Representative cryo - EM image where roughly half of the particles correspond to 40S ( blue ) and the other half to 80S ( orange ) .

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

Representative cryo-EM image where roughly half of the particles correspond to 40S (blue) and the other half to 80S (orange).


( H , I ) Induction of Ctcflos and Ucp1 in vivo during cold - induced iWAT browning . Relative expression levels of ( H ) Ctcflos transcript 1 and ( I ) Ucp1 in iWAT of C57BL / 6J mice held at 5 ° C for one week compared to age and weight matched mice held at 30 ° C or 23 ° C , assessed by quantitative PCR ( qPCR ) . Mean and individual values , n = 7 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * p < 0 . 01 , * * * * 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

(H, I) Induction of Ctcflos and Ucp1 in vivo during cold-induced iWAT browning. Relative expression levels of (H) Ctcflos transcript 1 and (I) Ucp1 in iWAT of C57BL/6J mice held at 5°C for one week compared to age and weight matched mice held at 30°C or 23°C, assessed by quantitative PCR (qPCR). Mean and individual values, n=7 (biological replicates), one-way ANOVA (Šídák-test), **p< 0.01, ****p<0.0001.


( D ) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 1 ( biological replicate ) for Ucp1n = 3 ( biological replicates ) for Ctcflos .

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

(D) Relative transcript levels of Ctcflos and Ucp1 in various tissues of 129S6 mice, assessed by qPCR. Mean values ± SD, n=1 (biological replicate) for Ucp1n=3 (biological replicates) for Ctcflos .


( E ) Ctcflos , 45SrRNA and 12SrRNA transcript abundance in nuclear , cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice , assessed by qPCR . Mean values ± SD , n = 6 ( biological replicates ) , two - way ANOVA ( Tukey - test ) , * * * * 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

(E) Ctcflos, 45SrRNA and 12SrRNA transcript abundance in nuclear, cytosolic and mitochondrial subfractions of primary brite adipocytes of 129S6 mice, assessed by qPCR. Mean values ± SD, n=6 (biological replicates), two-way ANOVA (Tukey-test), ****p< 0.0001.


( E ) Relative Ucp1 transcript levels in response to Ctcflos tr1 , 3 and 4 KD by ASO 1 , 3 , 4 and 2 , 5 6 , respectively , compared to nontargeting controls , assessed by qPCR . Mean values ± SD , n = 3 - 4 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , * * * p < 0 . 001 , * * * * 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

(E) Relative Ucp1 transcript levels in response to Ctcflos tr1, 3 and 4 KD by ASO 1, 3, 4 and 2, 5 6, respectively, compared to nontargeting controls, assessed by qPCR. Mean values ± SD, n=3-4 (biological replicates), one-way ANOVA (Šídák-test), ***p<0.001, ****p<0.0001.


( F ) Relative expression levels of cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) and cytochrome c oxidase subunit 7a1 ( Cox7a1 ) in Ctcflos tr1 , 3 and 4 KD compared to control samples , assessed by qPCR . Mean values ± SD , n = 3 ( biological replicates ) - 4 , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Relative expression levels of cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea) and cytochrome c oxidase subunit 7a1 (Cox7a1) in Ctcflos tr1, 3 and 4 KD compared to control samples, assessed by qPCR. Mean values ± SD, n=3 (biological replicates)-4, unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( H ) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 ( UCP1 ( red ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) ) .

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

(H) Immunocytochemistry of UCP1 protein in response to Ctcflos tr1 KD by ASO 1 (UCP1 (red); DNA staining by DAPI (green); phase contrast (PC) (gray)).


Impact of Ctcflos KD on mitochondrial biogenesis . ( R ) Microscopic images of Mito Tracker stained brite adipocytes ( Mito tracker ( blue ) ; DNA staining by DAPI ( green ) ; phase contrast ( PC ) ( gray ) )

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

Impact of Ctcflos KD on mitochondrial biogenesis. (R) Microscopic images of Mito Tracker stained brite adipocytes (Mito tracker (blue); DNA staining by DAPI (green); phase contrast (PC) (gray))


( D ) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 ( Ucp1 ) , cell death - inducing DNA fragmentation factor alpha - like effector A ( Cidea ) , cytochrome c oxidase subunit 7a1 ( Cox7a1 ) , peroxisome proliferator activated receptor γ coactivator 1α ( Pgc1a ) and elongation of very long chain fatty acids - like 3 ( Elovl3 ) , comparing Ctcflos tr1 ( ASO 1 ) and tr 3 and 4 ( ASO 2 ) KD with their respective controls . Mean values ± SD , n = 6 ( biological replicates ) , unpaired t tests or in grey parenthesis two - way ANOVA ( Šídák - test ) n . s . p > 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * 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

(D) Relative expression levels of brite adipocyte marker genes uncoupling protein 1 (Ucp1), cell death-inducing DNA fragmentation factor alpha-like effector A (Cidea), cytochrome c oxidase subunit 7a1 (Cox7a1), peroxisome proliferator activated receptor γ coactivator 1α (Pgc1a) and elongation of very long chain fatty acids-like 3 (Elovl3), comparing Ctcflos tr1 (ASO 1) and tr 3 and 4 (ASO 2) KD with their respective controls. Mean values ± SD, n=6 (biological replicates), unpaired t tests or in grey parenthesis two-way ANOVA (Šídák-test) n.s. p>0.05, **p<0.01, ***p<0.001, ****p<0.0001.


( K - M ) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression . Primary iWAT cells infected with Prdm16 - or turbo green fluorescent protein ( GFP ) - expressing viral particles at the second day of proliferation , followed by reverse transfection at the first day of differentiation using nontargeting control ( NC ) or Ctcflos - targeting ASO1 . Gene expression analyzed 72 hours later by qPCR ( K ) Transcript levels of Ctcflos tr1 relative to Fabp4 ( L ) Transcript levels of Prdm16 relative to Fabp4 ( M ) Transcript levels of Ucp1 relative to Fabp4 . Mean and individual values . Each graph presents pooled data from two experiments slightly varying virus titers . RM One - way ANOVA ( Tukey - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

(K-M) Rescue of Ctcflos KD impact on Ucp1 gene transcription by Prdm16 overexpression. Primary iWAT cells infected with Prdm16- or turbo green fluorescent protein (GFP)-expressing viral particles at the second day of proliferation, followed by reverse transfection at the first day of differentiation using nontargeting control (NC) or Ctcflos-targeting ASO1. Gene expression analyzed 72 hours later by qPCR (K) Transcript levels of Ctcflos tr1 relative to Fabp4 (L) Transcript levels of Prdm16 relative to Fabp4 (M) Transcript levels of Ucp1 relative to Fabp4. Mean and individual values. Each graph presents pooled data from two experiments slightly varying virus titers. RM One-way ANOVA (Tukey-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001.


( B - D ) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis , ( B ) expressed as Ucp1 relative to Fabp4 mRNA levels , ( C ) Ucp1 relative to Gtf2b and ( D ) Fabp4 relative to Gtf2b . Mean values ± SD , n = 3 ( biological replicates ) , one - way ANOVA ( Šídák - test ) , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 , * * * * p < 0 . 0001 .

O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-D) Impact of splicing inhibition by different concentrations of general splicing inhibitor isoginkgetin on brite adipogenesis, (B) expressed as Ucp1 relative to Fabp4 mRNA levels, (C) Ucp1 relative to Gtf2b and (D) Fabp4 relative to Gtf2b. Mean values ± SD, n=3 (biological replicates), one-way ANOVA (Šídák-test), n.s. p>0.05, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.


Effect of Ctcflos KD on Serine / Arginine rich Splicing factor 2 ( SC35 / SRSF2 ) and nuclear speckle abundance in Ctcflos tr1 ( ASO 1 ) KD and control cells ( KD day 1 of differentiation , analysis after 48h ) . ( E ) Immunocytochemistry of SC35 . SC35 signal ( blue ) alone ( middle images ) and SC35 ( blue ) , DNA staining by DAPI ( green ) and phase contrast ( PC ) ( gray ) combined ( outer images ) .

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

Effect of Ctcflos KD on Serine/Arginine rich Splicing factor 2 (SC35/SRSF2) and nuclear speckle abundance in Ctcflos tr1 (ASO 1) KD and control cells (KD day 1 of differentiation, analysis after 48h). (E) Immunocytochemistry of SC35. SC35 signal (blue) alone (middle images) and SC35 (blue), DNA staining by DAPI (green) and phase contrast (PC) (gray) combined (outer images).


Influence of Ctcflos KD on Prdm16 alternative splicing . ( O ) Relative Prdm16 total , ( P ) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD , comparing Ctcflos tr1 ( ASO1 ) and tr3 , 4 ( ASO2 ) KD and control samples , assessed by qPCR , Mean values ± SD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Influence of Ctcflos KD on Prdm16 alternative splicing. (O) Relative Prdm16 total, (P) Relative Prdm16 long transcript levels 24 hours after Ctcflos KD, comparing Ctcflos tr1 (ASO1) and tr3, 4 (ASO2) KD and control samples, assessed by qPCR, Mean values ± SD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, * p<0.05, **p<0.01, ***p<0.001.


Influence of Ctcflos KD on Prdm16 alternative splicing . ( R , S ) Fold change of Prdm16 short and Prdm16 long ( R ) ASO1 to NC and ( S ) ASO2 and NC 24 hours after Ctcflos KD , n = 3 - 4 ( biological replicates ) , unpaired t tests , n . s . p > 0 . 05 , * * p < 0 . 01 .

