of current . luminal - side - out patch was exposed to Tyrode ' s solution . A Cs + - solution ( 147 mM Cs - methanesulphonate ( Cs - MSA ) ) was used as a pipette solution for both configurations .

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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 absence of divalent cations ( nominal divalent - free ) . e , Lowering pH potentiated lysosomal ITRPML1Va . f , IFe / TRPML1Va induced by 30 mM 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 . pipette ( lumen ) solution contained nominal divalent - free Tyrode solution . A Cs + - bath solution ( 147 mM Cs - MSA ) was used . h , Whole - lysosome ITRPML1Va . i , Whole - lysosome IFe / TRPML1 . pipette ( lumen ) solution contained 105 mM Fe2 + ( pH 4 . 6 ) .

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

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

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

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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.


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

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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.


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

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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.


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

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

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

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

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

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

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

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


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

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

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


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

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

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

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


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

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

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


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

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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 ) . of 53BP1 foci in EdU positive at different time points is shown ( F ) . of of - bound RPA2 ( G ) RAD51 foci ( H ) 24 hours after UV - irradiation was performed . number of analyzed is indicated in ( n ) on each graph . * * * p < 0 . 001 ( Kruskal - Wallis ) . presented data are representative of 2 repeats . Bars represent median ± interquartile 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.


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

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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 SeV - , but not cGAS STING - induced activation of NF - κB promoter . HEK293T ( 3 × 105 ) were transfected with NF - κB reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , expression plasmids for cGAS ( 50 ng ) STING ( 50 ng ) , or empty vector ( 100 ng ) , 0 , 20 , 50 or 100 ng ( wedges ) of RNF123 for 12 h , were then infected for 24 h with SeV or left uninfected , followed by 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 SeV - induced , but not cGAS STING - mediated RANTES IFN - β transcription . transfection treatment were as in A . Total was isolated RT - PCR ( C ) was performed using 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 SeV - induced , but not cGAS STING - mediated RANTES IFN - β transcription . transfection treatment were as in A . Total was isolated Q - PCR ( D ) was performed using 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 SeV - induced , but not cGAS STING - mediated IFN - β secretion . transfection treatment were as in A . Bioassays for type I interferon in supernatants of HEK293T were performed results are presented relative to untreated control .

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

E 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 ( 1 × 105 ) were transfected with expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , were infected with NDV - eGFP . At 30 h after infection , viral replication was determined by GFP expression visualized by microscopy 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 ( 1 × 105 ) were transfected with expression plasmids for Flag - RNF123 or empty vector ( 2 µg ) . At 24 h after transfection , were infected with EMCV or given DMEM with no . At 18 h after infection , viral replication was determined by VP1 expression , which was analyzed by western blot .

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

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

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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 activation of IFN - β NF - κB promoters induced by SeV EMCV but not by cGAS STING . Stable HEK293T with GFPsh or RNF123 shRNAs ( 2 × 105 ) were transfected with indicated reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , cGAS ( 50 ng ) , 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 as described in legend of Fig 1A .

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


D , E Knockdown of RNF123 enhances IFN - β RANTES transcription induced by SeV EMCV , but not that by cGAS STING . HEK293T were transfected with cGAS ( 1 μg ) STING ( 1 μg ) or empty vector for 12 h , then infected with SeV or EMCV for 12 h or left uninfected . Total was isolated Q - PCR ( E ) were performed using 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 were infected with SeV for 6 , 12 , 18 , or 24 h , total was isolated , RT - PCR was performed using 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 were infected with SeV for 6 , 12 , 18 , or 24 h , total was isolated . Bioassays of type - I interferon in supernatants of HEK293T were performed , results are presented relative to untreated control ( G ) .

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 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 - EMCV - induced IFN - β expression in HeLa . HeLa were infected with SeV or EMCV for 12 h , total was isolated , RT - PCR ( I ) was performed using indicated primers .

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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 - EMCV - induced IFN - β expression in HeLa . HeLa were infected with SeV or EMCV for 12 h , total was isolated , Q - PCR ( J ) was performed using 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 Q-PCR (J) was performed using the indicated primers.


K Knockdown of RNF123 suppresses NDV - eGFP replication . HEK293T ( 2 × 105 ) were infected with NDV - eGFP . At 30 h after infection , viral replication was determined by GFP expression visualized by microscopy analyzed by western blot with GFP antibody .

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K Knockdown of RNF123 suppresses NDV-eGFP replication. HEK293T cells (2×105) 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 with GFP antibody.


L Knockdown of RNF123 inhibits EMCV replication . HeLa ( 2 × 105 ) were infected with EMCV for 0 , 12 , or 18 h .

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L Knockdown of RNF123 inhibits EMCV replication. HeLa cells (2×105) were infected with EMCV for 0, 12, or 18 h.


A , B RNF123 inhibits activation of IFN - β ( A ) promoters induced by overexpression of RIG - I , RIG - I CARD , MDA5 CARD , VISA , but has no effect on TBK1 - IRF3 - 5D - induced activation of IFN - β promoter ( A ) . HEK293T ( 2 × 105 ) were transfected with indicated plasmids ( 100 ng ) , IFN - β promoter luciferase plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , together with an empty vector RNF123 construct ( 100 ng ) . Luciferase assays were performed 24 h after transfection .

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A, B RNF123 inhibits the activation of the IFN-β (A) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TBK1- and IRF3-5D-induced activation of the IFN-β promoter (A) . HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), IFN-β promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.


A , B RNF123 inhibits activation of NF - κB ( B ) promoters induced by overexpression of RIG - I , RIG - I CARD , MDA5 CARD , VISA , but has no effect on TAB2 - IKKβ - induced activation of NF - κB promoter ( B ) . HEK293T ( 2 × 105 ) were transfected with indicated plasmids ( 100 ng ) , NF - κB promoter luciferase plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , together with an empty vector RNF123 construct ( 100 ng ) . Luciferase assays were performed 24 h after transfection .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 RNF123 inhibits the activation of the NF-κB (B) promoters induced by the overexpression of RIG-I, RIG-I CARD, MDA5 CARD, and VISA, but has no effect on the TAB2- and IKKβ-induced activation of the NF-κB promoter (B). HEK293T cells (2×105) were transfected with the indicated plasmids (100 ng), NF-κB promoter luciferase plasmid (100 ng), and pRL-TK Renilla luciferase plasmid (100 ng), together with an empty vector and RNF123 construct (100 ng). Luciferase assays were performed 24 h after transfection.


