Gene - targeting efficiency using Cas9 / mClover - LMNA1 homologous recombination of ( D ) siRNA PALB2 ( n = 4 , one - way ANOVA ) or ( E ) with no PALB2 depletion complemented with wild type p . M296Nfs siRNA - resistant constructs ( n = 7 , one - way ANOVA ) . Western blots of PALB2 wild type PALB2 p . M296Nfs for each condition are shown . Error bars indicate from independent experiments .

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Gene-targeting efficiency using Cas9/mClover-LMNA1 homologous recombination assay of (D) siRNA PALB2 cells (n=4, one-way ANOVA) or (E) cells with no PALB2 depletion complemented with wild type and p.M296Nfs siRNA-resistant constructs (n=7, one-way ANOVA). Western blots of PALB2 wild type and PALB2 p.M296Nfs for each condition are shown. Error bars indicate SEM from independent experiments.


( A ) of geminin - positive , RAD51 foci - containing detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate from independent tumors ( n ≥ 2 ) . PARPi - response is shown in summary underneath : box : PD ; black box : PR / CR . Immunofluorescence of RAD51 foci in PARPi - vehicle - treated PDX tumors is shown . Alterations in HRR - related genes are also summarized : hBRCA1 : BRCA1 promoter hypermethylation lack of BRCA1 expression BRCA1 foci formation .

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(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells detected by immunofluorescence in FFPE samples from PDX tumors treated with vehicle or PARPi Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: white box: PD; black box: PR/CR. Immunofluorescence staining of RAD51 foci in PARPi- and vehicle-treated PDX tumors is shown. Alterations in HRR-related genes are also summarized: hBRCA1: BRCA1 promoter hypermethylation and lack of BRCA1 expression and BRCA1 nuclear foci formation.


( B ) Restoration of RAD51 foci formation in PARPi acquired - resistant PDXs . Immunofluorescence of BRCA1 RAD51 foci in PARPi - treated tumors from STG201 , PDX302 corresponding PARPi acquired - resistant models ( STG201OR PDX302OR ) . Scale bars : 10µm .

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(B) Restoration of RAD51 foci formation in PARPi acquired-resistant PDXs. Immunofluorescence staining of BRCA1 and RAD51 foci in PARPi-treated tumors from STG201, PDX302 and the corresponding PARPi acquired-resistant models (STG201OR and PDX302OR). Scale bars: 10µm.


( C ) of geminin - positive that exhibit γH2AX foci following treatment with vehicle olaparib . Paired t in PARPi - sensitive ( CR / PR ) vs . PARPi - resistant ( PD ) PDXs .

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(C) Quantification of geminin-positive cells that exhibit γH2AX nuclear foci following treatment with vehicle and olaparib. Paired t test in PARPi-sensitive (CR/PR) vs. PARPi-resistant (PD) PDXs.


( A ) of geminin - positive , RAD51 foci - containing in FFPE samples from untreated PDX tumors . Color bars indicate presence of pathogenic variants in indicated genes . Error bars indicate from independent tumors ( n ≥ 2 ) . PARPi - response is shown in summary underneath : black box : PR / CR ; box : PD . Box colors indicate PARP inhibitor treatment . Boxes with two colors indicate same response to both treatments . Olaparib 100mg / kg & 50mg / kg indicates that both doses were tested resulted in same response categorization .

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(A) Percentage of geminin-positive, RAD51 nuclear foci-containing cells in FFPE samples from untreated PDX tumors. Color bars indicate the presence of pathogenic variants in the indicated genes. Error bars indicate SEM from independent tumors (n ≥ 2). PARPi-response is shown in the summary underneath: black box: PR/CR; white box: PD. Box colors indicate the PARP inhibitor treatment. Boxes with two colors indicate the same response to both treatments. Olaparib 100mg/kg & 50mg/kg indicates that both doses were tested and resulted in the same response categorization.


( B ) ROC curves of RAD51 score HRD score , for PARPi response prediction capacity in PDX cohort - 2 . Bootstrap statistical .

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(B) ROC curves of the RAD51 score and HRD score, for PARPi response prediction capacity in the PDX cohort-2. Bootstrap statistical test.


( C ) Immunofluorescence of 53BP1 BRCA1 foci [ with an antibody towards N - terminus ( B1 - NT ) or C - terminus ( B1 - CT ) of BRCA1 ] in three BRCA1 - mutant , PARPi - resistant models from PDX cohort - 2 . location of mutation within gene is indicated . Scale bars : 10µm .

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(C) Immunofluorescence staining of 53BP1 and BRCA1 nuclear foci [with an antibody towards the N-terminus (B1-NT) or C-terminus (B1-CT) of BRCA1] in three BRCA1-mutant, PARPi-resistant models from PDX cohort-2. The location of the mutation within the gene is indicated. Scale bars: 10µm.


( B ) of geminin - positive , RAD51 or γH2AX foci - containing in FFPE samples from with HBOC syndrome . box underneath summarizes ' s young onset ( < 35y : < 35 years ) , her family history ( FH , purple box for PALB2 - related tumors ) presence of BRCA1 foci in analyzed samples .

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(B) Percentage of geminin-positive, RAD51 or γH2AX nuclear foci-containing cells in FFPE tumor samples from patients with HBOC syndrome. The box underneath summarizes the patient's young onset (<35y: <35 years), her family history (FH, purple box for PALB2-related tumors) and the presence of BRCA1 nuclear foci in the analyzed tumor samples.


( C ) Immunofluorescence of γH2AX , BRCA1 RAD51 foci in three representative FFPE tumors from : one RAD51 - BRCA1 - positive ( Pt03 ) , one RAD51 - BRCA1 - negative ( Pt11 ) one PALB2 - related , with BRCA1 but not RAD51 foci ( Pt20 . 1 ) . Scale bars : 10µm .

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(C) Immunofluorescence staining of γH2AX, BRCA1 and RAD51 foci in three representative FFPE tumors from patients: one RAD51- and BRCA1-positive tumor (Pt03), one RAD51- and BRCA1-negative tumor (Pt11) and one PALB2-related tumor, with BRCA1 but not RAD51 nuclear foci (Pt20.1). Scale bars: 10µm.


( a - f ) Muscle biopsy from subject 4 . 1 subjected to electron microscopy ( transverse sections ) . In many , there is material between layers of basal lamina ( a , arrows ; scale bar , 500 nm ) or overt exocytic vacuoles ( b , arrow ; scale bar , 2 μm ) . Some show a single centralized nucleus ( c ; scale bar , 2 μm ) . Mitochondria are of variable distribution . In some , they form a loop around nucleus in periphery of fiber , resembling a necklace as in c ; in others , they form clusters ( d ; scale bar , 2 μm ) . appearance of cristae is often abnormal ( e ; scale bar , 2 μm ; f ; scale bar , 500 nm ) . N , nucleus ; M , mitochondrion .

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(a-f) Muscle biopsy from subject 4.1 subjected to electron microscopy (transverse sections). In many fibers, there is material between the layers of basal lamina (a, arrows; scale bar, 500 nm) or overt exocytic vacuoles (b, arrow; scale bar, 2 μm). Some fibers show a single centralized nucleus (c; scale bar, 2 μm). Mitochondria are of variable size and distribution. In some fibers, they form a loop around the nucleus in the periphery of the fiber, resembling a necklace as in c; in others, they form clusters (d; scale bar, 2 μm). The appearance of cristae is often abnormal (e; scale bar, 2 μm; f; scale bar, 500 nm). N, nucleus; M, mitochondrion.


Transverse sections from muscle biopsies from subject 3 . 1 ( top ) a normal control ( bottom ) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 counterstained with antibody to M - band titin , titin M8 . Accumulation of NBR1 in ( autophagosomes , arrowheads ) of MURF2 , as well as fiber inhomogeneity with marked fiber atrophy , characterizes Vici syndrome muscle . Numerous of very small cross - sectional ( arrows ) with high content of MURF2 NBR1 are frequently seen . Scale bar , 10 μm .