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

Influence of Ctcflos KD on Prdm16 alternative splicing. (R, S) Fold change of Prdm16 short and Prdm16 long (R) ASO1 to NC and (S) ASO2 and NC 24 hours after Ctcflos KD, n=3-4 (biological replicates), unpaired t tests, n.s. p>0.05, ** p<0.01.


B Chromatin accessibility as measured by the number of all ( TSS and distal ) ATAC - Seq peaks ( read count ≥ 10 ) detected in DN2 ( n = 5 ) , DN3 ( n = 5 ) , ISP ( n = 5 ) , DPCD3 - ( n = 6 ) , DPCD3 + ( n = 6 ) , SPCD4 + ( n = 5 ) , SPCD8 + ( n = 5 ) populations . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 0 . 035 ( Kruskal - Wallis Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatin accessibility as measured by the number of all (TSS and distal) ATAC-Seq peaks (read count ≥10) detected in DN2 (n=5), DN3 (n=5), ISP (n=5), DPCD3- (n=6), DPCD3+ (n=6), SPCD4+ (n=5), SPCD8+ (n=5) populations. Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=0.035 (Kruskal-Wallis Test).


C Fraction of OCRs having increasing , decreasing , steady and fluctuating accessibility pattern during T - cell development ( x - axis ) . Average number of normalized reads in OCRs ( y - axis ) .

O O O O O O O O O O O O O O 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 Fraction of OCRs having increasing, decreasing, steady and fluctuating accessibility pattern during T-cell development (x-axis). Average number of normalized reads in OCRs (y-axis).


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7 stages

O O O O O O O O O O O O O O O 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 Unsupervised hierarchical clustering based on the variance stabilizing transformation (VST) normalized read counts (each row is also normalized by the row mean) of the 2,021 signature open chromatin regions of 7 stages


A Unsupervised hierarchical clustering based on the variance stabilizing transformation ( VST ) normalized read counts ( each row is also normalized by the row mean ) of the 2 , 021 signature open chromatin regions of 7


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements database

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ATAC-Seq genome tracks showing three signature peaks located near the genes SAE1, RAG1, and CD8A. Framed regions indicate cell-type-specific signature ATAC-Seq peaks. Blue triangles indicate known promoter or enhancer regions, which are annotated in the GeneHancer (GH) Regulatory Elements database


B ATAC - Seq genome tracks showing three signature peaks located near the genes SAE1 , RAG1 , and CD8A . Framed regions indicate cell - type - specific signature ATAC - Seq peaks . Blue triangles indicate known promoter or enhancer regions , which are annotated in the GeneHancer ( GH ) Regulatory Elements

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 The rationale of footprinting analysis as illustrated by actual ATAC - Seq peaks , HINT - ATAC footprint hits , and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6 .

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

A The rationale of footprinting analysis as illustrated by actual ATAC-Seq peaks, HINT-ATAC footprint hits, and example TF motif instances near RAG1 gene in DN2 population of thymus donor 6.


B Heatmap of ln ( odds ratio ) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions . TFs with an SD of the motif counts < 10 are excluded .

B Heatmap of ln(odds ratio) of the 50 TFs showing the most differential abundance of binding motifs in the open chromatin regions. TFs with an SD of the motif counts <10 are excluded.


D Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T - cell precursors and in T - ALLs , respectively ( n = 10 / 19 patients have two biological replicates , n = 9 / 19 patients have one replicate ) . Horizontal lines of the box plot indicate the median , lower and upper limits of each box correspond to the first and third quartiles ( the 25th and 75th percentiles ) and the lower and upper whiskers extend from min to max ; pval = 4 . 4e - 08 ( Kruskal - Wallis Test ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Chromatin accessibility as assessed by the number of ATAC peaks identified in the sorted populations of normal thymic T-cell precursors and in T-ALLs, respectively (n=10/19 patients have two biological replicates, n=9/19 patients have one replicate). Horizontal lines of the box plot indicate the median, lower and upper limits of each box correspond to the first and third quartiles (the 25th and 75th percentiles) and the lower and upper whiskers extend from min to max; pval=4.4e-08 (Kruskal-Wallis Test).


E Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T - ALLs in comparison to healthy T - cell precursors . Patients on the x - axis ; same order as in panel ( C ) . Gray cells represent TFs having non - significant enrichment or depletion .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap representing the enrichment and depletion of binding motifs of top 50 TFs in OCRs of T-ALLs in comparison to healthy T-cell precursors. Patients on the x-axis; same order as in panel (C). Gray cells represent TFs having non-significant enrichment or depletion.


C log2 fold changes of 292 genes which are differentially expressed ( x - axis , RNA - Seq : non - sorted bulk thymi vs . T - ALLs ) and differentially accessible ( y - axis , ATAC - Seq : sorted T - cell populations vs . T - ALLs ) in T - ALLs in comparison to healthy T - cell precursors . Dot size and color indicate the frequency ( number of patients having dysregulation of the gene ) . Genes with ≥ 5 / 19 frequency are shown on the plot . Protein coding genes with ≥ 12 / 19 frequency are labelled . For plotting the average LFC values of ATAC and RNA are used per gene per quartile . Q1 - 4 stands for the quadrant 1 - 4 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 log2 fold changes of 292 genes which are differentially expressed (x-axis, RNA-Seq: non-sorted bulk thymi vs. T-ALLs) and differentially accessible (y-axis, ATAC-Seq: sorted T-cell populations vs. T-ALLs) in T-ALLs in comparison to healthy T-cell precursors. Dot size and color indicate the frequency (number of patients having dysregulation of the gene). Genes with ≥5/19 frequency are shown on the plot. Protein coding genes with ≥12/19 frequency are labelled. For plotting the average LFC values of ATAC and RNA are used per gene per quartile. Q1 - 4 stands for the quadrant 1 - 4.


D Heatmap of VST normalized read counts ( each row is also normalized by the row mean ) of differentially expressed genes ( pval < 0 . 1 ; DESeq2 ) in three patients in comparison to remaining 16 . Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts .

D Heatmap of VST normalized read counts (each row is also normalized by the row mean) of differentially expressed genes (pval<0.1; DESeq2) in three patients in comparison to remaining 16. Orange triangles indicate three patients with no DAB1 but high SPI1 expression and motif counts.


( B ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs without heparin .

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) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs without heparin.


( C ) Bar graph representing residual thrombin activity ( ΔOD / min ) in presence of the sdAbs and heparin .

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

(C) Bar graph representing residual thrombin activity (ΔOD/min) in presence of the sdAbs and heparin.


( D ) Bar graph representing residual activated factor X ( FXa ) activity ( ΔOD / min ) in presence of the sdAbs and heparin .

O O O 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) Bar graph representing residual activated factor X (FXa) activity (ΔOD/min) in presence of the sdAbs and heparin.


( B - C ) Graphs reporting the recovery of thrombin - and FXa - mediated substrate conversion in the presence of increasing doses of KB - AT - 23 , in presence of antithrombin and heparin .

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

(B-C) Graphs reporting the recovery of thrombin- and FXa-mediated substrate conversion in the presence of increasing doses of KB-AT-23, in presence of antithrombin and heparin.


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


( D - E ) Thrombin generation profiles of FVIII - deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB - AT - 23 .

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

(D-E) Thrombin generation profiles of FVIII-deficient plasma spiked with different concentrations of recombinant FVIII or 2 doses of KB-AT-23.


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


( B ) Purified KB - AT - 23 - fus and KB - hFX - 11 - fus were given intravenously to wild - type C57B6 mice ( 10 mg / kg ) . At indicated time - points , blood was collected and residual protein was measured . Plotted is residual protein versus time after injection . Green circles : KB - AT - 23 - fus ; Red circles : KB - hFX - 11 - fus .

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

(B) Purified KB-AT-23-fus and KB-hFX-11-fus were given intravenously to wild-type C57B6 mice (10 mg/kg). At indicated time-points, blood was collected and residual protein was measured. Plotted is residual protein versus time after injection. Green circles: KB-AT-23-fus; Red circles: KB-hFX-11-fus.


( D ) Graph reporting the volume of blood loss ( µL ) in treated mice ( n = 4 - 9 per group ) observed during 30 minutes post TVT . Dosing was 10 mg / kg for KB - AT - 23 and 1 mg / kg for FVIIa . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) .

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

(D) Graph reporting the volume of blood loss (µL) in treated mice (n=4-9 per group) observed during 30 minutes post TVT. Dosing was 10 mg/kg for KB-AT-23 and 1 mg/kg for FVIIa. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016).


( B ) Western blot analysis of KB - AT - 23 protein secretion in conditioned media 72 hours post - transfection of HuH7 cells . In the lower panel the western blot quantification via densitometry analysis is depicted . Data are presented as mean ± SD , and were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons . * * * * p < 0 . 001 .

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

(B) Western blot analysis of KB-AT-23 protein secretion in conditioned media 72 hours post-transfection of HuH7 cells. In the lower panel the western blot quantification via densitometry analysis is depicted. Data are presented as mean ±SD, and were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons. ****p<0.001.


( C ) HuH7 - celles were collected 72 hours post - transfection . Presented is the Western blot analysis of lysed cells for the presence of KB - AT - 23 . Cntr - refers to non - transfected cells , while Cntr + refers to purified recombinant KB - AT - 23 .

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

(C) HuH7-celles were collected 72 hours post-transfection. Presented is the Western blot analysis of lysed cells for the presence of KB-AT-23. Cntr- refers to non-transfected cells, while Cntr+ refers to purified recombinant KB-AT-23.