C Knockdown of RNF123 potentiates activation of IFN - β promoter induced by RIG - I , RIG - I CARD , MDA5 , MDA5 CARD , but not that by VISA , TBK1 , cGAS / STING . 293T - GFPsh , shRNF123 # 1 , # 3 , # 4 stable pools ( 2 × 105 ) were transfected with indicated plasmids . Luciferase assays were performed 24 h after transfection .

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C Knockdown of RNF123 potentiates the activation of the IFN-β promoter induced by RIG-I, RIG-I CARD, MDA5, and MDA5 CARD, but not that by VISA, TBK1, and cGAS/STING. 293T-GFPsh, shRNF123 #1, #3, and #4 stable cell pools (2×105) were transfected with the indicated plasmids. Luciferase assays were performed 24 h after transfection.


D RNF123 interacted with RIG - I MDA5 , but not VISA , TBK1 , cGAS . HEK293T ( 1 × 106 ) were transfected with Flag - RNF123 ( F - RNF123 ) together with HA - RIG - I , HA - MDA5 , HA - VISA , HA - TBK1 , or HA - cGAS plasmids ( 8 μg each ) . lysates were immunoprecipitated ( IP ) with anti - HA ( αHA ) . immunoprecipitates were analyzed by western blotting ( immunoblotting [ IB ] ) with anti - Flag ( αF ) or anti - HA antibody ( αHA ) . Whole - lysates ( WCL ) were analyzed by western blotting with anti - HA anti - Flag antibodies to determine expression of transfected plasmids .

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D RNF123 interacted with RIG-I and MDA5, but not VISA, TBK1, and cGAS. HEK293T cells (1×106) were transfected with Flag-RNF123 (F-RNF123) together with HA-RIG-I, HA-MDA5, HA-VISA, HA-TBK1, or HA-cGAS plasmids (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-HA (αHA). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-Flag (αF) or anti-HA antibody (αHA). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies to determine the expression levels of transfected plasmids.


E Endogenous interaction of RNF123 with RIG - I upon SeV infection . HEK293T ( 3 × 106 ) were treated with SeV for 12 h or left untreated . lysates were immunoprecipitated with rabbit anti - RNF123 antibody or control IgG . immunoprecipitates were analyzed by western blotting with rabbit anti - RIG - I antibody . expression of endogenous were analyzed by western blotting with anti - RIG - I anti - RNF123 antibodies .

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E Endogenous interaction of RNF123 with RIG-I upon SeV infection. HEK293T cells (3×106) were treated with SeV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-RIG-I antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-RIG-I and anti-RNF123 antibodies.


F Endogenous interaction of RNF123 with MDA5 upon EMCV infection . HeLa ( 3 × 106 ) were treated with EMCV for 12 h or left untreated . lysates were immunoprecipitated with rabbit anti - RNF123 antibody or control IgG . immunoprecipitates were analyzed by western blotting with rabbit anti - MDA5 antibody . expression of endogenous were analyzed by western blotting with anti - MDA5 anti - RNF123 antibodies . Each histogram shows mean ± SD of three independent experiments done in triplicate . * * * P < 0 . 001 ; * * P < 0 . 01 ; * P < 0 . 1 ; ns , P > 0 . 05 .

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F Endogenous interaction of RNF123 with MDA5 upon EMCV infection. HeLa cells (3×106) were treated with EMCV for 12 h or left untreated. Cell lysates were immunoprecipitated with rabbit anti-RNF123 antibody or control IgG. The immunoprecipitates were analyzed by western blotting with rabbit anti-MDA5 antibody. The expression levels of the endogenous proteins were analyzed by western blotting with anti-MDA5 and anti-RNF123 antibodies. Each histogram shows the mean ± SD of three independent experiments done in triplicate. ***P <0.001; **P <0.01; *P <0.1; ns, P >0.05.


B SPRY coiled - coil domains are both indispensable for inhibiting IFN - β promoter activation . HEK293T ( 3 × 105 ) were transfected with IFN - β reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , expression plasmid for RNF123 its mutants ( 100 ng ) , empty vector or RIG - I CARD / MDA5 CARD ( 100 ng ) . Reporter assays were performed 24 h after transfection .

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B The SPRY and coiled-coil domains are both indispensable for inhibiting IFN-β promoter activation. 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 RNF123 and its mutants (100 ng), and the empty vector or RIG-I CARD/MDA5 CARD (100 ng). Reporter assays were performed 24 h after transfection.


C SPRY domain two coiled - coil domains of RNF123 are both required for interaction with RIG - I CARD . HEK293T ( 1 × 106 ) were transfected with HA - RIG - I CARD together with F - RNF123 or its mutants ( 8 μg each ) . lysates were immunoprecipitated ( IP ) with anti - Flag ( α - F ) . immunoprecipitates were analyzed by western blotting ( immunoblotting [ IB ] ) with anti - HA ( α - HA ) or anti - Flag ( α - F ) . Whole - lysates ( WCL ) were analyzed by western blotting with anti - HA anti - Flag antibodies .

C The SPRY domain and two coiled-coil domains of RNF123 are both required for interaction with RIG-I CARD. HEK293T cells (1×106) were transfected with HA-RIG-I CARD together with F-RNF123 or its mutants (8 μg each). Cell lysates were immunoprecipitated (IP) with anti-Flag (α-F). The immunoprecipitates were analyzed by western blotting (immunoblotting [IB]) with anti-HA (α-HA) or anti-Flag (α-F). Whole-cell lysates (WCL) were analyzed by western blotting with anti-HA and anti-Flag antibodies.


D KPC2 has no effect on activation of IFN - β reporter induced by RIG - I CARD MDA5 CARD . HEK293T ( 3 × 105 ) were transfected with IFN - β reporter plasmid ( 100 ng ) , pRL - TK Renilla luciferase plasmid ( 100 ng ) , indicated expression plasmids . Reporter assays were performed 24 h after transfection .

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D KPC2 has no effect on the activation of the IFN-β reporter induced by RIG-I CARD and MDA5 CARD. HEK293T cells (3×105) were transfected with the IFN-β reporter plasmid (100 ng), the pRL-TK Renilla luciferase plasmid (100 ng), and the indicated expression plasmids. Reporter assays were performed 24 h after transfection.


E Effect of shKPC2 plasmids on endogenous KPC2 . RT - PCR was performed with indicated primers after HEK293T were infected with six lentiviruses encoding an shRNA control ( GFPsh ) five shRNAs targeting kpc2 ( Kpc2 # 1 - # 5 ) .

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E Effect of shKPC2 plasmids on endogenous KPC2. RT-PCR was performed with the indicated primers after HEK293T cells were infected with six lentiviruses encoding an shRNA control (GFPsh) and five shRNAs targeting kpc2 (Kpc2 #1-#5).