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Transverse sections from muscle biopsies from subject 3.1 (top) and a normal control (bottom) were stained with monoclonal antibody against NBR1 or polyclonal antibody to MURF2 and counterstained with antibody to the M-band titin, titin M8. Accumulation of NBR1 in puncta (autophagosomes, arrowheads) and of MURF2, as well as fiber inhomogeneity with marked fiber atrophy, characterizes Vici syndrome muscle. Numerous fibers of very small cross-sectional area (arrows) with high content of MURF2 and NBR1 puncta are frequently seen. Scale bar, 10 μm.


Accumulation of autophagy adaptors NBR1 SQSTM1 phagophore component LC3 is induced in fibroblasts from two controls a subject with Vici syndrome ( subject 4 . 1 ) after 12 h of treatment with rapamycin or dual treatment with rapamycin bafilomycin .

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Accumulation of autophagy adaptors NBR1 and SQSTM1 and the phagophore membrane component LC3 is induced in fibroblasts from two controls and a subject with Vici syndrome (subject 4.1) after 12 h of treatment with rapamycin or dual treatment with rapamycin and bafilomycin.


In control fibroblasts subjected to 6 h of bafilomycin treatment , lysosomal structures were detected by with monoclonal antibody to LAMP1 . Numerous LC3 - positive autophagosomes are found engulfed by LAMP1 - positive vesicular structures ( arrowheads ) . In contrast , fibroblasts from individuals with Vici syndrome consistently show smaller LC3 - positive that only sporadically colocalize with LAMP1 , with many isolated LC3 - positive ( arrows ) . Note that LC3 signal in Vici syndrome occurs mostly at rim of LAMP1 - positive structures , not centrally . Scale bar , 5 μm .

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In control fibroblasts subjected to 6 h of bafilomycin treatment, lysosomal structures were detected by staining with monoclonal antibody to LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, fibroblasts from individuals with Vici syndrome consistently show smaller LC3-positive puncta that only sporadically colocalize with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici syndrome cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar, 5 μm.


C , D . Immunolabeling of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody ( in green in C , in brown in D , A375P : GFP ) , an anti - HNK1 antibody ( in , migrating early post - migrating NCCs ) , an anti - Ki67 antibody ( in red in C , cycling ) , an anti - SOX10 antibody ( in red in D , also stains chick endogenous NCCs ) an anti - MITF antibody ( in yellow in D ) . Nuclei were stained with Hoechst ( in blue ) . In C , upper left photo shows a grafted prior to cryosection . In D , right panels are enlargements of lower left panel . Scale bar : 200 µm .

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C,D. Immunolabeling of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody (in green in C, in brown in D, A375P:GFP cells), an anti-HNK1 antibody (in white, migrating and early post-migrating NCCs), an anti-Ki67 antibody (in red in C, cycling cells), an anti-SOX10 antibody (in red in D, also stains chick endogenous NCCs) and an anti-MITF antibody (in yellow in D). Nuclei were stained with Hoechst (in blue). In C, the upper left photo shows a grafted embryo prior to cryosection. In D, right panels are enlargements of the lower left panel. Scale bar: 200 µm.


F . 3D views ( - sheet imaging ) of HH25 chick engrafted with different melanoma samples , showing different patterns of localization 50 hours post - engraftment .

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F. 3D views (light-sheet imaging) of HH25 chick embryos engrafted with different melanoma patient samples, showing different patterns of tumor cell localization 50 hours post-engraftment.


G . Immunofluorescent labeling of HH25 chick sections 50 hours post - engraftment of OF - MEL - 020 sample , labeled with CFSE ( in green ) prior to graft . An anti - SOX10 antibody ( in red ) an anti - MITF antibody ( in yellow ) were used . Nuclei were stained with Hoechst ( in blue ) . Right panels are enlargements of dotted in left panel .

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G. Immunofluorescent labeling of HH25 chick embryo sections 50 hours post-engraftment of OF-MEL-020 patient sample, labeled with CFSE (in green) prior to the graft. An anti-SOX10 antibody (in red) and an anti-MITF antibody (in yellow) were used. Nuclei were stained with Hoechst (in blue). Right panels are enlargements of the dotted area in the left panel.


A , B . Immunofluorescent labeling ( A ) of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody , an anti - MITF antibody ( pink ) an anti - SOX10 ( ) antibody . Nuclei were stained with Hoechst . Right panels are enlargements of dotted in left panel . In B , mean fraction of MITF - negative ( MITFneg ) , - low ( MITFlow ) - positive ( MITFpos ) A375P : : GFP in mass in migrating was quantified ( n = 16 slices from 3 ) . Error bars show . Mann - Whitney comparing tumors vs migrating for each class of MITF expression was performed , p - values are indicated on graph .

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A,B. Immunofluorescent labeling (A) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody, an anti-MITF antibody (pink) and an anti-SOX10 (white) antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in left panel. In B, the mean fraction of MITF-negative (MITFneg), -low (MITFlow) and -positive (MITFpos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 16 slices from 3 embryos). Error bars show SEM. Mann-Whitney test comparing tumors vs migrating cells for each class of MITF expression was performed, p-values are indicated on the graph.


C , D . Immunofluorescent labeling ( C ) of HH25 chick sections 50 hours post - engraftment of A375P : : GFP , using an anti - GFP antibody an anti - Ki67 antibody . Nuclei were stained with Hoechst . Right panels are enlargements of dotted in left panel . In D , mean fraction of Ki67 - positive ( Ki67pos ) A375P : : GFP in mass in migrating was quantified ( n = 25 slices from 3 ) . Arrows point at having a mesenchymal . Error bars show . Student t - , * * p = 0 . 0070 .

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C,D. Immunofluorescent labeling (C) of HH25 chick embryo sections 50 hours post-engraftment of A375P::GFP cells, using an anti-GFP antibody and an anti-Ki67 antibody. Nuclei were stained with Hoechst. Right panels are enlargements of the dotted area in the left panel. In D, the mean fraction of Ki67-positive (Ki67pos) A375P::GFP cells in the tumor mass and in migrating cells was quantified (n = 25 slices from 3 embryos). Arrows point at cells having a mesenchymal morphology. Error bars show SEM. Student t-test, **p = 0.0070.


H - K . 3D views ( H , J ) of volumes ( I , K ) of HH25 chick engrafted with GLO ( H , I ) or GLO - R ( J , K ) treated with a combination of Vemurafenib Cobimetinib , or Vorinostat alone , or a combination of three molecules . number of analyzed for each experimental condition are indicated on graphs . Bars error bars indicate mean + . Student t - , * * : p < 0 . 01 , ns : not significant . Exact p - values are indicated on graphs .

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H-K. 3D views (H, J) and quantification of tumor volumes (I, K) of HH25 chick embryos engrafted with GLO (H, I) or GLO-R (J, K) cells and treated with a combination of Vemurafenib and Cobimetinib, or Vorinostat alone, or a combination of the three molecules. The number of embryos analyzed for each experimental condition are indicated on the graphs. Bars and error bars indicate mean + SEM. Student t-tests, **: p < 0.01, ns: not significant. Exact p-values are indicated on the graphs.


A , B . 3D views ( A ) of variations in mean volume ( B ) of HH25 chick engrafted with BRAFwt ( OF - MEL - 028 ) or BRAFV600E ( OF - MEL - 020 , OF - MEL - 027 ) samples treated with excipient or a combination of Vemurafenib Cobimetinib . number of analyzed for each sample is indicated on graphs . Student t - for OF - MEL - 020 ; Mann - Whintey for OF - MEL - 028 , * : p < 0 . 05 , ns : not significant . Exact p - values are indicated on graphs .