( B ) Representative western blot analyses of KB - AT - 23 and antithrombin expression on plasma samples ( n = 4 ) collected from animals 27 weeks post - AAV injection . kDa , molecular weight marker .

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

(B) Representative western blot analyses of KB-AT-23 and antithrombin expression on plasma samples (n=4) collected from animals 27 weeks post-AAV injection. kDa, molecular weight marker.


( C ) Measurement of D - dimers ( D2D ) plasma concentration 27 weeks post vector administration . Data are presented as mean ± SD ( n = 6 - 9 / group ) .

O O O O O O O O O O O O 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) Measurement of D-dimers (D2D) plasma concentration 27 weeks post vector administration. Data are presented as mean ±SD (n=6-9/group).


( D ) Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 6 - 9 / group ) 8 weeks post - AAV administration . Data represent mean ± SD .

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

(D) Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=6-9/group) 8 weeks post-AAV administration. Data represent mean±SD.


( E ) TVT test on males and females ( n = 4 - 5 per group ) performed 29 weeks post vector administration . The volume of blood loss ( µL ) during 30 minutes observation time is reported . The grey area represents the range of blood loss in FVIII - treated mice ( 33 - 245 μL ) , which is similar to that of wild - type C57BL / 6 mice ( 49 - 308 μL ; Johansen et al . 2016 ) . Boxes represent 25 - 75 % quartiles ; the central line indicates the median . Whiskers span min to max . Data were analysed via 1 - way ANOVA with Dunnett ' s correction for multiple comparisons .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TVT test on males and females (n=4-5 per group) performed 29 weeks post vector administration. The volume of blood loss (µL) during 30 minutes observation time is reported. The grey area represents the range of blood loss in FVIII-treated mice (33-245 μL), which is similar to that of wild-type C57BL/6 mice (49-308 μL; Johansen et al. 2016). Boxes represent 25-75% quartiles; the central line indicates the median. Whiskers span min to max. Data were analysed via 1-way ANOVA with Dunnett's correction for multiple comparisons.


( F ) Vector genome copy number ( VGCN ) per cell in liver samples collected at sacrifice ( n = 8 / group ) . Data represent mean ± SD .

O O O O O O O O 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) Vector genome copy number (VGCN) per cell in liver samples collected at sacrifice (n=8/group). Data represent mean±SD.


( C ) Measurement of anti - hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS ( n = 5 / group ) at different time points . Data represent mean ± SEM .

O O O O O O O O O O O O O O O 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) Measurement of anti-hFIX mouse IgG in plasma samples collected from mice injected with FIX or PBS (n=5/group) at different time points. Data represent mean±SEM.


( D ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 8 weeks post - AAV injection .

O 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) Representative western blot analyses on plasma samples (n=4) collected from animals 8 weeks post-AAV injection.


( E ) Antithrombin activity in plasma samples collected from mice at different time points . Data are presented as mean ± SD ( n = 3 - 4 per datapoint ) . Data were analyzed in a 2 - way ANOVA with Tukey ' s correction for multiple comparisons . * * * p < 0 . 01 * * * * p < 0 . 001 ( 2 - way ANOVA with Tukey ' s multiple comparison test )

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

(E) Antithrombin activity in plasma samples collected from mice at different time points. Data are presented as mean±SD (n=3-4 per datapoint). Data were analyzed in a 2-way ANOVA with Tukey's correction for multiple comparisons. ***p<0.01 ****p<0.001 (2-way ANOVA with Tukey's multiple comparison test)


( F ) Graph reporting the volume of blood loss ( µL ) over 30 minutes following the tail clip . WT : wild - type littermates of F9 - / - mice . The grey area represents the range of blood loss of F9 - / - mice that received AAV - vectors expressing human FIX . Data are presented as mean ± SD ( n = 3 - 4 / group ) , and were analyzed in a 1 - way ANOVA with Tukey ' s correction for multiple comparisons . * * p < 0 . 01 Boxes represent 25 % - 75 % quartiles , with the line indicating the median . Whiskers span min to max .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Graph reporting the volume of blood loss (µL) over 30 minutes following the tail clip. WT: wild-type littermates of F9-/- mice. The grey area represents the range of blood loss of F9-/- mice that received AAV-vectors expressing human FIX. Data are presented as mean±SD (n=3-4/group), and were analyzed in a 1-way ANOVA with Tukey's correction for multiple comparisons. **p<0.01 Boxes represent 25%-75% quartiles, with the line indicating the median. Whiskers span min to max.


G Measurement of anti - KB - AT - 23 mouse IgG in plasma samples collected from mice ( n = 5 / group ) 8 weeks post - AAV administration . Data represent mean ± SD .

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

G Measurement of anti-KB-AT-23 mouse IgG in plasma samples collected from mice (n=5/group) 8 weeks post-AAV administration. Data represent mean±SD.


( C ) Representative western blot analyses on plasma samples ( n = 4 ) collected from animals 4 weeks post - AAV injection . Cntrl + : positive controls represented by the loading of the purified KB - AT - 23 or KB - AT - 113 proteins .

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

(C) Representative western blot analyses on plasma samples (n=4) collected from animals 4 weeks post-AAV injection. Cntrl +: positive controls represented by the loading of the purified KB-AT-23 or KB-AT-113 proteins.


( D - H ) Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 ( 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O

(D-H) Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 (D)


Measurement of anti - sdAbs mouse IgG in plasma samples collected at days 28 and 57 post - AAV injection . The ELISA plate was coated with the purified KB - AT - 01 , - 02 ( E ) , - 03 ( F ) , - 23 ( G ) or - 113 ( H ) sdAbs .

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

Measurement of anti-sdAbs mouse IgG in plasma samples collected at days 28 and 57 post-AAV injection. The ELISA plate was coated with the purified KB-AT-01 , -02 (E), -03 (F), -23 (G) or -113 (H) sdAbs.


( D ) RipAC overexpression inhibits flg22 - triggered MAPK activation in Arabidopsis . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points . Immunoblots were analysed using anti - pMAPK and anti - RipAC antibodies . Coomassie brilliant blue ( CBB ) staining and a non - specific band were used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) RipAC overexpression inhibits flg22-triggered MAPK activation in Arabidopsis. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points. Immunoblots were analysed using anti-pMAPK and anti-RipAC antibodies. Coomassie brilliant blue (CBB) staining and a non-specific band were used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( A - B ) Interaction between RipAC and SlPUB4 / AtPUB4 was confirmed by Split - LUC assay qualitatively ( A ) and quantitatively ( B ) in Nicotiana benthamiana . Values are means ± SE ( n = 8 biological replicates ) . Asterisks indicate significant differences compared to RipAC - nLUC / cLUC - AtPIP2A negative control ( Student ' s t test , * * * * p < 0 . 0001 ) . RLU : relative luminescence units .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Interaction between RipAC and SlPUB4/AtPUB4 was confirmed by Split-LUC assay qualitatively (A) and quantitatively (B) in Nicotiana benthamiana. Values are means ± SE (n=8 biological replicates). Asterisks indicate significant differences compared to RipAC-nLUC/cLUC-AtPIP2A negative control (Student's t test, **** p<0.0001). RLU: relative luminescence units.


( C ) Co - immunoprecipitation of SlPUB4 / AtPUB4 - GFP and RipAC - nLUC after co - expression in N . benthamiana . Two days after Agrobacterium infiltration , the plant tissues were collected and then subjected to anti - GFP immunoprecipitation . Immunoblots were analyzed using anti - LUC and anti - GFP antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

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

(C) Co-immunoprecipitation of SlPUB4/AtPUB4-GFP and RipAC-nLUC after co-expression in N. benthamiana. Two days after Agrobacterium infiltration, the plant tissues were collected and then subjected to anti-GFP immunoprecipitation. Immunoblots were analyzed using anti-LUC and anti-GFP antibodies. Molecular weight (kDa) marker bands are indicated for reference.


( E ) flg22 - triggered MAPK activation in pub4 T - DNA mutants . 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots . Immunoblots were analyzed using anti - pMAPK and anti - FLS2 antibodies . Coomassie brilliant blue ( CBB ) staining was used as loading control . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) flg22-triggered MAPK activation in pub4 T-DNA mutants. 100 nM flg22Pto was used to treat Arabidopsis seedlings and the samples were collected at indicated time points for western blots. Immunoblots were analyzed using anti-pMAPK and anti-FLS2 antibodies. Coomassie brilliant blue (CBB) staining was used as loading control. Molecular weight (kDa) marker bands are indicated for reference.


( B ) Co - immunoprecipitation of PUB4 and BIK1 in N . benthamiana . PUB4 - GFP or GFP - LTI6b were transiently co - expressed with BIK1 - HA in the leaves of N . benthamiana . After treatment with mock or 1 μM flg22Paer for 10 min , total proteins ( input ) were extracted and subjected for immunoprecipitation with anti - GFP beads .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Co-immunoprecipitation of PUB4 and BIK1 in N. benthamiana. PUB4-GFP or GFP-LTI6b were transiently co-expressed with BIK1-HA in the leaves of N. benthamiana. After treatment with mock or 1 μM flg22Paer for 10 min, total proteins (input) were extracted and subjected for immunoprecipitation with anti-GFP beads.


( C ) Co - immunoprecipitation of PUB4 with PRR complex members in Arabidopsis . PUB4 co - immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment , and constantly with BIK1 . Total protein extracts ( input ) from Col - 0 and PUB4 - FLAG plants were subjected to immunoprecipitation with anti - FLAG beads .