F KPC2 is not required for inhibitory action of RNF123 . HEK293T ( 2 × 105 ) with GFPsh or KPC2 sh ( # 1 , # 2 , or # 4 ) were transfected with IFN - β reporter plasmid ( 100 ng ) pRL - TK Renilla luciferase plasmid ( 100 ng ) with empty vector or RNF123 ( 100 ng ) . Twenty - four hours after transfection , were infected with SeV or left uninfected . Reporter assays were performed 18 h after 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

F KPC2 is not required for the inhibitory action of RNF123. HEK293T cells (2×105) with GFPsh or KPC2 sh (#1, #2, or #4) were transfected with the IFN-β reporter plasmid (100 ng) and the pRL-TK Renilla luciferase plasmid (100 ng) with the empty vector or RNF123 (100 ng). Twenty-four hours after transfection, cells were infected with SeV or left uninfected. Reporter assays were performed 18 h after infection.


G Knockdown of KPC2 had no effect on SeV - induced IFN - β expression . HEK293T with GFPsh or KPC2 sh # 4 were infected with SeV for indicated times , total was isolated , RT - PCR experiments were performed .

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G Knockdown of KPC2 had no effect on SeV-induced IFN-β expression. HEK293T cells with GFPsh or KPC2 sh #4 were infected with SeV for the indicated times, total RNA was isolated, and RT-PCR experiments were performed.


A , B RNF123 interacts with RIG - I / MDA5 CARD . HEK293T ( 1 × 106 ) were transfected with expression plasmids for Flag - RNF123 together with HA - RIG - I or its truncations ( 8 μg each ) . Co - immunoprecipitation whole - lysates were analyzed as described in legend of Fig . 3D .

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A, B RNF123 interacts with RIG-I/MDA5 CARD. HEK293T cells (1×106) were transfected with expression plasmids for Flag-RNF123 together with HA-RIG-I or its truncations (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in the legend of Fig. 3D.


C , D RNF123 competes with VISA for RIG - I / MDA5 CARD binding . HEK293T ( 1 × 106 ) were transfected with expression plasmids for HA - RIG - I CARD / MDA5 CARD alone or together with Flag - VISA or with Flag - VISA Flag - RNF123 ( 8 μg each ) . Co - immunoprecipitation whole - lysates were analyzed as described in Fig . 3D .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, D RNF123 competes with VISA for RIG-I/MDA5 CARD binding. HEK293T cells (1×106) were transfected with expression plasmids for HA-RIG-I CARD/MDA5 CARD alone or together with Flag-VISA or with Flag-VISA and Flag-RNF123 (8 μg each). Co-immunoprecipitation and whole-cell lysates were analyzed as described in Fig. 3D.


E RNF123 does not compete with REUL for RIG - I CARD binding . methods of transfection co - immunoprecipitation were as described in Fig . 5C , D .

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E RNF123 does not compete with REUL for RIG-I CARD binding. The methods of transfection and co-immunoprecipitation were as described in Fig. 5C,D.


F Knockdown of RNF123 facilitates endogenous interaction between RIG - I VISA in 293T . 293T stably infected with SeV were treated for indicated times . lysates were immunoprecipitated with anti - VISA antibody . immunoprecipitates lysates were analyzed by western blotting with indicated antibody .

O O O O O O O O O O O O O O O O O O O O O O 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 Knockdown of RNF123 facilitates the endogenous interaction between RIG-I and VISA in 293T cells. 293T cells stably infected with SeV were treated for the indicated times. Cell lysates were immunoprecipitated with anti-VISA antibody. The immunoprecipitates and lysates were analyzed by western blotting with the indicated antibody.


G Knockdown of RNF123 facilitates endogenous interaction between MDA5 VISA in HeLa . HeLa were stably infected with EMCV for indicated times . Co - immunoprecipitation whole - lysates were analyzed as described in ( F ) .

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

G Knockdown of RNF123 facilitates the endogenous interaction between MDA5 and VISA in HeLa cells. HeLa cells were stably infected with EMCV for the indicated times. Co-immunoprecipitation and whole-cell lysates were analyzed as described in (F).


ELISA of fecal IgA1 ( A ) , IgA2 ( B ) , total IgA ( C ) , from non - IBD ( n = 7 ) , CD ( n = 18 ) , UC ( n = 12 ) ' stool . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( A ) * * : p = 0 , 0098 ; ( B ) * : p = 0 , 0406 ; ( C ) Non - IBD v . UC * : p = 0 , 0479 , CD vs . UC * : p = 0 , 0166 ;

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

ELISA assay of fecal IgA1 (A), IgA2 (B), total IgA (C), levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (A)** : p= 0,0098; (B) *: p=0,0406; (C) Non-IBD v. UC *: p=0,0479, CD vs. UC *: p=0,0166;


ELISA of secretory component ( D ) from non - IBD ( n = 7 ) , CD ( n = 18 ) , UC ( n = 12 ) ' stool .

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

ELISA assay of secretory component (D) levels from non-IBD (n= 7), CD (n=18), and UC (n=12) patients' stool.


Data were further separated according to disease activity for IgA1 ( E ) IgA2 ( F ) . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( F ) * : p = 0 ,

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

Data were further separated according to disease activity for IgA1 (E) and IgA2 (F). Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (F) *: p=0,0167


In vitro of purified IgA1 ( G ) IgA2 ( H ) reverse - transcytosis abilities on an inverted model of FAE from Caco2 Raji co - culture . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( G ) * : p = 0 , 0441 ; ( H ) * : p = 0 , 423 ; For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

In vitro assay of purified IgA1 (G) and IgA2 (H) reverse-transcytosis abilities on an inverted model of FAE from Caco2 and Raji cells co-culture. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (G) *: p=0,0441; (H) *: p=0,423 ; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


( I ) ELISA of IgA1 - Dectin - 1 binding , at a rate of one receptor per 10 IgA ; ( J ) ELISA of IgA2 - Dectin - 1 binding , at a rate of one receptor per 10 IgA . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( J ) Non - IBD vs . UC * : p = 0 , 0199 , CD vs . UC * : p = 0 , 0243 ; For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

(I) ELISA assay of IgA1-Dectin-1 binding, at a rate of one receptor per 10 IgA; (J) ELISA assay of IgA2-Dectin-1 binding, at a rate of one receptor per 10 IgA. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (J) Non-IBD vs. UC *: p=0,0199 , CD vs. UC *: p= 0,0243; For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


of IgA1 ( K , L ) IgA2 ( M , N ) reverse - transcytosis for weak ( K , M ) strong ( L , N ) Dectin - 1 binding . Data were analyzed using Kruskall - Wallis multiple comparisons with Dunn ' s correction , when possible , or a Mann - Whitney . P - values are as follows : ( K ) * : p = 0 , 0221 ; ( L ) * : p = 0 , 0486 . For some , antibody purification did not yield a high enough , so samples had to be excluded . N are thus as follows : IgA1 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 4 ; IgA2 : Non - IBD : n = 7 ; CD : n = 8 ; UC : n = 5 . samples ( biological replicates ) have been tested in technical duplicates meaning nx2 .