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A,B. 3D views (A) and quantification of variations in the mean tumor volume (B) of HH25 chick embryos engrafted with BRAFwt (OF-MEL-028) or BRAFV600E (OF-MEL-020, OF-MEL-027) patient samples and treated with excipient or a combination of Vemurafenib and Cobimetinib. The number of embryos analyzed for each patient sample is indicated on the graphs. Student t-test for OF-MEL-020; Mann-Whintey test for OF-MEL-028, *: p < 0.05, ns: not significant. Exact p-values are indicated on the graphs.


C , D . Survival rate ( left axis ) mean surface ( BSA , right axis ) of chick injected with increasing doses of Dabrafenib ( C ) Trametinib ( D ) . maximum tolerated dose ( MTD , in red on X - axis ) was defined as higher dose of drug associated with a survival rate higher than 75 % a mean BSA similar ( i . e . , non - statistically different ) from treated with excipient ( 17 . 1 0 . 0228 mg / kg respectively ) . N = 10 replicates per experimental group . Error bars indicate . ns , non - significant using Student ' s t - compared with excipient .

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C,D. Survival rate (left axis) and mean body surface area (BSA, right axis) of chick embryos injected with increasing doses of Dabrafenib (C) and Trametinib (D). The maximum tolerated dose (MTD, in red on the X-axis) was defined as the higher dose of drug associated with a survival rate higher than 75% and a mean BSA similar (i.e., non-statistically different) from embryos treated with excipient (17.1 and 0.0228 mg/kg respectively). N=10 replicates per experimental group. Error bars indicate SEM. ns, non-significant using Student's t-test compared with excipient.


( F - H ) of HH25 chick engrafted with 3 different samples ( OF - MEL - 033 ( F ) , OF - MEL - 034 ( G ) , OF - MEL - 035 ( H ) ) treated with excipient or a combination of Dabrafenib Trametinib . clinical response of each after a 3 months treatment with Dabrafenib / Trametinib is indicated above graphs . Error bars indicate . Mann - Whitney , exact p - values are indicated on graphs .

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(F-H) of HH25 chick embryos engrafted with 3 different patient samples (OF-MEL-033 (F), OF-MEL-034 (G), OF-MEL-035 (H)) and treated with excipient or a combination of Dabrafenib and Trametinib. The clinical response of each patient after a 3 months treatment with Dabrafenib/Trametinib is indicated above the graphs. Error bars indicate SEM. Mann-Whitney tests, exact p-values are indicated on the graphs.


( D , E ) Scatter plot visualization of YFP vs mCherry signals in GCG1 ORC2 promoter FPR screens . Each data point represents median signal of a deletion mutant normalized to plate median . Highlighted data points represent mutants of Hda1C ( purple ) , SAGA acetylation module ( blue ) , SAGA deubiquitination module ( orange ) , cac2 ∆ ( yellow ) . regression is marked in black . mutants favoring DNC transcription are found above regression . mutants favoring coding transcription are below regression . Technical repeat = 1 , biological observations = 50 , 000 before filtering .

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(D, E) Scatter plot visualization of YFP vs mCherry fluorescence signals in the GCG1 and ORC2 promoter FPR screens. Each data point represents the median signal of a deletion mutant normalized to individual plate median. Highlighted data points represent the mutants of Hda1C (purple), SAGA acetylation module (blue), SAGA deubiquitination module (orange), and cac2∆ (yellow). The regression line is marked in black. The mutants favoring DNC transcription are found above the regression line. The mutants favoring coding transcription are below the regression line. Technical repeat = 1, biological observations = 50,000 individual cells before filtering.


( C ) Flow cytometry of mCherry / YFP in mutants with GCG1pr FPR background . data are normalized to signal values of wild type ( FPR ) .

(C) Flow cytometry quantification of mCherry/YFP fluorescence in the mutants with GCG1pr FPR background. The data are normalized to the signal values of the wild type (FPR) strain.


( D ) Endogenous transcript by RT - qPCR in mutants . bars represent fold gene expression of coding divergent transcript normalized to expression of reference gene , UBC6 .

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(D) Endogenous transcript analysis by RT-qPCR in the mutants. The bars represent fold gene expression ratio of coding and divergent transcript normalized to the expression of the reference gene, UBC6.


. ( B ) NET - seq data at YPL172C locus . signal represents NET - seq reads showing nascent transcription at genomic position of divergent non - coding strand ( black ) coding strand ( grey ) . bar graph depicts NET - seq reads as FPKM values for coding DNC transcript in strains

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. (B) NET-seq data at the YPL172C locus. The signal represents NET-seq reads showing nascent transcription at the genomic position of the divergent non-coding strand (black) and the coding strand (grey). The bar graph depicts NET-seq reads as FPKM values for the coding and DNC transcript in the strains


. ( C ) NET - seq data at YDR261C locus with a novel DNC transcript . Annotations as in ( B )

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. (C) NET-seq data at the YDR261C locus with a novel DNC transcript. Annotations as in (B)


( C ) A representative image of transcription initiation observed as spots in recorded live - movie of wild type . movie output using Python were analyzed snapshot was obtained using Image J software . red green channel denotes GCG1 SUT098 transcript initiation , respectively . represent maximum projections , right bottom sidebars indicate sideviews in yz xz directions , respectively . Scale : 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 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

(C) A representative image of transcription initiation observed as fluorescence spots in the recorded live-cell movie of wild type strain. The movie output using Python were analyzed and the snapshot was obtained using Image J software. The red and green channel denotes GCG1 and SUT098 transcript initiation, respectively. Images represent maximum intensity projections, and the right and bottom sidebars indicate sideviews in the yz and xz directions, respectively. Scale: 5 µm.


( D , E ) of transcription dynamics by duration frequency ( time between initiation ) of transcription . Bar graphs show transcription parameters of SUT098 in candidate mutants compared to wild type . error bars represent calculated by live - image of 179 , 135 , 60 129 single - biological repeats for WT , hda1 ∆ , hda3 ∆ cac2 ∆ strains , respectively . Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python ( asterisk * denotes p - value < 0 . 05 ) .

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

(D, E) Quantification of transcription dynamics by the duration and frequency (time between initiation) of transcription. Bar graphs show the transcription parameters of SUT098 in candidate mutants compared to the wild type strain. The error bars represent SEM calculated by live-cell image analysis of 179, 135, 60 and 129 single-cell biological repeats for WT, hda1∆, hda3∆ and cac2∆ strains, respectively. Statistical significance was calculated by random sampling with replacement method using a bootstrapping algorithm in Python (the asterisk * denotes p-value <0.05).


( A ) Flow cytometry data . Bars represent mCherry / YFP of respective yeast mutants normalized to signal values of H3 wild type FPR . error bars show calculated from biological triplicates with three technical repeats . Unpaired two - student ' s t - indicates statistical significance of mutant compared to respective wild type . asterisks * , * * * * * indicate p - values < 0 . 05 , < 0 . 01 < 0 . 001 , respectively .

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(A) Flow cytometry data analysis. Bars represent the mCherry/YFP fluorescence ratio of the respective yeast mutants normalized to the signal values of the H3 wild type FPR strain. The error bars show SEM calculated from biological triplicates with three technical repeats. Unpaired two-tailed student's t-test analysis indicates the statistical significance of the mutant compared to the respective wild type. The asterisks *, ** and *** indicate p-values <0.05, <0.01 and <0.001, respectively.


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A )

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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment , metabolic activity was assessed by a WST - 1 ( B )

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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment, metabolic activity was assessed by a WST-1 assay (B)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment viral expression was analyzed by FACS ( C )

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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment viral protein expression was analyzed by FACS (C)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of 5 - AzadC or 5 - AzaC . At 72 h post - treatment ; initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR ( D ) .