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

(C) Co-immunoprecipitation of PUB4 with PRR complex members in Arabidopsis. PUB4 co-immunoprecipitated with FLS2 and BAK1 specifically after 10 min of 1 μM flg22Paer treatment, and constantly with BIK1. Total protein extracts (input) from Col-0 and PUB4-FLAG plants were subjected to immunoprecipitation with anti-FLAG beads.


( D ) PUB4 directly interacts with BIK1 in vitro . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference .

O O O O O O O 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) PUB4 directly interacts with BIK1 in vitro. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference.


RipAC does not affect PUB4 association with PRR complex members . PUB4 - FLAG , PUB4 - FLAG RipAC - GFP and Col - 0 plants were treated for 10 min with water ( as mock ) or 1 μM flg22Paer and elf18Ecol treatment . Total protein extracts ( input ) were subjected for immunoprecipitation with anti - FLAG beads . Immunoblots were analyzed using the indicated antibodies . Molecular weight ( kDa ) marker bands are indicated for reference . Data information : The experiment was performed 3 times with similar results .

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

RipAC does not affect PUB4 association with PRR complex members. PUB4-FLAG, PUB4-FLAG RipAC-GFP and Col-0 plants were treated for 10 min with water (as mock) or 1 μM flg22Paer and elf18Ecol treatment. Total protein extracts (input) were subjected for immunoprecipitation with anti-FLAG beads. Immunoblots were analyzed using the indicated antibodies. Molecular weight (kDa) marker bands are indicated for reference. Data information: The experiment was performed 3 times with similar results.


( A ) PUB4 has auto - ubiquitination activity in vitro . PUB4 autoubiquitination assay using His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) and MBP - PUB4 or MBP - PUB4 Cys239Ala inactive mutant reconstituted in bacteria . Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography ( IMAC ) . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates autoubiquitinated PUB4 .

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) PUB4 has auto-ubiquitination activity in vitro. PUB4 autoubiquitination assay using His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2) and MBP-PUB4 or MBP-PUB4 Cys239Ala inactive mutant reconstituted in bacteria. Ubiquitinated products were purified under denaturing conditions by immobilized metal ion chromatography (IMAC). Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates autoubiquitinated PUB4.


( B ) PUB4 ubiquitinates non - active BIK1 , and does not ubiquitinate active BIK1 . Operon containing His - Ubiquitin , UBA1 ( E1 ) , HA - UBC8 ( E2 ) , and MBP - PUB4 were co - transformed with GST , GST - BIK1 , or GST - BIK1 kinase dead ( BIK1 - KD ) in the presence of PUB4 . Ubiquitinated products were purified under denaturing conditions by IMAC , and modified proteins further enriched by GSH chromatography . Input and precipitated proteins were resolved in 10 % and 8 % acrylamide gels , respectively , and analyzed by immunoblot with indicated antibodies . The line indicates the ubiquitination products .

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

(B) PUB4 ubiquitinates non-active BIK1, and does not ubiquitinate active BIK1. Operon containing His-Ubiquitin, UBA1 (E1), HA-UBC8 (E2), and MBP-PUB4 were co-transformed with GST, GST-BIK1, or GST-BIK1 kinase dead (BIK1-KD) in the presence of PUB4. Ubiquitinated products were purified under denaturing conditions by IMAC, and modified proteins further enriched by GSH chromatography. Input and precipitated proteins were resolved in 10% and 8% acrylamide gels, respectively, and analyzed by immunoblot with indicated antibodies. The line indicates the ubiquitination products.


( A ) Analysis of BIK1 - HA protein accumulation in BIK1 - HA + / - , pub4 - / - BIK1 - HA + / - , and PUB4 - FLAG + / - BIK1 - HA + / - in 4 - to 5 - week - old plants . Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132 , 50 mM CHX , and for 10 min with water ( as mock ) or 1 mM flg22Paer . Proteins were extracted in SDS buffer and analysed by western blot . BAK1 was used as a loading control . Numbers correspond to the quantitation of the BIK1 - HA band , normalized to the quantitation of the BAK1 control band in the same sample .

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

(A) Analysis of BIK1-HA protein accumulation in BIK1-HA+/-, pub4-/- BIK1-HA+/-, and PUB4-FLAG+/- BIK1-HA+/- in 4- to 5-week-old plants. Prior to protein extraction leave disks were treated for 6 h with 100 mM MG132, 50 mM CHX, and for 10 min with water (as mock) or 1 mM flg22Paer. Proteins were extracted in SDS buffer and analysed by western blot. BAK1 was used as a loading control. Numbers correspond to the quantitation of the BIK1-HA band, normalized to the quantitation of the BAK1 control band in the same sample.


( B ) BIK1 - HA accumulation is reduced in PUB4 - FLAG + / - BIK1 - HA + / - line compared to BIK1 - HA + / - line in the absence of PAMP treatment . Total protein extracts were analysed by western blot . Tubulin was used as a loading 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 O O O O O O O O O O O O

(B) BIK1-HA accumulation is reduced in PUB4-FLAG+/- BIK1-HA+/- line compared to BIK1-HA+/- line in the absence of PAMP treatment. Total protein extracts were analysed by western blot. Tubulin was used as a loading control.


( E ) BIK1 - HA accumulation is reduced in RipAC - GFP / BIK1 - HA line compared to Col - 0 / BIK1 - HA line . Total protein extracts were analysed by western blot . Actin was used as a loading 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 O O

(E) BIK1-HA accumulation is reduced in RipAC-GFP/BIK1-HA line compared to Col-0/BIK1-HA line. Total protein extracts were analysed by western blot. Actin was used as a loading control.


Transfection of poly ( I : C ) induces ACE2 expression . HEK293 cells were cultured in six - well plates and transfected with poly ( I : C ) as indicated above using Lipofectamine 2000 . HEK293 cells were then collected for RNA isolation and RT - qPCR analysis at 9 hrs post - transfection . Results in each panel are representative of three independent experiments .

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

Transfection of poly (I:C) induces ACE2 expression.HEK293 cells were cultured in six-well plates and transfected with poly (I:C) as indicated above using Lipofectamine 2000. HEK293 cells were then collected for RNA isolation and RT-qPCR analysis at 9 hrs post-transfection. Results in each panel are representative of three independent experiments.


VSV - eGFP infection induces ACE2 expression . HEK293 cells were cultured in six - well plates and infected with VSV - eGFP as indicated above . 6 hrs later , HEK293 cells were collected for RNA isolation and RT - qPCR analysis . Results in each panel are representative of three independent experiments .

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

VSV-eGFP infection induces ACE2 expression.HEK293 cells were cultured in six-well plates and infected with VSV-eGFP as indicated above. 6 hrs later, HEK293 cells were collected for RNA isolation and RT-qPCR analysis. Results in each panel are representative of three independent experiments.


Overexpression of TBK1 or TRIF induces ACE2 expression . TBK1 is an essential activator of RNA - sensing pathway . HEK293 cells were cultured in 24 - well plates . 24 hrs later , plasmids as shown above were transfected into HEK293 cells . 36 hrs after transfection , total RNA was isolation for subsequent RT - qPCR analysis . Results in each panel are representative of three independent experiments .

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

Overexpression of TBK1 or TRIF induces ACE2 expression.TBK1 is an essential activator of RNA-sensing pathway. HEK293 cells were cultured in 24-well plates. 24 hrs later, plasmids as shown above were transfected into HEK293 cells. 36 hrs after transfection, total RNA was isolation for subsequent RT-qPCR analysis. Results in each panel are representative of three independent experiments.


ACE2 expression is upregulated in the lungs from SARS - CoV MA15 infected C57Bl / 6 mouse . Intranasal instillation of 102 , 103 PFU of SARS - CoV MA15 in 50 µl of PBS or mock - infected with PBS alone , lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis . ACE2 expression data was retrieved from Gene Expression Omnibus ( GEO ) microarray database ( NCBI Accession : PRJNA149057 ; GEO : GSE33266 ) .

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

ACE2 expression is upregulated in the lungs from SARS-CoV MA15 infected C57Bl/6 mouse.Intranasal instillation of 102, 103 PFU of SARS-CoV MA15 in 50 µl of PBS or mock-infected with PBS alone, lungs were harvested 24 hrs later and total RNA was isolated and subjected to microarray analysis. ACE2 expression data was retrieved from Gene Expression Omnibus (GEO) microarray database (NCBI Accession: PRJNA149057; GEO: GSE33266).


ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS - CoV ( icMERS - CoV EMC2012 ) . Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell . Infected samples were collected as indicated above . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA391962 ; GEO : GSE100504 ) .

ACE2 expression is upregulated in primary human airway epithelial cells in response to wild type MERS-CoV (icMERS-CoV EMC2012).Human airway epithelial cells were infected with a multiplicity of infection of 5 PFU per cell. Infected samples were collected as indicated above. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA391962; GEO: GSE100504).


ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection . Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only ( used as control ) . 24 hrs post - infection , gene expression was assessed using Affymetrix U133 plus 2 . 0 human GeneChips . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA137767 ; GEO : GSE27973 ) .

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

ACE2 expression is upregulated in human airway epithelial cells in response to rhinovirus infection.Primary epithelial cells from 4 donors were exposed to rhinovirus or medium only (used as control). 24 hrs post-infection, gene expression was assessed using Affymetrix U133 plus 2.0 human GeneChips. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA137767; GEO: GSE27973).