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

Percent of IgA1 (K, L) and IgA2 (M, N) reverse-transcytosis for weak (K, M) and strong (L, N) Dectin-1 binding. Data were analyzed using Kruskall-Wallis multiple comparisons with Dunn's correction, when possible, or a Mann-Whitney test. P-values are as follows: (K) *: p=0,0221 ; (L) *: p=0,0486 . For some patients, antibody purification did not yield a high enough concentration, so samples had to be excluded. N are thus as follows: IgA1: Non-IBD: n= 7; CD: n= 8; UC : n=4; IgA2 : Non-IBD: n=7; CD : n= 8; UC: n=5. All patient samples (biological replicates) have been tested in technical duplicates meaning nx2.


Heatmaps displaying standardized log2 - transformed signal intensities per glycan for IgA1 ( A ) IgA2 ( B ) in non - IBD , CD UC groups , plotted by hierarchical clustering of Euclidean .

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

Heatmaps displaying standardized log2-transformed signal intensities per glycan for IgA1 (A) and IgA2 (B) in non-IBD, CD and UC groups, plotted by hierarchical clustering of Euclidean distance.


Flow cytometry of total IgA1 - bound ( A ) IgA2 - bound ( B ) fecal microbiota . ( C ) Flow cytometry of stool IgA1 + IgA2 - ( grey dots ) , IgA1 - IgA2 + ( black dots ) IgA1 + IgA2 + ( empty dots ) . ( A - C ) Data were analysed with a Kruskall - Wallis multiple comparison . In ( C ) * : p = 0 , 0226 . Non - IBD : n = 7 , CD : n = 18 , UC : n = 12 .

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

Flow cytometry analysis of total IgA1-bound (A) and IgA2-bound (B) fecal microbiota. (C) Flow cytometry analysis of stool IgA1+ IgA2- bacteria (grey dots), IgA1- IgA2+ bacteria (black dots) and IgA1+ IgA2+ bacteria (empty dots). (A-C) Data were analysed with a Kruskall-Wallis multiple comparison test. In (C) *: p= 0,0226. Non-IBD: n= 7, CD: n=18, and UC: n=12.


( A - E ) Optical density ( 600nm ) variations during in vitro growth of Salmonella enterica Typhimurium ( SL1344 ) co - cultured with stool - purified IgA1 ( A ) or IgA2 ( B ) from non - IBD ( n = 4 ) , CD ( n = 6 ) UC ( n = 3 ) . Comparison between IgA1 IgA2 co - culture in non - IBD ( C ) , CD ( D ) , UC ( E ) . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality , p - values are as follows : ( A ) UC curve : 2h - p = 0 , 0024 , 3h - p = 0 , 003 ; CD curve : 2h 3h p < 0 , 0001 ( B ) UC curve : 4h - p = 0 , 0186 , 5h - p = 0 , 0393 , 6h - p = 0 , 039 , 8h - p < 0 , 0001 ; CD curve : 4h - p = 0 , 0001 , 5h - p = 0 , 0028 , 6h - p = 0 , 0024 , 8h - p < 0 , 0001 . ( C ) 3h - p = 0 , 0422 , 4h - p = 0 , 0422 , 6h - p = 0 , 0012 , 8h - p < 0 , 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Optical density (600nm) variations during in vitro growth assay of Salmonella enterica Typhimurium (SL1344) co-cultured with stool-purified IgA1 (A) or IgA2 (B) from non-IBD (n=4), CD (n=6) and UC (n=3). Comparison between IgA1 and IgA2 co-culture in non-IBD (C), CD (D), and UC (E). Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (A) UC curve: 2h - p=0,0024, 3h - p=0,003; CD curve: 2h and 3h p<0,0001 (B) UC curve: 4h - p=0,0186, 5h - p=0,0393, 6h - p=0,039, 8h - p<0,0001; CD curve: 4h - p=0,0001, 5h - p=0,0028, 6h - p=0,0024, 8h - p<0,0001. (C) 3h - p=0,0422, 4h - p=0,0422, 6h - p=0,0012, 8h - p<0,0001.


( F - I ) Optical density ( 600nm ) during in vitro growth of Escherichia coli ( 25922 from ATCC ) co - cultured with stool - purified IgA1 ( F ) or IgA2 ( G ) from non - IBD ( n = 2 ) CD ( n = 2 ) . samples ( biological replicates ) have been tested in technical duplicates . Comparison between IgA1 IgA2 co - culture in non - IBD ( H ) , CD ( I ) . These IgA samples were from same throughout each experiment . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality , p - values are as follows : ( F ) Grey curve : 4h - p = 0 , 0004 , 5h - p = 0 , 0014 ; Black curve : 4h 5h - p < 0 , 0001 . ( G ) De - glycosylated CD vs . De - glycosylated non - IBD : 4h - p = 0 , 0003 , 5h - p < 0 , 0001 . ( H ) Native vs . de - glycosylated : 4h 5h - p < 0 , 0001 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p = 0 , 0012 . ( H ) Native vs . de - glycosylated : 4h - p = 0 , 001 5h - p = 0 , 006 ; De - glycosylated IgA1 vs . De - glycosylated IgA2 : 5h - p < 0 , 0001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-I) Optical density (600nm) during in vitro growth assay of Escherichia coli (25922 strain from ATCC) co-cultured with stool-purified IgA1 (F) or IgA2 (G) from non-IBD (n=2) and CD (n=2). All patient samples (biological replicates) have been tested in technical duplicates. Comparison between IgA1 and IgA2 co-culture in non-IBD (H), and CD (I). These IgA samples were from the same patients throughout each experiment. Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test, p-values are as follows: (F) Grey curve: 4h - p=0,0004, 5h - p=0,0014; Black curve: 4h and 5h - p<0,0001. (G) De-glycosylated CD vs. De-glycosylated non-IBD: 4h - p=0,0003, 5h - p<0,0001. (H) Native vs. de-glycosylated: 4h and 5h - p<0,0001; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p=0,0012. (H) Native vs. de-glycosylated: 4h - p=0,001 and 5h - p= 0,006; De-glycosylated IgA1 vs. De-glycosylated IgA2: 5h - p<0,0001.