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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of 5-AzadC or 5-AzaC. At 72 h post-treatment; and initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR (D).


E J - Lat 8 . 4 were mock - treated or treated with 5 - AzadC ( 400nM ) or TNFα ( 10ng / ml ) as positive control . At 24 h , 48 h or 72 h post - treatment , initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR .

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

E J-Lat 8.4 cells were mock-treated or treated with 5-AzadC (400nM) or TNFα (10ng/ml) as positive control. At 24 h, 48 h or 72 h post-treatment, initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR.


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B )

O O O O O O O O O O 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

A-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)


A - D J - Lat 8 . 4 were mock - treated or treated with increasing concentrations of HDACIs . At 24 h post - treatment , metabolic activity was assessed by a WST - 1 ( C , D ) . Data information : Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value 100 % ( C , D ) . selected doses were indicated by " an arrow " . Plasmatic concentrations after usual dosage ( Cmax ) of each drug in human therapy are indicated below LRA names by a box in graph .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D J-Lat 8.4 cells were mock-treated or treated with increasing concentrations of HDACIs. At 24 h post-treatment, metabolic activity was assessed by a WST-1 assay (C, D).Data information: Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value 100% (C, D). The selected doses were indicated by "an arrow". Plasmatic concentrations after usual dosage (Cmax) of each drug in human therapy are indicated below the LRA names and by a box in the graph.


A - D J - Lat 8 . 4 was mock - treated or treated with 5 - AzadC / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . Viral production was measured by quantifying p24 antigen production in culture supernatants ( A , B )

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

A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. Viral production was measured by quantifying p24 antigen production in culture supernatants (A, B)


A - D J - Lat 8 . 4 was mock - treated or treated with 5 - AzadC / or SAHA for different periods of time as indicated . Samples were harvested at indicated times . metabolic activity was assessed by a WST - 1 ( C , D ) . Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value of 100 % ( C , D ) .

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

A-D J-Lat 8.4 cell line was mock-treated or treated with 5-AzadC and/or SAHA for different periods of time as indicated. Samples were harvested at indicated times. metabolic activity was assessed by a WST-1 assay (C, D). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 100% (C, D).


E From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from 24 HIV + presented in Appendix Table S2 , extracellular HIV - 1 genomic for each LRA treatment are represented . One night after purification , were mock - treated or simultaneously treated with 5 - AzadC ( 1µM ) / or SAHA ( 1µM ) . Six days after treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

E From data of ex vivo cultures of CD8+-depleted PBMCs isolated from 24 HIV+ patients presented in Appendix Table S2, the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented. One night after cell purification, cells were mock-treated or simultaneously treated with 5-AzadC (1µM) and/or SAHA (1µM). Six days after treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : viral p24 production in supernatant was measured ( A B )

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

A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: viral p24 production in cell supernatant was measured (A and B)


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : FACS analyses were performed percentages of GFP + are presented as histograms ( C D )

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

A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows:FACS analyses were performed and the percentages of GFP+ cells are presented as histograms (C and D)


A - F J - Lat 8 . 4 ( A , C , E ) 15 . 4 ( B , D , F ) lines were mock - treated or treated with 5 - AzadC . At 48 h post - treatment , HDACIs were then added for 24 h . At 72 h 5 - AzadC post - treatment samples were harvested analysed as follows : initiated ( primers TAR ) or elongated ( primers tat ) transcripts were quantified by RT - qPCR results were normalized using β - actin gene primers are presented as histograms representing fold - inductions compared to mock - treated condition ( E F ) . Means standard errors of means from three independent biological duplicates ( n = 6 ) are indicated . result obtained with mock - treated was arbitrarily set at a value of 1 ( E , 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 B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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

A-F J-Lat 8.4 (A, C, E) and 15.4 (B, D, F) cell lines were mock-treated or treated with 5-AzadC. At 48 h post-treatment, HDACIs were then added for 24 h. At 72 h 5-AzadC post-treatment samples were harvested and analysed as follows: initiated (primers TAR) or elongated (primers tat) transcripts were quantified by RT-qPCR and results were normalized using the β-actin gene primers and are presented as histograms representing fold-inductions compared to the mock-treated condition (E and F). Means and standard errors of the means from three independent biological duplicates (n=6) are indicated. The result obtained with mock-treated cells was arbitrarily set at a value of 1 (E, F).


One night after purification , CD8 + - depleted PBMCs from 12 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added to cultures . Six days after 5 - AzadC treatment , WST - 1 was performed . Mock value was arbitrary fixed at 100 % for each .

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

One night after cell purification, CD8+-depleted PBMCs from 12 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added to the cultures. Six days after 5-AzadC treatment, WST-1 assay was performed. Mock value was arbitrary fixed at 100% for each individual.


Figure 6 : Representation of reactivation status of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + . A - C From data of ex vivo cultures of CD8 + - depleted PBMCs isolated from HIV + ( Table 2 ) , extracellular HIV - 1 genomic for each LRA treatment are represented from cultures ( A ) , from cultures presenting no viral reactivation in mock condition ( C ) , from cultures exhibiting reactivation in mock condition ( B ) . One night after purification , were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . Six days after 5 - AzadC treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

Figure 6: Representation of reactivation status of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients.A-C From data of ex vivo cultures of CD8+-depleted PBMCs isolated from HIV+ patients (Table 2), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A Reactivation status of cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + ( shown in Appendix Table S2 ) . B Reactivation status of cultures presenting no viral reactivation in mock condition isolated from aviremic cART - treated HIV + ( shown in Table 2 ) . Data information : results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented by a .

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A Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Appendix Table S2).B Reactivation status of patient cell cultures presenting no viral reactivation in mock condition isolated from aviremic cART-treated HIV+ patients (shown in Table 2).Data information: The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented by a line.


Figure 8 : Representation of reactivation status of ex vivo cultures of resting CD4 + T . A - C From data of ex vivo cultures of resting CD4 + T isolated from HIV + ( Table 3 ) , extracellular HIV - 1 genomic for each LRA treatment are represented from cultures ( A ) , from cultures presenting no viral reactivation in mock condition ( C ) , from cultures exhibiting reactivation in mock condition ( B ) . One night after purification , were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . Six days after 5 - AzadC treatment , of viral in culture supernatants was determined ( in copies / ml ) . results were reported as actual HIV copy numbers / ml or as an estimated value calculated as 50 % of smallest value when HIV was not detected in order to assign a log value . Means are represented .

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

Figure 8: Representation of reactivation status of ex vivo cultures of resting CD4+ T cells.A-C From data of ex vivo cultures of resting CD4+ T cells isolated from HIV+ patients (Table 3), the extracellular HIV-1 genomic RNA levels for each LRA treatment are represented from all patient cell cultures (A), from patient cell cultures presenting no viral reactivation in mock condition (C), and from patient cell cultures exhibiting reactivation in mock condition (B). One night after cell purification, cells were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. Six days after 5-AzadC treatment, the concentration of viral RNA in culture supernatants was determined (in copies/ml). The results were reported as the actual HIV RNA copy numbers/ml or as an estimated value calculated as 50% of the smallest value when HIV RNA was not detected in order to assign a log value. Means are represented.


A - H One night after purification , CD8 + - depleted PBMCs or HLA DR - CD69 - CD25 - CD4 + T from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . activation status of CD4 + T subset was assessed 6 days after 5 - AzadC treatment by flow cytometry of activation markers relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

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

A-H One night after cell purification, CD8+-depleted PBMCs or HLA DR- CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The activation status of CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis of cellular activation markers relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.


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


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


One night after purification , HLA DR - CD69 - CD25 - CD4 + T from 4 HIV negative donors were mock - treated or treated with 5 - AzadC . Three days post - treatment , 1 / 3 of medium was replaced HDACIs were added in cultures . median of CD4 receptor of viable CD4 + T subset was assessed 6 days after 5 - AzadC treatment by flow cytometry relative to mock treatment before 5 - AzadC stimulation corresponding to day 0 . Means are represented .