ACE2 expression is upregulated in human bronchial epithelial cells ( HBECs ) after IFNβ and H1N1 influenza stimulation . HBECs were treated with IFNβ ( 1000U / ml ) or wild type H1N1 influenza ( A / PR / 8 / 34 ) . Control samples were incubated with fresh medium under the same conditions . HBECs were harvested at indicated time points ( 0 . 25 , 0 . 5 , 1 , 1 . 5 , 2 , 4 , 6 , 8 , 12 , and 18 hours post - treatment ) . Total RNA was extracted and analyzed using the Affymetrix High Throughput Array . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA121751 ; GEO : GSE19392 ) .

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

ACE2 expression is upregulated in human bronchial epithelial cells (HBECs) after IFNβ and H1N1 influenza stimulation.HBECs were treated with IFNβ (1000U/ml) or wild type H1N1 influenza (A/PR/8/34). Control samples were incubated with fresh medium under the same conditions. HBECs were harvested at indicated time points (0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, and 18 hours post-treatment). Total RNA was extracted and analyzed using the Affymetrix High Throughput Array. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA121751; GEO: GSE19392).


ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ . Primary keratinocytes from 3 donors were either untreated ( control ) or exposed to cytokines IFNα , IFNγ . 24 hrs after treatment , total RNA was extracted for subsequent microarray analysis . ACE2 expression data was retrieved from GEO microarray database ( NCBI Accession : PRJNA153023 ; GEO : GSE36287 ) .

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

ACE2 expression is upregulated in human primary keratinocytes exposed to IFNα and IFNγ.Primary keratinocytes from 3 donors were either untreated (control) or exposed to cytokines IFNα, IFNγ. 24 hrs after treatment, total RNA was extracted for subsequent microarray analysis. ACE2 expression data was retrieved from GEO microarray database (NCBI Accession: PRJNA153023; GEO: GSE36287).


( A ) NIH - 3T3 cells were transduced with retroviral vectors expressing M45 or GFP . Two days after transduction cells were stimulated with the TLR4 agonist LPS ( 10 µg / ml ) , the TLR2 agonist LTA - SA ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) . IκBα levels were determined by immunoblotting .

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

(A) NIH-3T3 cells were transduced with retroviral vectors expressing M45 or GFP. Two days after transduction cells were stimulated with the TLR4 agonist LPS (10 µg/ml), the TLR2 agonist LTA-SA (10 µg/ml), or IL-1β (20 ng/ml). IκBα levels were determined by immunoblotting.


( B ) NIH - 3T3 cells were transduced with an M45 - expressing or an empty retroviral vector . 30 min after stimulation with IL - 1β ( 10 ng / ml ) cells were fixed , and subcellular localization of the NF - κB p65 subunit was analyzed by immunofluorescence .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells were transduced with an M45-expressing or an empty retroviral vector. 30 min after stimulation with IL-1β (10 ng/ml) cells were fixed, and subcellular localization of the NF-κB p65 subunit was analyzed by immunofluorescence.


( C ) NIH - 3T3 cells expressing an NF - κB - dependent secreted alkaline phosphatase ( SEAP ) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 ( Pam . , 1 µg / ml ) , the TLR4 agonist LPS ( 0 . 1 µg / ml ) , or IL - 1β ( 5 ng / ml ) . SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non - stimulated cells ( mean ± SD )

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

(C) NIH-3T3 cells expressing an NF-κB-dependent secreted alkaline phosphatase (SEAP) reporter were transduced with retroviral vectors expressing M45 or GFP and stimulated 78 h later with the TLR2 agonist Pam3CSK4 (Pam.,1 µg/ml), the TLR4 agonist LPS (0.1 µg/ml), or IL-1β (5 ng/ml). SEAP activity in the supernatant was quantified 15 h after stimulation and is shown as fold induction of SEAP activity of stimulated cells compared to non-stimulated cells (mean ± SD)


( D ) NIH - 3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL - 1β ( 20 ng / ml , 15 min ) or TNFα ( 10 ng / ml , 5 min ) . Phosphorylated and total p38 in cell lysates was detected by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells transduced with retroviral vectors expressing M45 or GFP were stimulated with IL-1β (20 ng/ml, 15 min) or TNFα (10 ng/ml, 5 min). Phosphorylated and total p38 in cell lysates was detected by immunoblotting.


( A ) NIH - 3T3 cells were infected with wt MCMV - GFP , an M45 deletion mutant ( ΔM45 ) or a revertant virus ( RM45 ) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 ( Pam . , 0 . 1 µg / ml ) , the TLR4 agonist LPS ( 10 µg / ml ) , or IL - 1β ( 20 ng / ml ) for the indicated times . Levels of the indicated proteins were analyzed by immunoblotting .

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

(A) NIH-3T3 cells were infected with wt MCMV-GFP, an M45 deletion mutant (ΔM45) or a revertant virus (RM45) at an MOI of 10 and treated 5 h postinfection with the TLR2 agonist Pam3CSK4 (Pam., 0.1 µg/ml), the TLR4 agonist LPS (10 µg/ml), or IL-1β (20 ng/ml) for the indicated times. Levels of the indicated proteins were analyzed by immunoblotting.


( B ) BMDMs were infected with wt MCMV - GFP , ΔM45 , or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 ( 0 . 1 µM ) . TNFα and IL - 6 levels in the supernatant were determined by ELISA ( mean ± SD ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDMs were infected with wt MCMV-GFP, ΔM45, or RM45 at an MOI of 3 and stimulated 8 h postinfection for 16 h with the TLR7 agonist R848 (0.1 µM). TNFα and IL-6 levels in the supernatant were determined by ELISA (mean ± SD).


( C ) RAW264 . 7 macrophages were infected with wt MCMV - GFP or ΔM45 at an MOI of 0 . 1 , stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 ( Pam . ) or Malp - 2 ( TLR2 ) , LPS ( TLR4 ) , R837 ( TLR7 ) , or CpG ( TLR9 ) , in the presence of brefeldin A . Cells were fixed , permeabilized , and stained with a TNFα - specific antibody . The percentages of TNFα - positive cells within infected ( GFP - positive ) cell populations were determined by FACS analysis ( mean ± SD ) .

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

(C) RAW264.7 macrophages were infected with wt MCMV-GFP or ΔM45 at an MOI of 0.1, stimulated 24 h postinfection for 4 hours with TLR agonists Pam3CSK4 (Pam.) or Malp-2 (TLR2), LPS (TLR4), R837 (TLR7), or CpG (TLR9), in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SD).


( A ) 293A cells were transfected with plasmids expressing HA - tagged M45 or an unrelated MCMV control protein ( m143 ) and Flag - tagged IKK subunits . Lysates were subjected to immunoprecipitation ( IP ) using an anti - HA antibody . Immunoprecipitates were analyzed by immunoblotting ( IB ) using anti - Flag antibody . Whole cell lysates ( WCL ) were immunoblotted using Flag - , HA - , and actin - specific antibodies , respectively .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 293A cells were transfected with plasmids expressing HA-tagged M45 or an unrelated MCMV control protein (m143) and Flag-tagged IKK subunits. Lysates were subjected to immunoprecipitation (IP) using an anti-HA antibody. Immunoprecipitates were analyzed by immunoblotting (IB) using anti-Flag antibody. Whole cell lysates (WCL) were immunoblotted using Flag-, HA-, and actin-specific antibodies, respectively.


( B ) Nemo − / − MEFs were transfected with HA - tagged IKK subunits and HA - tagged M45 or an unrelated MCMV control protein ( m142 ) , respectively . IP and IB were performed using anti - HA and anti - Flag antibodies , respectively . WCL were immunoblotted with anti - NEMO antibody and as described for panel 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

(B) Nemo−/− MEFs were transfected with HA-tagged IKK subunits and HA-tagged M45 or an unrelated MCMV control protein ( m142), respectively. IP and IB were performed using anti-HA and anti-Flag antibodies, respectively. WCL were immunoblotted with anti-NEMO antibody and as described for panel A.


( C ) rip1 − / − MEFs were transfected with HA - tagged M45 or an unrelated MCMV control protein ( m142 ) and Flag - tagged NEMO . IP was done with an anti - HA antibody . Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) rip1−/− MEFs were transfected with HA-tagged M45 or an unrelated MCMV control protein (m142) and Flag-tagged NEMO. IP was done with an anti-HA antibody. Immunoprecipitates and WCL were analyzed by immunoblotting using the indicated antibodies.


( D ) 293A cells were transfected with plasmids expressing HA - tagged M45 or m143 ( as a control ) . Lysates were subjected to anti - HA immunoprecipitation . Immunoblotting of precipitates and the WCL was done with antibodies 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

(D) 293A cells were transfected with plasmids expressing HA-tagged M45 or m143 (as a control). Lysates were subjected to anti-HA immunoprecipitation. Immunoblotting of precipitates and the WCL was done with antibodies as indicated.


( E ) NIH - 3T3 cells were infected for 4 hours with RM45 ( MCMV expressing HA - tagged M45 ) , ΔM45 , or Rm142 ( MCMV expressing HA - tagged m142 ) at an MOI of 5 . Cell lysates were subjected to IP and IB with antibodies as indicated .

(E) NIH-3T3 cells were infected for 4 hours with RM45 (MCMV expressing HA-tagged M45), ΔM45, or Rm142 (MCMV expressing HA-tagged m142) at an MOI of 5. Cell lysates were subjected to IP and IB with antibodies as indicated.