( A ) Combined FISH - IF for each probe . ( B ) Total detected per probe per biopsy . Erec482 - * : p = 0 , 00233 ; Ato291 - * : p = 0 , 0268 ; Bac303 - * : p = 0 , 0196 . ( C ) of co - occurred IgA2 FISH - bound particles . Erec482 - * : p = 0 , 0073 . Two - way ANOVA with Holm - Sidak correction after D ' Agostino - Pearson nomality . Non - IBD : n = 3 ; Active CD : n = 6 ; Inactive CD : n = 4 . samples ( biological replicates ) have been tested in technical duplicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Combined FISH-IF staining for each probe. (B) Total fluorescence detected per probe and per biopsy. Erec482 - *: p=0,00233; Ato291 - *: p=0,0268; Bac303 - *: p=0,0196. (C) Percent of co-occurred IgA2 and FISH-bound particles. Erec482 - *: p=0, 0073.Two-way ANOVA with Holm-Sidak correction after D'Agostino-Pearson nomality test. Non-IBD: n = 3; Active CD: n= 6; Inactive CD: n=4. All patient samples (biological replicates) have been tested in technical duplicates.


( B ) Immunoblots from ( cyt ) ( nuc ) fractionations of RPE - 1 in early - G1 showing presence of endogenous ACTN4 in fractions . were detected with antibodies against ( α ) ACTN4 , α - Tubulin or Histone 3 as

O O O O O O O O O O O O O O O O O O O O O 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) Immunoblots from cytosolic (cyt) and nuclear (nuc) fractionations of RPE-1 cells in early-G1 showing the presence of endogenous ACTN4 in nuclear fractions. Proteins were detected with antibodies against (α) ACTN4, α-Tubulin or Histone 3 as indicated


( C ) Upper middle panels : immunoblots showing precipitation of ACTN4 β - actin from RPE - 1 lysates in early - G1 in presence or absence of biotin - phalloidin , as indicated . Lower panel : Immunoblot of ( cyt ) ( nuc ) fractions . were detected with antibodies against ( α ) ACTN4 , β - Actin , α - Tubulin or Histone 3 as indicated .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Upper and middle panels: immunoblots showing precipitation of ACTN4 and β-actin from RPE-1 nuclear lysates in early-G1 in the presence or absence of biotin-phalloidin, as indicated. Lower panel: Immunoblot of cytosolic (cyt) and nuclear (nuc) fractions. Proteins were detected with antibodies against (α) ACTN4, β-Actin, α-Tubulin or Histone 3 as indicated.


( D ) Immmunofluorescence of endogenous ACTN4 in non - dividing post - mitotic NIH3T3 RPE - 1 in as indicated . Shown are maximum projections . were fixed in 4 % PFA stained for ACTN4 nuclei ( DAPI ) as indicated . Scale bars 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

(D) Immmunofluorescence images of endogenous ACTN4 in non-dividing and post-mitotic NIH3T3 and RPE-1 cells in as indicated. Shown are maximum intensity projections. Cells were fixed in 4% PFA and stained for ACTN4 and nuclei (DAPI) as indicated. Scale bars 10 μm.


( A ) in early G1 stably expressing nAC - mCherry doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - 647SiR dye ( green ) . Different arrows mark dynamic ACTN4 clusters . square 1 is shown as a timeseries on right . square 2 is shown as a magnification below . square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . Scale bar overview ( nAC ) represents 10 µm . Scale bar time series magnification 5 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

(A) Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). Different arrows mark dynamic ACTN4 clusters. The white square 1 is shown as a timeseries on the right. The white square 2 is shown as a magnification below. The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. Scale bar overview (nAC) represents 10 µm. Scale bar time series and magnification 5 µm.


in early G1 stably expressing nAC - mCherry doxycycline - inducible ACTN4 - SNAP were analyzed by time - lapse microscopy . ACTN4 - SNAP was labeled by SNAP - 647SiR dye ( green ) . square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B . B ) Linescan of an actin filament with associated ACTN4 from square 3 in A .

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

Cells in early G1 stably expressing nAC-mCherry and doxycycline-inducible ACTN4-SNAP were analyzed by time-lapse microscopy. ACTN4-SNAP was labeled by SNAP-Cell 647SiR dye (green). The white square 3 shows an actin filament decorated with ACTN4 that was analyzed by linescan in Fig 2B. B) Linescan of an actin filament with associated ACTN4 from square 3 in A.


( C ) Automated tracking ( autoregressive motion ) of actin ACTN4 in nuclei by Imaris software . were treated like in A . Tracks are visualized by red ( actin ) or green ( ACTN4 ) lines in a representative image ( see corresponding Movie EV2 ) . Scale bar is 2 µm . track , track displacement ( between starting - end point ) , average speed maximum speed were quantified from 3 independent experiments . black shows median grey dashed lines show quartiles . Quantifications show similar motion characteristics for actin ACTN4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Automated tracking (autoregressive motion) of nuclear actin and ACTN4 in nuclei by Imaris software. Cells were treated like in A. Tracks are visualized by red (actin) or green (ACTN4) lines in a representative image (see corresponding Movie EV2). Scale bar is 2 µm. The track length, track displacement (distance between starting- and end point), average speed and maximum speed were quantified from 3 independent experiments. The black line shows the median and grey dashed lines show the quartiles. Quantifications and images show similar motion characteristics for actin and ACTN4.


( A ) NIH3T3 expressing nAC - mCherry were imaged for 120 min . Image frames represent maximum projections of 30 Z planes . Lower panel is a magnification of indicated ( orange box ) ; scale bars 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

(A) NIH3T3 cells expressing nAC-mCherry were imaged for 120 min. Image frames represent maximum projections of 30 Z planes. Lower panel is a magnification of the indicated area (orange box); scale bars 10 µm.


( B ) NIH3T3 stably expressing H2B - mCherry were transfected with siCtrl or siACTN1 + siACTN4 . After mitotic exit z - stacks ( 30 planes ) were acquired every 5 min . Nuclei were 3D - reconstructed volume was measured by Imaris . Data are mean ± from 3 biological replicates with 22 - 27 ( siCtrl ) 32 - 42 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * * * * p < 0 . 0001 by t - at 90 min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH3T3 cells stably expressing H2B-mCherry were transfected with siCtrl or siACTN1 + siACTN4. After mitotic exit z-stacks (30 planes) were acquired every 5 min. Nuclei were 3D-reconstructed and volume was measured by Imaris. Data are mean ± SEM from 3 biological replicates with 22-27 (siCtrl) and 32-42 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by t-test at 90 min.