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

One night after cell purification, HLA DR-CD69-CD25-CD4+T cells from 4 HIV negative donors were mock-treated or treated with 5-AzadC. Three days post-treatment, 1/3 of medium was replaced and HDACIs were added in the cultures. The median fluorescence intensity of CD4 receptor of viable CD4+T cell subset was assessed 6 days after 5-AzadC treatment by flow cytometry analysis relative to mock treatment before 5-AzadC stimulation corresponding to day 0. Means are represented.


( A ) RFP - ATG8g - labeled autophagosomes were quantified from syringe infected with mock or Xcv ΔxopQ ( oD600 = 0 . 2 ) at 2 dpi in presence or absence of ConA ( bars = 20 μm ) . were calculated from z - stacks ( 15 ) of n = 6 individuals using ImageJ . Central band of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Different letters indicate statistically different groups ( P < 0 . 05 ) as determined by one - way ANOVA . experiment was repeated twice with similar results .

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(A) RFP-ATG8g-labeled autophagosomes were quantified from plants syringe infected with mock or Xcv ΔxopQ (oD600=0.2) at 2 dpi in the presence or absence of ConA (bars = 20 μm). Puncta were calculated from z-stacks (15) of n=6 individuals using ImageJ. Central band of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically different groups (P < 0.05) as determined by one-way ANOVA. The experiment was repeated twice with similar results.


( B ) Immunoblot of NBR1 ATG8 in Xcv ΔxopQ or mock infected N . benthamiana at 1 2dpi . Agrobacterium - mediated transient expression of AIMp - RFP serves as a control for autophagy suppression . Ponceau ( PS ) served as a loading control . experiment was repeated three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ or mock infected N. benthamiana plants at 1 and 2dpi. Agrobacterium-mediated transient expression of AIMp-RFP serves as a control for autophagy suppression. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( C ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ was co - infiltrated with Agrobacteria containing luciferase reporter constructs . Coexpression of RFP - AIMp serves as a control for autophagy inhibition . Expression of latter was confirmed with western blot ( inset ) . Renilla ( RLUC ) Firefly ( FLUC ) luciferase activities were simultaneously measured in leaf extracts at 48 h post - infiltration using dual - luciferase system ( n = 4 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - . experiment was repeated more than 3 times with similar results .

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(C) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ was co-infiltrated with Agrobacteria containing the luciferase reporter constructs. Coexpression of RFP-AIMp serves as a control for autophagy inhibition. Expression of the latter was confirmed with western blot (inset). Renilla (RLUC) and Firefly (FLUC) luciferase activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P<0.001) was revealed by Student's t-test. The experiment was repeated more than 3 times with similar results.


( D ) RT - qPCR of NbATG8 - 1 . 1 / 1 . 2 , NbATG8 - 2 . 1 / 2 . 2 NbJoka2 transcript upon challenge of N . benthamiana with Xcv ΔxopQ for 1 2 dpi compared to mock infected . Values represent expression relative to mock control of respective time point were normalized to actin . Values are biological replicates ( n = 4 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 ) was revealed by Student ' s t - .

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(D) RT-qPCR analysis of NbATG8-1.1/1.2, NbATG8-2.1/2.2 and NbJoka2 transcript levels upon challenge of N. benthamiana plants with Xcv ΔxopQ for 1 and 2 dpi compared to mock infected plants. Values represent expression relative to mock control of respective time point and were normalized to actin. Values are biological replicates (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (*P < 0.05, **P < 0.01, *** P < 0.001) was revealed by Student's t-test.


( E ) density in leaves of N . benthamiana infected with Xcv in presence or absence autophagy suppressor AIMp - RFP . Leaves were syringe - infiltrated with OD600 = 0 . 0004 , colony - forming units were counted at 6 dpi . Compared to empty vector control ( EV ) , AIMp expressing ( n = 6 ) harbour significantly more . growth was repeated with same result in 12 over two independent experiments . Red yellow data points indicate independent repeats of experiment . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( * * * P < 0 . 001 ) was revealed by Student ' s t - . Expression of RFP - AIMp was verified at 6 dpi with an anti - RFP blot ( inset ) .

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(E) Bacterial density in leaves of N. benthamiana infected with Xcv in the presence or absence autophagy suppressor AIMp-RFP. Leaves were syringe-infiltrated with OD600 = 0.0004, and colony-forming units were counted at 6 dpi. Compared to empty vector control (EV), AIMp expressing plants (n=6) harbour significantly more bacteria. Bacterial growth was repeated with the same result in 12 plants over two independent experiments. Red and yellow data points indicate independent repeats of the experiment. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (***P < 0.001) was revealed by Student's t-test. Expression of RFP-AIMp was verified at 6 dpi with an anti-RFP blot (inset).


( A ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . XopL or GFP constructs were co - infiltrated . RLUC FLUC signals were simultaneously measured in leaf extracts at 48 h post - infiltration using dual - luciferase system . Values represent of RLUC - ATG8a FLUC activities to mean of control ( n = 4 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( P < 0 . 01 ) was shown by Student ' s t - . experiment was repeated more than 3 times by with similar results .

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(A) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. XopL or GFP constructs were co-infiltrated. RLUC and FLUC signals were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities to the mean of control (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (P < 0.01) was shown by Student's t-test. The experiment was repeated more than 3 times by with similar results.


( B ) Immunoblot of NBR1 ATG8 in N . benthamiana transiently expressing GFP - XopL or GFP control at 2dpi verified with an anti - GFP antibody ( same blot was split for visualization purpose ) . Ponceau ( PS ) served as a loading control . experiment was repeated at least three times with similar results .

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(B) Immunoblot analysis of NBR1 and ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL or GFP control at 2dpi verified with an anti-GFP antibody (same blot was split for visualization purpose). Ponceau Staining (PS) served as a loading control. The experiment was repeated at least three times with similar results.


( C ) Immunoblot of ATG8 in N . benthamiana transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment . Expression of GFP - XopL was verified with an anti - GFP antibody , while expression of GUS - HA was confirmed with an anti - HA antibody . Ponceau ( PS ) served as a loading control . experiment was repeated twice with similar results .

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(C) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing XopL or GUS control at 2dpi after ConA or DMSO treatment. Expression of GFP-XopL was verified with an anti-GFP antibody, while expression of GUS-HA was confirmed with an anti-HA antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( D ) GFP - ATG8g - labeled autophagosomes were quantified from infected with Xcv or Xcv ΔxopL at 2 dpi in presence or absence of ConA . were calculated from z - stacks ( 15 ) of n = 12 individuals using ImageJ . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( * * P < 0 . 01 , * * * P < 0 . 001 ) was determined by one way ANOVA . experiment was repeated twice with similar results .

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(D) GFP-ATG8g-labeled autophagosomes were quantified from plants infected with Xcv or Xcv ΔxopL at 2 dpi in the presence or absence of ConA. Puncta were calculated from z-stacks (15) of n=12 individuals using ImageJ. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (** P < 0.01, *** P < 0.001) was determined by one way ANOVA. The experiment was repeated twice with similar results.


( E ) Immunoblot of NBR1 ATG8 in Xcv ΔxopQ , Xcv ΔxopQ ΔxopL or mock infected N . benthamiana at 2dpi . Ponceau ( PS ) served as a loading control . experiment was repeated twice with similar results .