( B ) NIH - 3T3 cells were transduced with retroviruses expressing full length M45 , truncated M45 proteins , or GFP . After stimulation with IL - 1β ( 20 ng / ml , 15 min ) , IκBα levels were determined by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells were transduced with retroviruses expressing full length M45, truncated M45 proteins, or GFP. After stimulation with IL-1β (20 ng/ml, 15 min), IκBα levels were determined by immunoblotting.


( C ) 293A cells were transfected with plasmids encoding Flag - tagged NEMO and HA - tagged full - length M45 , truncated M45 , or an unrelated MCMV control protein ( m142 ) , respectively . Lysates were subjected to immunoprecipitation ( IP ) with an anti - HA antibody . Immunoprecipitates and the whole cell lysates ( WCL ) were analyzed by immunoblotting ( IB ) with the indicated antibodies .

(C) 293A cells were transfected with plasmids encoding Flag-tagged NEMO and HA-tagged full-length M45, truncated M45, or an unrelated MCMV control protein (m142), respectively. Lysates were subjected to immunoprecipitation (IP) with an anti-HA antibody. Immunoprecipitates and the whole cell lysates (WCL) were analyzed by immunoblotting (IB) with the indicated antibodies.


( D ) Primary BMDMs were mock infected or infected with GFP - expressing wt MCMV ( wt ) , ΔM45 mutant ( ΔM45 ) , M45 revertant ( RM45 ) , or MCMVs expressing Ct or Nt3 ( RCt and RNt3 ) at an MOI of 1 . 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG ( 0 . 5 µM ) or TLR7 agonist R838 ( 0 . 1 µM ) in the presence of brefeldin A . Cells were fixed , permeabilized , and stained with a TNFα - specific antibody . The percentages of TNFα - positive cells within infected ( GFP - positive ) cell populations were determined by FACS analysis ( mean ± SEM ) .

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

(D) Primary BMDMs were mock infected or infected with GFP-expressing wt MCMV (wt), ΔM45 mutant (ΔM45), M45 revertant (RM45), or MCMVs expressing Ct or Nt3 (RCt and RNt3) at an MOI of 1. 17 h postinfection cells were stimulated for 4 hours with TLR9 agonist CpG (0.5 µM) or TLR7 agonist R838 (0.1 µM) in the presence of brefeldin A. Cells were fixed, permeabilized, and stained with a TNFα-specific antibody. The percentages of TNFα-positive cells within infected (GFP-positive) cell populations were determined by FACS analysis (mean ± SEM).


( A ) NIH - 3T3 cells were infected with MCMV ΔM45 or the revertant virus , RM45 , at an MOI of 3 . Cells were harvested at indicated time points postinfection , and the levels of IKK subunits , M45 , IE1 , and actin in lysates were analyzed by immunoblotting .

(A) NIH-3T3 cells were infected with MCMV ΔM45 or the revertant virus, RM45, at an MOI of 3. Cells were harvested at indicated time points postinfection, and the levels of IKK subunits, M45, IE1, and actin in lysates were analyzed by immunoblotting.


( B ) Murine fibroblasts ( NIH - 3T3 , 10 . 1 ) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP , harvested 72 hours later , and analyzed by immunoblotting .

O O O O O O O O O O O O O O O O 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) Murine fibroblasts (NIH-3T3, 10.1) or human embryonic kidney 293A cells were transduced with retroviral vectors expressing M45 or GFP, harvested 72 hours later, and analyzed by immunoblotting.


( C ) ts20 cells harboring a immunoblotting - sensitive E1 enzyme were incubated for 14 h at a permissive ( 35 ° C ) or a restrictive ( 40 . 5 ° C ) temperature . Cells were then mock - infected or infected with RM45 , and analyzed 10 h postinfection by

(C) ts20 cells harboring a immunoblotting-sensitive E1 enzyme were incubated for 14 h at a permissive (35°C) or a restrictive (40.5°C) temperature. Cells were then mock-infected or infected with RM45, and analyzed 10 h postinfection by immunoblotting


( D ) NIH - 3T3 cells were mock infected or infected with RM45 ( MOI of 5 ) and treated 2 hpi with 10 mM ammonium chloride ( NH4Cl ) or a mix of lysosomal protease inhibitors ( PI - mix ) . 9 hpi , levels of IKK subunits , M45 , IE1 , and actin were determined by immunoblotting . (

O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O 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) NIH-3T3 cells were mock infected or infected with RM45 (MOI of 5) and treated 2 hpi with 10 mM ammonium chloride (NH4Cl) or a mix of lysosomal protease inhibitors (PI-mix). 9 hpi, levels of IKK subunits, M45, IE1, and actin were determined by immunoblotting. (


( E ) NIH - 3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP , and treated for the indicated times with PI - mix , 10 mM NH4Cl , or 5 µM lactacystin , respectively . Levels of the indicated proteins were analyzed by immunoblotting .

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

(E) NIH-3T3 cells were stably transduced with retroviral vectors expressing M45 or GFP, and treated for the indicated times with PI-mix, 10 mM NH4Cl, or 5 µM lactacystin, respectively. Levels of the indicated proteins were analyzed by immunoblotting.


( A ) NIH - 3T3 cells stably expressing Flag - tagged NEMO were mock infected or infected with wt MCMV - GFP or MCMV - GFP - ΔM45 at an MOI of 6 . Cells were fixed 6 hpi , and NEMO distribution was analyzed by immunofluorescence . The arrow indicates the region which is shown in higher magnification in the right corner of the picture .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells stably expressing Flag-tagged NEMO were mock infected or infected with wt MCMV-GFP or MCMV-GFP-ΔM45 at an MOI of 6. Cells were fixed 6 hpi, and NEMO distribution was analyzed by immunofluorescence. The arrow indicates the region which is shown in higher magnification in the right corner of the picture.


( B ) NIH - 3T3 cells were transfected with Flag - NEMO and M45 - HA expressing plasmids as indicated . 24 h later , cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags . Regions shown in higher magnification in the left corner of the pictures are indicated by arrows .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells were transfected with Flag-NEMO and M45-HA expressing plasmids as indicated. 24 h later, cells were fixed and double immunofluorescence staining was performed using antibodies against the HA and Flag tags. Regions shown in higher magnification in the left corner of the pictures are indicated by arrows.


( C ) NIH - 3T3 cells stably expressing Flag - NEMO were mock infected or infected with MCMV - M45 - HA or MCMV - ΔM45 at an MOI of 7 . Seven hpi , cells were fixed and analyzed as described for panel 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

(C) NIH-3T3 cells stably expressing Flag-NEMO were mock infected or infected with MCMV-M45-HA or MCMV-ΔM45 at an MOI of 7. Seven hpi, cells were fixed and analyzed as described for panel B.


( D ) NIH - 3T3 cells were transfected with expression plasmids for Flag - NEMO and M45 - HA or empty vector as indicated . Plasmids encoding GFP - tagged LC3 , Rab5 , or Rab7 were cotransfected . 24 hours posttransfection cells were fixed and used for anti - Flag immunofluorescence staining .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells were transfected with expression plasmids for Flag-NEMO and M45-HA or empty vector as indicated. Plasmids encoding GFP-tagged LC3, Rab5, or Rab7 were cotransfected. 24 hours posttransfection cells were fixed and used for anti-Flag immunofluorescence staining.


( A ) atg5 − / − and atg5 + / + MEFs were mock infected or infected with MCMVΔM45 or the revertant virus , RM45 , at an MOI of 10 . Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) atg5−/− and atg5+/+ MEFs were mock infected or infected with MCMVΔM45 or the revertant virus, RM45, at an MOI of 10. Eight hpi cells were harvested and levels of the indicated proteins were analyzed by immunoblotting.


( B ) 10 . 1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5 . Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3 - II . Cells were harvested at the indicated time points , and LC3 - II levels in cell lysates were analyzed by immunoblotting using an LC3 - II - specific antibody . LC3 - II levels were quantified by densitometric analysis and normalized using the corresponding actin levels . Fold increases are shown relative to mock - infected cells .

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

(B) 10.1 fibroblasts were mock infected or infected with wt MCMV or ΔM45 at an MOI of 5. Cells were left untreated or treated 30 min after infection with 10 mM NH4Cl to block lysosomal degradation of LC3-II. Cells were harvested at the indicated time points, and LC3-II levels in cell lysates were analyzed by immunoblotting using an LC3-II-specific antibody. LC3-II levels were quantified by densitometric analysis and normalized using the corresponding actin levels. Fold increases are shown relative to mock-infected cells.


( A ) NIH - 3T3 cells stably expressing GFP - LC3 were transduced with retroviral vectors expressing M45 or Ct3 . Three days later , cells were analyzed by confocal laser scanning microscopy . GFP - LC3 dots were counted in 50 cells per sample . Results are shown as box and whisker plots . The bottom and top of the box represent the first and third quartile , respectively . The horizontal line within the box represents the median . The minima and maxima within 1 . 5 - fold interquartile range are shown as whiskers . Dots represent outliers . Significance was determined using ANOVA ( * * * , p < 0 . 001 ) . M45 and Ct3 were detected by

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) NIH-3T3 cells stably expressing GFP-LC3 were transduced with retroviral vectors expressing M45 or Ct3. Three days later, cells were analyzed by confocal laser scanning microscopy. GFP-LC3 dots were counted in 50 cells per sample. Results are shown as box and whisker plots. The bottom and top of the box represent the first and third quartile, respectively. The horizontal line within the box represents the median. The minima and maxima within 1.5-fold interquartile range are shown as whiskers. Dots represent outliers. Significance was determined using ANOVA (***, p<0.001). M45 and Ct3 were detected by immunoblot


( B ) Fluorescence images of cells described above . Large GFP - LC3 aggregates are indicated by arrow heads .