( C ) RPE - 1 were analyzed as in B . Data are mean ± from 5 biological replicates with 32 - 37 ( siCtrl ) 31 - 39 ( siACTN1 + 4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * * * * p < 0 . 0001 by Mann Whitney at 90 min .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RPE-1 cells were analyzed as in B. Data are mean ± SEM from 5 biological replicates with 32-37 (siCtrl) and 31-39 (siACTN1+4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and ****p < 0.0001 by Mann Whitney test at 90 min.


( E ) NIH3T3 stably expressing H2B - mCherry siRNA - resistant ACTN4 - NES were transfected with indicated siRNAs analyzed as described in B . Data are mean ± from 5 biological replicates with 63 - 71 ( siCtrl ) , 37 - 41 ( siACTN1 + 4 ) , 52 - 58 ( siACTN1 ) 52 - 55 ( siACTN4 ) analyzed nuclei ; * * * * p ( siACTN4 ) < 0 . 0001 ; * * * * p ( siACTN1 + 4 ) < 0 . 0001 by One Way ANOVA at 90 min .

(E) NIH3T3 cells stably expressing H2B-mCherry and siRNA-resistant ACTN4-NES were transfected with the indicated siRNAs and analyzed as described in B. Data are mean ± SEM from 5 biological replicates with 63-71 (siCtrl), 37-41 (siACTN1+4), 52-58 (siACTN1) and 52-55 (siACTN4) analyzed nuclei; ****p(siACTN4) < 0.0001; ****p(siACTN1+4) < 0.0001 by One Way ANOVA at 90 min.


( F ) NIH3T3 stably expressing H2B - mCherry indicated ACTN4 mutants were transfected with indicated siRNAs analyzed as in B . Data are mean ± from 3 biological replicates with 63 - 69 ( ACTN4 wt + siACTN4 ) , 63 - 71 ( ACTN4 - NES + siCtrl ) 54 - 58 ( ACTN4 - NES + siACTN4 ) analyzed nuclei ; * * * * p ( ACTN4 - NES + siACTN4 ) < 0 . 0001 by One Way ANOVA at 90 min .

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(F) NIH3T3 cells stably expressing H2B-mCherry and the indicated ACTN4 mutants were transfected with the indicated siRNAs and analyzed as in B. Data are mean ± SEM from 3 biological replicates with 63-69 (ACTN4 wt + siACTN4), 63-71 (ACTN4-NES + siCtrl) and 54-58 (ACTN4-NES + siACTN4) analyzed nuclei; ****p(ACTN4-NES + siACTN4) < 0.0001 by One Way ANOVA at 90 min.


( G ) NIH3T3 expressing siRNA - resistant ACTN4 - NES were transfected with siCtrl or siACTN4 ; density was calculated from H2B - mCherry intensities by total volumes ( / μm3 ) normalized to t = 0 ( mitotic exit ) ; Data are mean ± from 5 biological replicates with 44 - 48 ( siCtrl ) 44 - 47 ( siACTN4 ) analyzed nuclei ; * * * p < 0 . 001 by Wilcoxon * p < 0 . 05 by t - at 90 min .

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(G) NIH3T3 cells expressing siRNA-resistant ACTN4-NES were transfected with siCtrl or siACTN4; chromatin density was calculated from H2B-mCherry fluorescence intensities by total nuclear volumes (intensity/μm3) and normalized to t=0 (mitotic exit); Data are mean ± SEM from 5 biological replicates with 44-48 (siCtrl) and 44-47 (siACTN4) analyzed nuclei; ***p < 0.001 by Wilcoxon test and *p < 0.05 by t-test at 90 min.


( B ) Upper panel shows expression of ACTN4 constructs ( green , confocal image ) phalloidin - AF647 in same ( orange , dSTORM image ) scale bar 10 µm ; lower panel represents zoom of indicated areas ( boxes ) ; scale bars 5 µm .

(B) Upper panel shows expression of ACTN4 constructs (green, confocal image) and phalloidin-AF647 staining in the same cells (orange, dSTORM image) scale bar 10 µm; lower panel represents zoom of the indicated areas (white boxes); scale bars 5 µm.


( C ) Phalloidin - AF647 localizations per post - mitotic nuclei ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei 40 nuclei of non - dividing ( ACTN4 - wt ) from 5 biological replicates . * * * p ( ACTN4ΔCH1 - NLS post - mitotic ) < 0 . 001 , * * * * p ( ACTN4 - wt non - dividing ) < 0 . 0001 , * p ( ACTN4ΔCH1 - NLS post - mitotic vs . ACTN4 - wt non - dividing ) < 0 . 05 by One Way ANOVA .

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(C) Phalloidin-AF647 localizations per post-mitotic nuclei; data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei and 40 nuclei of non-dividing cells (ACTN4-wt) from 5 biological replicates. ***p(ACTN4ΔCH1-NLS post-mitotic) < 0.001, ****p(ACTN4-wt non-dividing) < 0.0001, *p (ACTN4ΔCH1-NLS post-mitotic vs. ACTN4-wt non-dividing) < 0.05 by One Way ANOVA.


( D ) Number of filaments in post mitotic nuclei ; as in ( C ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile : 52 ACTN4 - wt nuclei , 55 ACTN4ΔCH1 - NLS nuclei from 5 biological replicates , * * * p < 0 . 001 by t - .

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(D) Number of filaments in the post mitotic nuclei; as in (C); data shown as mean ± 2.5-97.5 percentile: 52 ACTN4-wt nuclei, 55 ACTN4ΔCH1-NLS nuclei from 5 biological replicates, ***p < 0.001 by t-test.


( E ) Actin filament widths for ACTN4 - wt ACTN4ΔCH1 - NLS in early G1 nuclei ; ) ; data shown as mean ± 2 . 5 - 97 . 5 percentile with 96 filaments ( ACTN4 - wt ) 36 filaments ( ACTN4ΔCH1 - NLS ) from 5 biological replicates , * * p < 0 . 01 by t - .

(E) Actin filament widths for ACTN4-wt and ACTN4ΔCH1-NLS in early G1 nuclei; ); data shown as mean ± 2.5-97.5 percentile with 96 filaments (ACTN4-wt) and 36 filaments (ACTN4ΔCH1-NLS) from 5 biological replicates, **p < 0.01 by t-test.