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(E) Immunoblot analysis of NBR1 and ATG8 protein levels in Xcv ΔxopQ, Xcv ΔxopQ ΔxopL or mock infected N. benthamiana plants at 2dpi. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( F ) RLUC - ATG8a or RLUC - NBR1 constructs were coexpressed with internal control FLUC in N . benthamiana . Xcv ΔxopQ Xcv ΔxopQ ΔxopL were co - infiltrated with Agrobacteria containing respective constructs . RLUC FLUC activities were simultaneously measured in leaf extracts at 48 h post - infiltration using dual - luciferase system . Values represent of RLUC - ATG8a FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance comparing Xcv ΔxopQ Xcv ΔxopQ ΔxopL values ( * * * P < 0 . 001 ) was revealed by Student ' s t - . experiment was repeated 3 times with similar results .

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(F) RLUC-ATG8a or RLUC-NBR1 constructs were coexpressed with internal control FLUC in N. benthamiana. Xcv ΔxopQ and Xcv ΔxopQ ΔxopL were co-infiltrated with Agrobacteria containing the respective constructs. RLUC and FLUC activities were simultaneously measured in leaf extracts at 48 h post- infiltration using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance comparing Xcv ΔxopQ and Xcv ΔxopQ ΔxopL values (***P < 0.001) was revealed by Student's t-test. The experiment was repeated 3 times with similar results.


( A ) Interaction of XopL with SH3P2 in yeast two - hybrid assays . XopL fused to GAL4 - binding domain was expressed in combination with SH3P2 fused to GAL4 activation domain ( AD ) in yeast Y190 . were grown on selective media before a LacZ filter was performed . pSV40 / p53 served as positive control , while empty AD or BD vector served as negative control . NtSH3P2 = Nicotiana tabacum SH3P2 . - LT = yeast growth on medium without Leu Trp , - HLT = yeast growth on medium lacking His , Leu , Trp , indicating expression of HIS3 reporter gene . LacZ , activity of lacZ reporter gene .

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(A) Interaction of XopL with SH3P2 in yeast two-hybrid assays. XopL fused to the GAL4 DNA-binding domain was expressed in combination with SH3P2 fused to the GAL4 activation domain (AD) in yeast strain Y190. Cells were grown on selective media before a LacZ filter assay was performed. pSV40/p53 served as positive control, while the empty AD or BD vector served as negative control. NtSH3P2 = Nicotiana tabacum SH3P2. -LT = yeast growth on medium without Leu and Trp, -HLT = yeast growth on medium lacking His, Leu, and Trp, indicating expression of the HIS3 reporter gene. LacZ, activity of the lacZ reporter gene.


( B ) Coimmunoprecipitation of GFP - XopL with AtSH3P2 - HA . GFP - XopL or GFP were transiently coexpressed with AtSH3P2 - HA in leaves of N . benthamiana . After 48 h , total ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot using either anti - GFP or anti - HA antibodies . GFP blot was split for visualization purpose . AtSH3P2 = Arabidopsis thaliana SH3P2 . Two repetitions with similar results have been conducted .

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(B) Coimmunoprecipitation of GFP-XopL with AtSH3P2-HA. GFP-XopL or GFP were transiently coexpressed with AtSH3P2-HA in leaves of N. benthamiana. After 48 h, total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis using either anti-GFP or anti-HA antibodies. GFP blot was split for visualization purpose. AtSH3P2 = Arabidopsis thaliana SH3P2. Two repetitions with similar results have been conducted.


( C ) Visualization of interactions in planta by bimolecular complementation . Yellow ( YFP ) confocal microscopy show Nicotiana benthamiana leaf epidermal transiently expressing VenusN173 - XopL in combination with AtSH3P2 - VenusC155 . A positive control showing dimerization of fructose - 1 , 6 - bisphosphatase ( FBPase ) within cytosol . arrows indicate reconstitution of YFP . red structures indicate autofluorescence of chloroplasts . combination of VenusN173 - XopL with FBPase - VenusC155 or VenusN173 - FBPase with AtSH3P2 - VenusC155 do not induce YFP serve as negative controls . Bars = 20 μm .

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(C) Visualization of protein interactions in planta by the bimolecular fluorescence complementation assay. Yellow fluorescent protein (YFP) confocal microscopy images show Nicotiana benthamiana leaf epidermal cells transiently expressing VenusN173-XopL in combination with AtSH3P2-VenusC155. A positive control showing the dimerization of fructose-1,6-bisphosphatase (FBPase) within the cytosol. White arrows indicate reconstitution of YFP fluorescence. The red structures indicate autofluorescence of chloroplasts. The combination of VenusN173-XopL with FBPase-VenusC155 or VenusN173-FBPase with AtSH3P2-VenusC155 do not induce YFP fluorescence and serve as negative controls. Bars = 20 μm.


( D ) Colocalization of GFP - XopL with SH3P2 - RFP , RFP - ATG8e RFP - ATG8g in N . benthamiana leaves . Imaging was performed 2 d after transient expression represent single confocal planes from abaxial epidermal ( scale bars = 20 μm 10 μm , lower panel ) . arrows indicate colocalization of GFP RFP signals . experiment was repeated twice with similar results .

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(D) Colocalization analysis of GFP-XopL with SH3P2-RFP, RFP-ATG8e and RFP-ATG8g in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (scale bars = 20 μm and 10 μm, lower panel). White arrows indicate colocalization of GFP and RFP signals. The experiment was repeated twice with similar results.


( E ) Total were extracted 48 hpi with A . tumefaciens harboring respective GFP - XopL , HA - XopL SH3P2 - GFP expression constructs . SH3P2 - GFP ( lower band ) were detected using an anti - GFP antibody . Expression of XopL was verified using an anti - HA or anti - GFP antibody . Expression of GUS - HA served as a control . Ponceau S serves as a loading control . experiment was repeated three times with similar results .

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(E) Total proteins were extracted 48 hpi with A. tumefaciens harboring the respective GFP-XopL, HA-XopL and SH3P2-GFP expression constructs. SH3P2-GFP protein levels (lower band) were detected using an anti-GFP antibody. Expression of the XopL was verified using an anti-HA or anti-GFP antibody. Expression of GUS-HA served as a control. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( F ) Growth of Xcv Xcv ΔxopL strains in roq1 N . benthamiana silenced for SH3P2 ( pTRV2 - SH3P2 ) compared to control ( pTRV2 ) . Leaves were dip - inoculated with a suspension at OD600 = 0 . 2 were quantified at 3 6 dpi . Experimental repeats are indicated by data points in red ( n = 8 ) yellow ( n = 6 ) data points . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Different letters indicate statistically significant differences ( P < 0 . 05 ) as determined by one - way ANOVA .

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(F) Growth of Xcv and Xcv ΔxopL strains in roq1 N. benthamiana plants silenced for SH3P2 (pTRV2-SH3P2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2 and bacteria were quantified at 3 and 6 dpi. Experimental repeats are indicated by data points in red (n=8) and yellow (n=6) data points. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Different letters indicate statistically significant differences (P < 0.05) as determined by one-way ANOVA.


( A ) SH3P2 - GFP was transiently coexpressed together with GUS - HA GFP - XopL in N . benthamiana using agroinfiltration . At 42 hpi , 200 μM MG132 was infiltrated into A . tumefaciens - inoculated leaves , leaf material was collected 48 hpi . Expression of SH3P2 - GFP ( lower band ) GFP - XopL ( upper band ) was detected using an anti - GFP antibody . GUS - HA expression was confirmed with an anti - HA antibody . Ponceau S serves as a loading control . experiment was repeated three times with similar results .

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(A) SH3P2-GFP was transiently coexpressed together with GUS-HA and GFP-XopL in N. benthamiana using agroinfiltration. At 42 hpi, 200 μM MG132 was infiltrated into A. tumefaciens-inoculated leaves, and leaf material was collected 48 hpi. Expression of SH3P2-GFP (lower band) and GFP-XopL (upper band) was detected using an anti-GFP antibody. GUS-HA expression was confirmed with an anti-HA antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( B ) In vitro ubiquitination reveals ubiquitination of SH3P2 by XopL . GST - XopL , MBP - SH3P2 were tested using Arabidopsis His - AtUBA1 His - AtUBC8 . Lanes 2 to 4 are negative controls . were separated by SDS - PAGE detected by immunoblotting using indicated antibodies . Asterisks indicate bands that are not ubiquitinated an present in controls . experiment was repeated twice with similar results .