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

(B) Fluorescence images of cells described above. Large GFP-LC3 aggregates are indicated by arrow heads.


( C ) Electron micrograph of a double - membrane autophagosome in an M45 - expressing cell .

(C) Electron micrograph of a double-membrane autophagosome in an M45-expressing cell.


( D ) Autophagosomes were counted in 10 randomly selected M45 - and Ct3 - expressing cells ( 2 - 3 TEM sections per cell ) . Results are shown in box and whisker plots as described for panel A . Significance was determined using the student ' s t - test ( * * * , p < 0 . 001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Autophagosomes were counted in 10 randomly selected M45- and Ct3-expressing cells (2-3 TEM sections per cell). Results are shown in box and whisker plots as described for panel A. Significance was determined using the student's t-test (***, p<0.001).


( E ) Epifluorescence image of an M45 - expressing cell with large GFP - LC3

(E) Epifluorescence image of an M45-expressing cell with large GFP-LC3 structures


( F ) TEM image of the same cell reveals large aggregates marked with ' A ' . ( G , H ) Successive enlargement of the region marked in panel F shows an example of an aggregate - associated autophagosome .

O O O O O O O O O O O O O O O O O O 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) TEM image of the same cell reveals large aggregates marked with 'A'. (G, H) Successive enlargement of the region marked in panel F shows an example of an aggregate-associated autophagosome.


( A ) Clone P15 induces HA - LC3 lipidation . 293T cells were transfected with P15 plus plasmids expressing HA - LC3A and / or the apoptotic inhibitor p35 ( as shown ) , and lysed for western blotting against the indicated molecules ( anti ‐ HA for HA - LC3A ) . The figure shows that expression of P15 induces HA - LC3A conversion to a lower molecular weight form indicative of protein lipidation . This activity remains unchanged by p35 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Clone P15 induces HA-LC3 lipidation. 293T cells were transfected with P15 plus plasmids expressing HA-LC3A and/or the apoptotic inhibitor p35 (as shown), and lysed for western blotting against the indicated molecules (anti‐HA for HA-LC3A). The figure shows that expression of P15 induces HA-LC3A conversion to a lower molecular weight form indicative of protein lipidation. This activity remains unchanged by p35.


( B ) P15 induces GFP - LC3 translocation to a vesiculated pattern . 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP - LC3A and p35 . The known autophagic inducer bNIP3L constituted a positive control . Representative confocal microscopy pictures are shown .

(B) P15 induces GFP-LC3 translocation to a vesiculated pattern. 293T cells were transfected with the indicated plasmids mixed with vectors expressing GFP-LC3A and p35. The known autophagic inducer bNIP3L constituted a positive control. Representative confocal microscopy pictures are shown.


( C ) TMEM59 induces HA - LC3 lipidation in different cell lines lacking the SV40 large T − antigen plasmid amplification system . Cells were transfected with the shown plasmids , vectors expressing HA - LC3A ( left ) or HA - LC3B ( right ) and GST ( as transfection control ) , and lysed for western blotting against the indicated molecules .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TMEM59 induces HA-LC3 lipidation in different cell lines lacking the SV40 large T−antigen plasmid amplification system. Cells were transfected with the shown plasmids, vectors expressing HA-LC3A (left) or HA-LC3B (right) and GST (as transfection control), and lysed for western blotting against the indicated molecules.


( D ) TMEM59 induces GFP - LC3 activation . 293 cells were transfected with the indicated plasmids and GFP - LC3A ( top ) or GFP - LC3B ( bottom ) . Representative confocal microscopy pictures are shown .

(D) TMEM59 induces GFP-LC3 activation. 293 cells were transfected with the indicated plasmids and GFP-LC3A (top) or GFP-LC3B (bottom). Representative confocal microscopy pictures are shown.


( E ) Quantification of the phenotype in D . The number of GFP - LC3 ‐ positive vesicles per transfected cell was scored for at least fifty cells . Data are expressed as means ± s . d . of one representative experiment of three repetitions . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the phenotype in D. The number of GFP-LC3‐positive vesicles per transfected cell was scored for at least fifty cells. Data are expressed as means ±s.d. of one representative experiment of three repetitions.Source data for this figure is available on the online supplementary information page.


( A ) TMEM59 ID is required for HA - LC3 lipidation . 293 cells were transfected with full ‐ length TMEM59 ( FL ) or a deleted version lacking the ID ( ΔID , Δ263 - 323 ) , HA - LC3A ( left ) or HA - LC3B ( right ) and GST . Cells were lysed for western blotting against the shown molecules .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TMEM59 ID is required for HA-LC3 lipidation. 293 cells were transfected with full‐length TMEM59 (FL) or a deleted version lacking the ID (ΔID, Δ263-323), HA-LC3A (left) or HA-LC3B (right) and GST. Cells were lysed for western blotting against the shown molecules.


( B ) The ID is necessary for GFP - LC3 activation . 293 cells were transfected with the indicated TMEM59 constructs and GFP - LC3A or GFP - LC3B ( as indicated ) . Representative confocal pictures are shown .

O O O O O O O O O O O O O O O O O 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) The ID is necessary for GFP-LC3 activation. 293 cells were transfected with the indicated TMEM59 constructs and GFP-LC3A or GFP-LC3B (as indicated). Representative confocal pictures are shown.


( C ) Quantification of the phenotype in B . Scoring and data expression were as in Figure 1E .

(C) Quantification of the phenotype in B. Scoring and data expression were as in Figure 1E.


( D ) TMEM59 ID ( amino acids 263 - 323 ) suffices for HA - LC3A lipidation . 293 cells were transfected with the indicated CD16 : 7 chimera ( Control : empty chimera ) and vectors encoding HA - LC3A and GST , subjected to aggregation with the shown amounts of anti ‐ CD16 antibody and lysed for western blotting . The right panel shows control WBs demonstrating equal loading ( ACTIN ) , transfection ( GST ) and chimera expression ( CD16 ) in unaggregated samples , as all experimental points per chimera derive from a single 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

(D) TMEM59 ID (amino acids 263-323) suffices for HA-LC3A lipidation. 293 cells were transfected with the indicated CD16:7 chimera (Control: empty chimera) and vectors encoding HA-LC3A and GST, subjected to aggregation with the shown amounts of anti‐CD16 antibody and lysed for western blotting. The right panel shows control WBs demonstrating equal loading (ACTIN), transfection (GST) and chimera expression (CD16) in unaggregated samples, as all experimental points per chimera derive from a single transfection.


( E ) Comparable surface levels of CD16 : 7 constructs . Transfected 293 cells were processed for anti ‐ CD16 flow cytometry . The graph displays percentages of positive cells ( left axis ) and means of fluorescence of positive cells ( MF , right axis ) obtained from triplicates . Data are expressed as means ± s . d . of the triplicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparable surface levels of CD16:7 constructs. Transfected 293 cells were processed for anti‐CD16 flow cytometry. The graph displays percentages of positive cells (left axis) and means of fluorescence of positive cells (MF, right axis) obtained from triplicates. Data are expressed as means ±s.d. of the triplicates.


( F ) The ID suffices for HA - LC3B lipidation .

(F) The ID suffices for HA-LC3B lipidation.


( G ) The ID suffices for GFP - LC3 activation . 293 cells were transfected with the indicated chimeras and GFP - LC3A or GFP - LC3B , aggregated and mounted . Representative confocal microscopy pictures are shown . Activated GFP - LC3 appears as a collapsed mass of indiscernible vacuoles .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) The ID suffices for GFP-LC3 activation. 293 cells were transfected with the indicated chimeras and GFP-LC3A or GFP-LC3B, aggregated and mounted. Representative confocal microscopy pictures are shown. Activated GFP-LC3 appears as a collapsed mass of indiscernible vacuoles.


( H ) Quantification of the phenotype in G . As individual vesicles could not be counted , quantification was done as the percentage of transfected cells showing redistributed GFP - LC3 . At least 10 different fields were counted ( about 400 cells ) . The experiment was repeated three times . Data are expressed as means ± s . d . of the triplicates . Source data for this figure is available on the online supplementary information page .

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

(H) Quantification of the phenotype in G. As individual vesicles could not be counted, quantification was done as the percentage of transfected cells showing redistributed GFP-LC3. At least 10 different fields were counted (about 400 cells). The experiment was repeated three times. Data are expressed as means ±s.d. of the triplicates.Source data for this figure is available on the online supplementary information page.


( A , B ) Ability of serial C ‐ terminal deletions of TMEM59 to activate LC3 . 293 cells were transfected with the indicated TMEM59 C ‐ terminal deletions , HA - LC3A ( A ) or HA - LC3B ( B ) and GST , and lysed for western blotting against the indicated molecules .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Ability of serial C‐terminal deletions of TMEM59 to activate LC3. 293 cells were transfected with the indicated TMEM59 C‐terminal deletions, HA-LC3A (A) or HA-LC3B (B) and GST, and lysed for western blotting against the indicated molecules.


( C ) Amino acids 263 - 281 retain the full potential of TMEM59 ID to promote HA - LC3 conversion . 293 cells were transfected with the shown CD16 : 7 chimeras , HA - LC3A or HA - LC3B ( as indicated ) and GST , and subjected to anti ‐ CD16 aggregation before lysing them for western blotting . The lower panels show control WBs ( unaggregated samples ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Amino acids 263-281 retain the full potential of TMEM59 ID to promote HA-LC3 conversion. 293 cells were transfected with the shown CD16:7 chimeras, HA-LC3A or HA-LC3B (as indicated) and GST, and subjected to anti‐CD16 aggregation before lysing them for western blotting. The lower panels show control WBs (unaggregated samples).