( a ) siRNA screening . MDA - MB - 231 reverse transfected with siRNA pools against MT1 - MMP , TKS5 , or each human RAB GTPase were plated on fluorescein - labeled - gelatin coated optical microplates . Degradation activity was measured by counting number of degrading gelatin identified as black foci ( similar results were obtained by measuring of degradation / number of ) . inset shows representative of degraded Oregon green gelatin ( upper panels ) merged with TRITC - phalloidin ( red ) DAPI ( blue ) in scrambled siRNA ( NC ) , or siRNA against RAB28 or RAB42 transfected ( lower panel ) . Data are means ± of 3 independent experiments . 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 B-MEASURED_VAR O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O 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

(a) Primary siRNA screening analysis. MDA-MB-231 cells reverse transfected with siRNA pools against MT1-MMP, TKS5, or each human RAB GTPase were plated on fluorescein-labeled-gelatin coated optical microplates. Degradation activity was measured by counting the number of cells degrading gelatin identified as dark black foci (similar results were obtained by measuring the area of degradation/number of cells). The inset shows representative images of degraded Oregon green gelatin (upper panels) and merged images with TRITC-phalloidin (red) and DAPI (blue) in scrambled siRNA (NC), or siRNA against RAB28 or RAB42 transfected cells (lower panel). Data are means ± SEM of 3 independent experiments. Bar, 50 µm.


( b ) Secondary siRNA screening was performed on candidate RAB GTPases selected from screen using 3 deconvoluted siRNAs . Data are mean ± SD ( n > 5 different fields / experiment ) .

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(b) Secondary siRNA screening analysis was performed on candidate RAB GTPases selected from the primary screen using 3 individual deconvoluted siRNAs. Data are mean ± SD (n> 5 different fields/experiment).


( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com . plated onto fluorescently conjugated gelatin ( red ) were stimulated with 100 ng / ml HGF overnight in presence or absence of GM6001 , followed by with FITC - phalloidin ( green ) DAPI ( blue ) ( c ) . Data are mean ± ( d , error bars ; n > 100 / experiment in at least 4 independent ones ) .

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(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Cells plated onto fluorescently conjugated gelatin (red) were stimulated with 100 ng/ml HGF overnight in the presence or absence of GM6001, followed by staining with FITC-phalloidin (green) and DAPI (blue) (c). Data are the mean ± SEM (d, error bars; n > 100 cells/experiment in at least 4 independent ones).


( c - e ) Tertiary siRNA screening . Pooled siRNAs against candidate RAB GTPases identified from secondary screen or TKS5 , used as control , were transfected into MCF10 . DCIS . com . Efficacy of targeted gene silencing was verified by qPCR ( e ) . Bar , 50 µm .

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(c-e) Tertiary siRNA screening. Pooled siRNAs against candidate RAB GTPases identified from the secondary screen or TKS5, used as control, were transfected into MCF10.DCIS.com cells. Efficacy of targeted gene silencing was verified by qPCR (e). Bar, 50 µm.


( f - h ) Inverse invasion . An equal number of MDA - MB - 231 was reverse transfected with a mixture of three siRNA oligonucleotides targeting interest genes . Invasive activity is shown as average number of / field that migrate into fibronectin - supplemented ( 25 µg / ml ) Matrigel further than 20 µm ( f , g ) . Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed .

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(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Invasive activity is shown as the average number of cells/field that migrate into fibronectin-supplemented (25 µg/ml) Matrigel further than 20 µm (f, g). Data are mean ± SD from three different fields from one representative experiments out of 5 that were performed.


( f - h ) Inverse invasion . An equal number of MDA - MB - 231 was reverse transfected with a mixture of three siRNA oligonucleotides targeting interest genes . Efficacy of targeted gene silencing was verified by qPCR ( h ) . Bar , 250 µm . * P < 0 . 05 . * * * , P < 0 . 001 .

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(f-h) Inverse invasion assay. An equal number of MDA-MB-231 cells was reverse transfected with a mixture of three individual siRNA oligonucleotides targeting interest genes. Efficacy of targeted gene silencing was verified by qPCR (h). Bar, 250 µm. *P < 0.05. ***, P < 0.001.


( a ) Doxycycline - inducible empty vector ( EV ) or RAB2A - shRNA MDA - MB - 231 were cultured in absence ( - ) or presence ( + ) of doxycycline for three days , immunoblotted with anti - RAB2A anti - actin antibodies . of RAB2A relative to actin is shown on bottom . Relative density in uninduced EV was set to 1 .

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(a) Doxycycline-inducible empty vector (EV) or RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (-) or presence (+) of doxycycline for three days, and immunoblotted with anti-RAB2A and anti-actin antibodies. The intensity of RAB2A relative to actin is shown on the bottom. Relative density in uninduced EV cells was set to 1.


( b ) Doxycycline - inducible RAB2A - shRNA MDA - MB - 231 were cultured in absence ( CTR ) or presence ( shRAB2A ) of doxycycline for three days . Invasion of MDA - MB - 231 through Matrigel - coated transwells toward HGF was assessed after 24 hours . Representative are shown . Data are mean ± SD ( n = 10 field / condition in at least 3 experiments ) . Bar , 200 µm .

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(b) Doxycycline-inducible RAB2A-shRNA MDA-MB-231 cells were cultured in the absence (CTR) or presence (shRAB2A) of doxycycline for three days. Invasion of MDA-MB-231 cells through Matrigel-coated transwells toward HGF was assessed after 24 hours. Representative images are shown. Data are the mean ± SD (n=10 field/condition in at least 3 experiments). Bar, 200 µm.


( c - e ) EV shRAB2AMDA - MB - 231 were embedded in Spheroid Formation ECMR in absence ( - ) or presence ( + ) of doxycycline / or GM6001 . After 3 days , were transferred into Invasive MatrixR . Phase contrast microscopy of representative spheroids are shown after 9 days ( c ) . Dashed circle indicates of spheroids at day 0 . Bars , 500 µm . Invasive activity into ECM was expressed as total spheroid ( relative to control at day 0 ) quantified by ImageJ ( d ) . number of at indicated days was quantified by CyQUANTR Proliferation Kit ( e ) . Data are mean ± SD ( n = 3 independent experiments ) .

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(c-e) EV and shRAB2AMDA-MB-231 cells were embedded in Spheroid Formation ECMR in the absence (-) or presence (+) of doxycycline and/or GM6001. After 3 days, the cells were transferred into Invasive MatrixR. Phase contrast light microscopy images of representative spheroids are shown after 9 days (c). Dashed circle indicates the size of spheroids at day 0. Bars, 500 µm. Invasive activity into ECM was expressed as total spheroid area (relative to control cells at day 0) quantified by ImageJ (d). The number of cells at the indicated days was quantified by CyQUANTR Cell Proliferation Assay Kit (e). Data are the mean ± SD (n=3 independent experiments).