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(B) In vitro ubiquitination assay reveals ubiquitination of SH3P2 by XopL. GST-XopL, and MBP-SH3P2 were tested using the Arabidopsis His-AtUBA1 and His-AtUBC8. Lanes 2 to 4 are negative controls. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. Asterisks indicate bands that are not ubiquitinated an present in controls. The experiment was repeated twice with similar results.


( C ) SH3P2 - GFP was transiently coexpressed together with GFP , GFP - XopL GFP - XopL ΔE3 in N . benthamiana using agroinfiltration . GFP were detected with an anti - GFP antibody . Ponceau S serves as a loading control . experiment was repeated three times with similar results .

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(C) SH3P2-GFP was transiently coexpressed together with GFP, GFP-XopL and GFP-XopL ΔE3 in N. benthamiana using agroinfiltration. GFP protein levels were detected with an anti-GFP antibody. Ponceau S staining serves as a loading control. The experiment was repeated three times with similar results.


( D ) Immunoblot of ATG8 in N . benthamiana transiently expressing GFP - XopL , GFP - XopL ΔE3 or GFP control at 2dpi . Expression of binary constructs was verified with an anti - GFP antibody . GFP blot was split for visualization purpose . Ponceau ( PS ) served as a loading control . experiment was repeated twice with similar results .

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(D) Immunoblot analysis of ATG8 protein levels in N. benthamiana plants transiently expressing GFP-XopL, GFP-XopL ΔE3 or GFP control at 2dpi. Expression of binary constructs was verified with an anti-GFP antibody. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( E ) RLUC - ATG8a constructs were coexpressed with internal control FLUC in N . benthamiana . GFP - XopL , GFP - XopL ΔE3 or GFP control were co - infiltrated together with RLUC / FLUC mixture . Renilla Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using dual - luciferase system . Values represent of RLUC - ATG8a FLUC activities ( n = 4 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Statistical significance ( * P < 0 . 5 , * * P < 0 . 01 ) was revealed by Student ' s t - . experiment was repeated 3 times .

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(E) RLUC-ATG8a constructs were coexpressed with internal control FLUC in N. benthamiana. GFP-XopL, GFP-XopL ΔE3 or GFP control were co-infiltrated together with RLUC/FLUC mixture. Renilla and Firefly luciferase activities were simultaneously measured in leaf extracts at 48 hpi using the dual-luciferase system. Values represent the ratio of RLUC-ATG8a and FLUC activities (n=4). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Statistical significance (* P < 0.5, ** P < 0.01) was revealed by Student's t-test. The experiment was repeated 3 times.


( A ) GFP - XopL was coexpressed with GFP or AIMp - RFP . were separated by SDS - PAGE detected by immunoblotting using indicated antibodies . GFP blot was split for visualization purpose . Ponceau ( PS ) served as a loading control . experiment was repeated three times with similar results .

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(A) GFP-XopL was coexpressed with GFP or AIMp-RFP. Proteins were separated by SDS-PAGE and detected by immunoblotting using the indicated antibodies. GFP blot was split for visualization purpose. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( B ) Colocalization of RFP - XopL with GFP - Joka2 in N . benthamiana leaves . Imaging was performed 2 d after transient expression represent single confocal planes from abaxial epidermal ( bars = 20 μm ) . experiment was repeated twice with similar results .

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(B) Colocalization of RFP-XopL with GFP-Joka2 in N. benthamiana leaves. Imaging was performed 2 d after transient expression and images represent single confocal planes from abaxial epidermal cells (bars = 20 μm). The experiment was repeated twice with similar results.


( C ) FRET FLIM measurements of GFP - Joka2 RFP - XopL in N . benthamiana leaves . freeRFP construct served as a negative control RFP - ATG8E ( n = 9 ) as a positive control . Scattered points show data points , color indicates biological repeats . lifetime ( in ns ) of GFP - Joka2 ( donor , n = 41 ) was significantly reduced in presence of RFP - XopL ( n = 40 ) but not in presence of freeRFP ( n = 35 ) . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Significant differences were calculated using Wilcoxon rank sum , with significantly different groups denoted by different letters . experiment was repeated three times with similar results .

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(C) FRET FLIM measurements of GFP-Joka2 and RFP-XopL in N. benthamiana leaves. The freeRFP construct served as a negative control and RFP-ATG8E (n = 9) as a positive control. Scattered points show individual data points, color indicates biological repeats. The lifetime (in ns) of GFP-Joka2 (donor, n = 41) was significantly reduced in the presence of RFP-XopL (n = 40) but not in the presence of freeRFP (n = 35). Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Wilcoxon rank sum test, with significantly different groups denoted by different letters. The experiment was repeated three times with similar results.


( D ) Immunoprecipitation ( IP ) of GFP - XopL reveals association with NBR1 . Immunoblots of input IP samples from N . benthamiana transiently expressing GFP or GFP - XopL were probed with anti - GFP anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(D) Immunoprecipitation (IP) of GFP-XopL reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP or GFP-XopL were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( E ) Immunoprecipitation ( IP ) of GFP - XopL GFP - XopL ΔE3 reveals association with NBR1 . Immunoblots of input IP samples from N . benthamiana transiently expressing GFP , GFP - XopL GFP - XopL ΔE3 were probed with anti - GFP anti - NBR1 antibodies . GFP blot was split for visualization purpose .

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(E) Immunoprecipitation (IP) of GFP-XopL and GFP-XopL ΔE3 reveals association with NBR1. Immunoblots of input and IP samples from N. benthamiana plants transiently expressing GFP, GFP-XopL and GFP-XopL ΔE3 were probed with anti-GFP and anti-NBR1 antibodies. GFP blot was split for visualization purpose.


( F ) GFP - XopL was transiently expressed in pTRV2 , pTRV2 - Joka2 N . benthamiana WT . Expression of binary constructs was verified with an anti - GFP antibody . Joka2 silencing was verified using an anti - NBR1 antibody . Ponceau ( PS ) served as a loading control . experiment was repeated twice with similar results .

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(F) GFP-XopL was transiently expressed in pTRV2, pTRV2-Joka2 and N. benthamiana WT plants. Expression of binary constructs was verified with an anti-GFP antibody. Joka2 silencing was verified using an anti-NBR1 antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated twice with similar results.


( G ) Growth of Xcv ΔxopQ in N . benthamiana silenced for Joka2 ( pTRV2 - Joka2 ) compared to control ( pTRV2 ) . Leaves were dip - inoculated with a suspension at OD600 = 0 . 2 . were quantified at 3 6 dpi . Red , blue , green data points represent repeats of experiments . Middle horizontal bars of boxplots represent median , bottom top represent 25th 75th percentiles , whiskers extend to at most 1 . 5 times interquartile range . Significant differences were calculated using Student ' s t - are indicated by : * * , P < 0 . 01 . experiment was repeated three times with similar trends .

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(G) Growth of Xcv ΔxopQ in N. benthamiana plants silenced for Joka2 (pTRV2-Joka2) compared to control plants (pTRV2). Leaves were dip-inoculated with a bacteria suspension at OD600 = 0.2. and bacteria were quantified at 3 and 6 dpi. Red, blue, and green data points represent repeats of the experiments. Middle horizontal bars of boxplots represent the median, the bottom and top represent the 25th and 75th percentiles, whiskers extend to at most 1.5 times the interquartile range. Significant differences were calculated using Student's t-test and are indicated by: **, P < 0.01. The experiment was repeated three times with similar trends.