D Amino acids 263 - 281 suffice for GFP - LC3 activation and retain the full potential of TMEM59 ID to promote GFP - LC3 activation . 293 cells were transfected with the indicated CD16 : 7 chimeras and GFP - LC3A or GFP - LC3B ( as indicated ) , and subjected to anti ‐ CD16 aggregation before fixing them for microscopy . The graph shows percentages of transfected cells exhibiting redistributed GFP - LC3 . Quantification and data expression were done as in Figure 2H .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Amino acids 263-281 suffice for GFP-LC3 activation and retain the full potential of TMEM59 ID to promote GFP-LC3 activation. 293 cells were transfected with the indicated CD16:7 chimeras and GFP-LC3A or GFP-LC3B (as indicated), and subjected to anti‐CD16 aggregation before fixing them for microscopy. The graph shows percentages of transfected cells exhibiting redistributed GFP-LC3. Quantification and data expression were done as in Figure 2H.


( E ) Amino acids 282 - 323 of TMEM59 lack LC3 activation potential . 293 cells were transfected with the shown chimeras , HA - LC3A or HA - LC3B and GST , and processed for western blotting against the indicated molecules . Control WBs of unaggregated samples are shown in the lower panels .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Amino acids 282-323 of TMEM59 lack LC3 activation potential. 293 cells were transfected with the shown chimeras, HA-LC3A or HA-LC3B and GST, and processed for western blotting against the indicated molecules. Control WBs of unaggregated samples are shown in the lower panels.


( F ) Surface expression levels of CD16 : 7 chimeras . Procedures and data expression were as in Figure 2E . The figure shows that the functional differences observed between CD16 : 7 chimeras ( C - E ) are not caused by differential surface expression .

O O O O O O O O O O O O O O O O O O O O O O O O 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) Surface expression levels of CD16:7 chimeras. Procedures and data expression were as in Figure 2E. The figure shows that the functional differences observed between CD16:7 chimeras (C-E) are not caused by differential surface expression.


( A ) Endocytic vesicles containing aggregated CD16 : 7 - 263 - 281 become labelled with GFP - LC3 . JAR cells were transfected with vectors expressing the indicated CD16 : 7 chimeras and GFP - LC3A , and subjected to anti ‐ CD16 aggregation for 8 h before staining for the endocytosed chimera ( red ) . Representative confocal pictures are shown . The inset highlights a vesicle where the peripheral GFP - LC3 staining is particularly distinguishable . The right panel displays control WBs showing that chimera aggregation for 8 h activates HA - LC3A conversion in JAR cells .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Endocytic vesicles containing aggregated CD16:7-263-281 become labelled with GFP-LC3. JAR cells were transfected with vectors expressing the indicated CD16:7 chimeras and GFP-LC3A, and subjected to anti‐CD16 aggregation for 8 h before staining for the endocytosed chimera (red). Representative confocal pictures are shown. The inset highlights a vesicle where the peripheral GFP-LC3 staining is particularly distinguishable. The right panel displays control WBs showing that chimera aggregation for 8 h activates HA-LC3A conversion in JAR cells.


( B ) Colocalization of endocytosed CD16 : 7 - 263 - 281 with EEA1 and CD63 . JAR cells were transfected with CD16 : 7 - 263 - 281 and subjected to aggregation before staining them for the endocytosed chimera ( red ) and EEA1 or CD63 ( green ; FITC ‐ coupled primary antibodies ) , as indicated . Representative confocal microscopy pictures are shown .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Colocalization of endocytosed CD16:7-263-281 with EEA1 and CD63. JAR cells were transfected with CD16:7-263-281 and subjected to aggregation before staining them for the endocytosed chimera (red) and EEA1 or CD63 (green; FITC‐coupled primary antibodies), as indicated. Representative confocal microscopy pictures are shown.


( C ) Immunoelectron microscopy assay showing that endocytosed CD16 : 7 - 263 - 281 localizes to LC3 − labelled , single − membrane vesicles . JAR cells were transfected with the CD16 : 7 - 263 - 281 chimera and a construct expressing human IgG1 fused to LC3A . Cells were subjected to anti − CD16 aggregation for 8 h and processed for immunoelectron microscopy . Thick gold signal ( 18 nm ) : aggregated , endocytosed chimera ; thin gold signal ( 12 nm ) : IgG1 - LC3 . Arrows indicate single membrane ( black ) or IgG1 - LC3A ( white ) . Scale bar : 400 nm .

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

(C) Immunoelectron microscopy assay showing that endocytosed CD16:7-263-281 localizes to LC3−labelled, single−membrane vesicles. JAR cells were transfected with the CD16:7-263-281 chimera and a construct expressing human IgG1 fused to LC3A. Cells were subjected to anti−CD16 aggregation for 8 h and processed for immunoelectron microscopy. Thick gold signal (18 nm): aggregated, endocytosed chimera; thin gold signal (12 nm): IgG1-LC3. Arrows indicate single membrane (black) or IgG1-LC3A (white). Scale bar: 400 nm.


( D ) Lysosomal inhibition increases HA - LC3II levels promoted by CD16 : 7 - 263 - 281 . 293 cells were transfected with the indicated chimera and HA - LC3A , and subjected to anti − CD16 aggregation in the absence or presence of bafilomycin ( 200 nM , added 4 h post aggregation ) before lysing them for western blotting against the indicated molecules .

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

(D) Lysosomal inhibition increases HA-LC3II levels promoted by CD16:7-263-281. 293 cells were transfected with the indicated chimera and HA-LC3A, and subjected to anti−CD16 aggregation in the absence or presence of bafilomycin (200 nM, added 4 h post aggregation) before lysing them for western blotting against the indicated molecules.


( E ) Aggregation of CD16 : 7 - 263 - 281 promotes its own degradation . 293 cells were transfected with the indicated constructs , aggregated and lysed for western blotting . Shown are overexposed anti ‐ CD16 WBs .

(E) Aggregation of CD16:7-263-281 promotes its own degradation. 293 cells were transfected with the indicated constructs, aggregated and lysed for western blotting. Shown are overexposed anti‐CD16 WBs.


( F ) Degradation of CD16 : 7 - 263 - 281 is inhibited by ATG5 depletion . 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16 : 7 constructs , aggregated and lysed for anti ‐ CD16 western blotting ( left panel ) . The right panel displays control WBs showing ATG5 depletion . Source data for this figure is available on the online supplementary information page .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Degradation of CD16:7-263-281 is inhibited by ATG5 depletion. 293 cells were transfected with the indicated siRNAs and subsequently with the shown CD16:7 constructs, aggregated and lysed for anti‐CD16 western blotting (left panel). The right panel displays control WBs showing ATG5 depletion.Source data for this figure is available on the online supplementary information page.


( A ) Identification of critical amino acids for HA - LC3 conversion . 293 cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and HA - LC3A , subjected to anti ‐ CD16 aggregation and lysed for western blotting against the indicated molecules . Asterisks mark mutations with reduced activity . Shown is one representative experiment of six repetitions .

(A) Identification of critical amino acids for HA-LC3 conversion. 293 cells were transfected with the indicated CD16:7-263-281 mutants and HA-LC3A, subjected to anti‐CD16 aggregation and lysed for western blotting against the indicated molecules. Asterisks mark mutations with reduced activity. Shown is one representative experiment of six repetitions.


( B ) Identification of critical amino acids for GFP - LC3 activation and colocalization with the endocytosed chimera . JAR cells were transfected with the indicated CD16 : 7 - 263 - 281 mutants and GFP - LC3A , aggregated and stained for the endocytosed chimera ( red ) . Preparations were scored by blindly counting the number of red vesicles ( chimera ) and green vesicles ( GFP - LC3A ) per cell , as well as the number of red vesicles colocalizing with green ones . The percentage of green vesicles colocalizing with red ones was close to 100 % for all mutants , that is , virtually no GFP - LC3A vesicles were unrelated to endocytosed chimera ( not shown ) . At least 50 cells were scored per experimental point . The experiment was repeated three times . The graph shows the number of GFP - LC3A vesicles per cell expressed as the percentage of the value obtained for the wild − type chimera ( left axis ) , and the percentage of chimera vesicles labelled with GFP - LC3A ( right axis ) . Data are expressed as means ± s . d . of the triplicates . Asterisks indicate significant differences with respect to wild − type values ( paired Student ' s t − test ; P < 0 . 01 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Identification of critical amino acids for GFP-LC3 activation and colocalization with the endocytosed chimera. JAR cells were transfected with the indicated CD16:7-263-281 mutants and GFP-LC3A, aggregated and stained for the endocytosed chimera (red). Preparations were scored by blindly counting the number of red vesicles (chimera) and green vesicles (GFP-LC3A) per cell, as well as the number of red vesicles colocalizing with green ones. The percentage of green vesicles colocalizing with red ones was close to 100% for all mutants, that is, virtually no GFP-LC3A vesicles were unrelated to endocytosed chimera (not shown). At least 50 cells were scored per experimental point. The experiment was repeated three times. The graph shows the number of GFP-LC3A vesicles per cell expressed as the percentage of the value obtained for the wild−type chimera (left axis), and the percentage of chimera vesicles labelled with GFP-LC3A (right axis). Data are expressed as means ±s.d. of the triplicates. Asterisks indicate significant differences with respect to wild−type values (paired Student's t−test; P<0.01).