( f - h ) Doxycycline - inducible murine RAB2A ( mRAB2A ) - expression MCF10 . DCIS . com were transfected with scrambled siRNA or siRNA against human RAB2A ( siRAB2A ) in absence ( - ) or presence ( + ) of doxycycline . Serum - starved were plated onto fluorescently conjugated gelatin ( red ) , stimulated with HGF ( 100 ng / ml ) overnight , stained with phalloidin ( green ) DAPI ( blue ) ( f ) . Bar , 50 μm . Data are mean ± ( error bars ; n > 100 / experiment in 4 independent ones ) ( g ) . Silencing of endogenous RAB2A ( lower band ) ectopic RAB2A expression ( upper band ) was verified by immunoblotting ( h ) . * P < 0 . 05 , * * , P < 0 . 01 , * * * , P < 0 . 001 , NS , not significant .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-h) Doxycycline-inducible murine RAB2A (mRAB2A)-expression MCF10.DCIS.com cells were transfected with scrambled siRNA or siRNA against human RAB2A (siRAB2A) in the absence (-) or presence (+) of doxycycline. Serum-starved cells were plated onto fluorescently conjugated gelatin (red), stimulated with HGF (100 ng/ml) overnight, and stained with phalloidin (green) and DAPI (blue) (f). Bar, 50 μm. Data are the mean ± SEM (error bars; n > 100 cells/experiment in 4 independent ones) (g). Silencing of endogenous RAB2A protein (lower band) and ectopic RAB2A expression (upper band) was verified by immunoblotting (h). * P < 0.05, **, P < 0.01, ***, P < 0.001, NS, not significant.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( a ) are representative of RAB2A expression scoring according to in TMA . In tissues signals were associated with component not with adjacent or infiltrating stroma .

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Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(a) Images are representative of RAB2A expression scoring according to intensity staining in TMA. In tumor tissues the IHC signals were associated with the tumor cell component and not with the adjacent or infiltrating stroma.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( b ) Correlation of RAB2A expression with clinicopathological parameters . Only for 520 of 622 samples expression data for RAB2A was available . Note that number of scored cases is lower than total number of cases since : i ) in some cases , cores detached from slides during manipulations ; ii ) clinical information was not available for . In tissues signal was associated with component not with adjacent or infiltrating stroma .

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Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(b) Correlation of RAB2A expression with clinicopathological parameters. Only for 520 of 622 samples expression data for RAB2A was available. Note that the number of scored cases is lower than the total number of cases since: i) in some cases, individual cores detached from the slides during the manipulations; ii) clinical information was not available for all patients. In tumor tissues the IHC signal was associated with the tumor cell component and not with the adjacent or infiltrating stroma.


RAB2A expression was performed on Invasive Breast Cancer Consecutive Cohort ( n = 622 ) operated in European Institute of Oncology , Milano , Italy , in year 2000 . Expression was measured by on TMA . ( c ) Kaplan - Meier Distant Recurrence Free Interval survival ( DRFI [ 70 ] ) ( d ) Univariate Multivariate Cox - regression , shows that in BC ' high of RAB2A predict a poor prognosis , that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence .

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Analysis RAB2A expression was performed on the Invasive Breast Cancer Consecutive Cohort (n = 622) operated in the European Institute of Oncology, Milano, Italy, in the year 2000. Expression was measured by IHC on TMA.(c) Kaplan-Meier Distant Recurrence Free Interval survival (DRFI [70]) and (d) Univariate and Multivariate Cox-regression analysis, shows that in BC patients' high levels of RAB2A protein predict a poor prognosis, and that RAB2A expression is an independent predictor of higher risk of developing a distant metastatic recurrence.


( a , b ) MCF10 . DCIS . com were transfected with FLAG - empty vector ( EV ) , constitutively active RAB2A - Q65L , or dominant negative RAB2A - S20N mutant . were immunostained with anti - FLAG ( red ) RAB7 ( a , green ) or anti - EEA1 ( b ) antibodies . Merged with DAPI ( blue ) are shown on right . Enlarged endosomes in boxed region are highly magnified in insets . Diameters of RAB7 positive vesicles are also shown . n , number of scored for each population . RGB profiler plugin from ImageJ was used to plot colocalization in lines drawn in inset . Bars , 10 μm , ( inset 2 μm ) .

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(a, b) MCF10.DCIS.com cells were transfected with FLAG-empty vector (EV), constitutively active RAB2A-Q65L, or dominant negative RAB2A-S20N mutant. Cells were immunostained with anti-FLAG (red) and RAB7 (a, green) or anti-EEA1 (b) antibodies. Merged images with DAPI (blue) are shown on the right. Enlarged endosomes in the boxed region are highly magnified in the insets. Diameters of RAB7 positive vesicles are also shown. n, number of cells scored for each cell population. The RGB profiler plugin from ImageJ was used to plot colocalization in the lines drawn in the inset images. Bars, 10 μm, (inset 2 μm).


( c ) MDA - MB - 231 were transfected with EGFP - fused EV , RAB2A - Q65L , or RAB2A - S20N . are immunostained with anti - RAB7 ( blue ) anti - MT1 - MMP ( red ) antibodies , their merged are shown . Merged are shown on right . Enlarged endosomes containing MT1 - MMP in boxed region are highly magnified in insets . Note that RAB2A - Q65L RAB7 - positive vesicles contain MT1 - MMP ( arrows ) . Bars , 10 μm , ( inset 2 μm ) .

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(c) MDA-MB-231 cells were transfected with EGFP-fused EV, RAB2A-Q65L, or RAB2A-S20N. Cells are immunostained with anti-RAB7 (blue) and anti-MT1-MMP (red) antibodies, and their merged images are shown. Merged images are shown on the right. Enlarged endosomes containing MT1-MMP in the boxed region are highly magnified in the insets. Note that RAB2A-Q65L and RAB7-positive vesicles contain MT1-MMP (arrows). Bars, 10 μm, (inset 2 μm).


( d - f ) MDA - MB - 231 expressing MT1 - MMP - pHluorin transfected with scrambled siRNA ( siNC ) or siRNA against RAB2A were seeded on type I collagen , were acquired for 20 min period by TIRF microscopy . Frequency of MT1 - MMP - pHluorin exocytic events was quantified ( e ) . corresponding time - lapse series are shown in Movie 1 , while an entire shown in Movie 2 . Efficacy of RAB2A gene silencing was verified by qPCR ( f ) . Bars , 2 μm . * * , P < 0 . 01 .

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(d-f) MDA-MB-231 cells expressing MT1-MMP-pHluorin transfected with scrambled siRNA (siNC) or siRNA against RAB2A were seeded on type I collagen, and images were acquired for 20 min period by TIRF microscopy. Frequency of MT1-MMP-pHluorin exocytic events was quantified (e). The corresponding time-lapse series are shown in Movie 1, while an entire cell shown in Movie 2. Efficacy of RAB2A gene silencing was verified by qPCR (f). Bars, 2 μm. **, P < 0.01.