( H ) GFP - XopL , GFP - XopL ΔE3 were transiently expressed in N . benthamiana . RFP - AIMp was co - infiltrated to stabilize both XopL variants . Samples were taken 48 hpi , total ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot of precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a negative control . RFP - AIMp expression was verified by an anti - RFP antibody . GFP blot was split for visualization purpose . Asterisk indicates GFP - XopL full - . experiment was repeated three times with similar results .

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(H) GFP-XopL, GFP-XopL ΔE3 were transiently expressed in N. benthamiana. RFP-AIMp was co-infiltrated to stabilize both XopL variants. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a negative control. RFP-AIMp expression was verified by an anti-RFP antibody. GFP blot was split for visualization purpose. Asterisk indicates the GFP-XopL full-length protein. The experiment was repeated three times with similar results.


( I ) GFP - XopL was transiently expressed in N . benthamiana . Samples were taken 48 hpi , total ( Input ) were subjected to immunoprecipitation ( IP ) with ubiquitin pan selector , followed by immunoblot of precipitates using either anti - GFP or anti - ubiquitin antibodies . GFP served as a control . Asterisk indicates GFP - XopL full - . GFP blot was split for visualization purpose . experiment was repeated two times with similar results .

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(I) GFP-XopL was transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with the ubiquitin pan selector, followed by immunoblot analysis of the precipitates using either anti-GFP or anti-ubiquitin antibodies. GFP served as a control. Asterisk indicates the GFP-XopL full-length protein. GFP blot was split for visualization purpose. The experiment was repeated two times with similar results.


( J ) Immunoblot GFP - XopL GFP - XopLK191A at 1 2 dpi using an anti - GFP antibody . Ponceau ( PS ) served as a loading control . experiment was repeated three times with similar results .

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(J) Immunoblot analysis GFP-XopL and GFP-XopLK191A at 1 and 2 dpi using an anti-GFP antibody. Ponceau Staining (PS) served as a loading control. The experiment was repeated three times with similar results.


( K ) HA - XopL GFP - Joka2 its variants were transiently expressed in N . benthamiana . Samples were taken 48 hpi , total ( Input ) were subjected to immunoprecipitation ( IP ) with GFP - Trap beads , followed by immunoblot of precipitates using either anti - GFP , anti - ubiquitin anti - NBR1 antibodies . GFP served as a control .

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(K) HA-XopL and GFP-Joka2 and its variants were transiently expressed in N. benthamiana. Samples were taken 48 hpi, and total proteins (Input) were subjected to immunoprecipitation (IP) with GFP-Trap beads, followed by immunoblot analysis of the precipitates using either anti-GFP, anti-ubiquitin and anti-NBR1 antibodies. GFP served as a control.


C ) ELISA binding results of mAbs specific for hRSV F ( left panels ) or hMPV F ( right panels ) with indicated at left . mAbs specific of hRSV F bind epitopes of antigenic site II ( Mota 47F ) or antigenic site IV ( 101F ) . mAs specific of hMPV F bind epitopes of antigenic site II ( MF9 , MF12 , MF14 MF15 ) or antigenic site IV ( MF16 ) .

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C) ELISA binding results of mAbs specific for hRSV F (left panels) or hMPV F (right panels) with the proteins indicated at left. mAbs specific of hRSV F bind epitopes of antigenic site II (Mota and 47F) or antigenic site IV (101F). mAs specific of hMPV F bind epitopes of antigenic site II (MF9, MF12, MF14 and MF15) or antigenic site IV (MF16).


Binding of Mz 47F Fabs to immobilized indicated at left . Sensorgrams of eight different concentrations are shown in each panel . calculated binding parameters are shown in Table . RUs , response units .

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Binding of Mz and 47F Fabs to the immobilized proteins indicated at left. Sensorgrams of eight different concentrations are shown in each panel. The calculated binding parameters are shown in the Table. RUs, response units.


Groups of BALB / c mice ( n = 5 ) were inoculated twice ( four apart ) i . m . with 10 µg / dose of indicated at top of each panel , folded in their respective postfusion conformations . One week after last dose , mice were sacrificed their blood collected . A ) Each mouse serum ( identified by a number ) was tested in ELISA for antibody binding to postfusion F indicated at bottom . results represented in y axis for each mouse serum correspond to inverse dilution that gave 50 % of maximal binding , expressed in log10 units ( EC50 ( log10 ) ) . Each number represents an mouse . Short horizontal bars indicate mean values for each group . P values were calculated as indicated under Statistical . Only relevant P values are shown . * P = 0 . 00045 . Differences were considered significant when P < 0 . 05 .

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Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. A) Each mouse serum (identified by a number) was tested in ELISA for antibody binding to the postfusion F proteins indicated at the bottom. The results represented in the y axis for each individual mouse serum correspond to the inverse dilution that gave 50% of maximal binding, expressed in log10 units (EC50 (log10)). Each number represents an individual mouse. Short horizontal bars indicate mean values for each group. P values were calculated as indicated under Statistical Analysis. Only relevant P values are shown. *P = 0.00045. Differences were considered significant when P < 0.05.


Groups of BALB / c mice ( n = 5 ) were inoculated twice ( four apart ) i . m . with 10 µg / dose of indicated at top of each panel , folded in their respective postfusion conformations . One week after last dose , mice were sacrificed their blood collected . B ) Mouse sera were also tested in microneutralization assays with hRSV hMPV , as indicated at bottom . results represented in y axis correspond to inverse dilution that inhibited 50 % of viral infectivity ( IC50 ( log10 ) ) . P values : * P = 0 . 01001 * * P = 0 . 00271 . Long horizontal lines in A B indicate detection limits .

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Groups of BALB/c female mice (n = 5) were inoculated twice (four weeks apart) i.m. with 10 µg/dose of the proteins indicated at the top of each panel, folded in their respective postfusion conformations. One week after the last dose, mice were sacrificed and their blood collected. B) Mouse sera were also tested in microneutralization assays with hRSV and hMPV, as indicated at the bottom. The results represented in the y axis correspond to the inverse dilution that inhibited 50% of the viral infectivity (IC50 (log10)). P values: *P = 0.01001 and **P = 0.00271. Long horizontal lines in A and B indicate detection limits.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four apart , with 20 µg of postfusion F indicated above each panel . One group ( n = 5 ) was inoculated only with CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of panels . A ) Mouse sera , collected just before challenging with hRSV ( A2 ) , were used in microneutralization assays against indicated at bottom . Statistical results are shown as in Fig . 3B . * P = 0 . 00085 * * P = 0 . 01986 .

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Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. A) Mouse sera, collected just before challenging with hRSV (A2 strain), were used in microneutralization assays against the viruses indicated at bottom. Statistical results are shown as in Fig. 3B. *P = 0.00085 and **P = 0.01986.


Groups of BALB / c mice ( n = 5 ) were inoculated three times , four apart , with 20 µg of postfusion F indicated above each panel . One group ( n = 5 ) was inoculated only with CpG adjuvant , as indicated . Two other groups of mice ( n = 8 ) were similarly inoculated with either F - 415 or F - 416 , as indicated at top of panels . B ) Five days after inoculation , mice were sacrificed amount of in lung extracts was measured in a plaque . Mean titer for each group is indicated by short horizontal bar .

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Groups of BALB/c mice (n = 5) were inoculated three times, four weeks apart, with 20 µg of the postfusion F proteins indicated above each panel. One group (n = 5) was inoculated only with the CpG adjuvant, as indicated. Two other groups of mice (n = 8) were similarly inoculated with either F-415 or F-416, as indicated at top of the panels. B) Five days after virus inoculation, mice were sacrificed and the amount of virus in lung extracts was measured in a plaque assay. Mean virus titer for each group is indicated by short horizontal bar.