( E ) Lengths of A - C linkers in wild - type ( cw92 ) , bld12 , bld10bld12 : : ∆ N3 , and bld10bld12 : : ∆ C2 centrioles . Centrioles containing nine triplets and eight triplets were separately analyzed . n , number of A - Clinkers measured . ( F ) Angles between adjacent triplets in centrioles analyzed in panel € . The angle ( θ ) measured was defined as indicated in the schematic diagram ; the angle between the line connecting the centers of B - tubules of the two adjacent triplets ( black line ) and the line connecting the centers of A - and C - tubules of the triplet of the two ( brown line ) . * , P < 0 . 01 . n , number of triplet angles measured . Data information : Data were analyzed by unpaired t - test . The graphs show 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Lengths of A-C linkers in wild-type (cw92), bld12, bld10bld12::∆N3, and bld10bld12::∆C2 centrioles. Centrioles containing nine triplets and eight triplets were separately analyzed. n, number of A-Clinkers measured. (F) Angles between adjacent triplets in centrioles analyzed in panel €. The angle (θ) measured was defined as indicated in the schematic diagram; the angle between the line connecting the centers of B-tubules of the two adjacent triplets (black line) and the line connecting the centers of A- and C-tubules of the triplet of the two (brown line). *, P<0.01. n, number of triplet angles measured. Data information: Data were analyzed by unpaired t-test. The graphs show mean ± SEM.


( A ) LC - MS / MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA . Signal intensities of the modifications are scaled to that for m1A , the ribonucleoside with the highest MS

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

(A) LC-MS/MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA. Signal intensities of the modifications are scaled to that for m1A, the ribonucleoside with the highest MS signal


( A ) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC - MS / MS in total tRNA extracted from parasites across the IDC time course . The average fold - change values ( relative to an arbitrary average control ) were subjected to hierarchical clustering analysis ( log2 - transformed data ) . The scale of the heat map has saturated the fold - change values of some modifications . Refer to Appendix Table S6 . ( B ) Three groups of modifications show differential regulation during the course of IDC . The majority ( 22 ) increase synchronously across the IDC ( blue ) , while 4 show asynchronous or irregular behavior ( purple ) and 2 ( orange ) are uniquely up - regulated in the ring stage . Log2Fold - change values plotted in the three graphs represent mean ± SEM ( N = 3 ) . Differences between the highest value ( denoted as peak ) and the lowest value ( denoted as trough ) were subjected to a two - tailed Student ' s t - test : NS , not

O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC-MS/MS in total tRNA extracted from parasites across the IDC time course. The average fold-change values (relative to an arbitrary average control) were subjected to hierarchical clustering analysis (log2-transformed data). The scale of the heat map has saturated the fold-change values of some modifications. Refer to Appendix Table S6. (B) Three groups of modifications show differential regulation during the course of IDC. The majority (22) increase synchronously across the IDC (blue), while 4 show asynchronous or irregular behavior (purple) and 2 (orange) are uniquely up-regulated in the ring stage. Log2Fold-change values plotted in the three graphs represent mean ± SEM (N = 3). Differences between the highest value (denoted as peak) and the lowest value (denoted as trough) were subjected to a two-tailed Student's t-test: NS, not significant


iTRAQ proteomics analysis ( Appendix Fig . S2 ) resulted in 2 , 100 proteins consistently quantified at every point across the IDC . The heat map shows hierarchical clustering analysis of fold - changes ( log2 - transformed ) in the levels of the proteins relative to an arbitrary average across the IDC . The 100 most up - regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P . falciparum 3D7 . The significantly ( P < 0 . 05 ) enriched pathways among the up - regulated proteins at each time point are summarized in tables for each time

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

iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point


LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of the unmodified ( 3055 . 425 Da ) and modified ( 3143 . 43 Da ) oligos for Glu - tRNAGAA (

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

LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (B


Collision - induced dissociation of the parent molecular ions for m / z 1046 . 477 - 3 from Glu - tRNAGA produced expected - c , - w and - y ions for the sequences CU [ mcm5s2U ]

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

Collision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCC


LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of th unmodified ( 1835 . 20 Da ) and modified ( 1923 . 262 Da ) oligos for Gln - tRNACAA ( E )

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

LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E)


Collision - induced dissociation of the parent molecular ions fo m / z 1923 . 262 from Gln - tRNACAA produced expected - c , - w and - y ions for the sequence CC [ mcm5s2U ]

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

Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAA


Ratios of modified oligonucleotides from the anticodon stem - loop o ( H ) GluGA isoacceptors expressed as percentages of their sum

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

Ratios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum total


Ratios of modified oligonucleotides from the anticodon stem - loop o ( G )

Ratios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCA


Ratios of modified oligonucleotides from the anticodon stem - loop o ( I ) GlyGGA isoacceptors expressed as percentages of their sum

Ratios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total


( A , B ) Comparison of arbitrary translation efficiency ( TE ) values of ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) proteins across all experimental time points . The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system . The top 5 % up - regulated proteins ( n = 100 ) at ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) were used for this analysis . Differences among all groups were first tested by one - way ANOVA analysis , followed by Dunnett ' s test as multiple comparison post - test to find significant differences between pairs of ( A ) TP 40 or ( B ) TP 46 and other time points . P < 0 . 01 is denoted as * * . P < 0 . 0001 is denoted as * * * * . Scatterplots of mRNA and protein fold - changes ( log - transformed ) at time points ( C ) 40 h and ( D ) 46 h in the erythrocytic cycle . Quadrants are designated with roman numerals . Data points falling into quadrant I are designated as high translationally efficient ( HTE ) proteins while those falling into quadrant IV are designated low translationally efficient ( LTE )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Comparison of arbitrary translation efficiency (TE) values of (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) proteins across all experimental time points. The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system. The top 5% up-regulated proteins (n = 100) at (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) were used for this analysis. Differences among all groups were first tested by one-way ANOVA analysis, followed by Dunnett's test as multiple comparison post-test to find significant differences between pairs of (A) TP 40 or (B) TP 46 and other time points. P<0.01 is denoted as **. P<0.0001 is denoted as ****. Scatterplots of mRNA and protein fold-changes (log-transformed) at time points (C) 40 h and (D) 46 h in the erythrocytic cycle. Quadrants are designated with roman numerals. Data points falling into quadrant I are designated as high translationally efficient (HTE) proteins while those falling into quadrant IV are designated low translationally efficient (LTE) proteins


( C ) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study . The relevant markers are listed to the left of the micrographs . Scale bar : 5 µm . ( D ) Pie charts showing the proportion of specific phenotype mutants ( SPMs ) that have one or more distinct aberrant phenotypes , and non - phenotype - specific mutants for each of the compartments screened .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Example micrographs of yeast cells for each of the 21 subcellular endocytic phenotypes identified in this study. The relevant markers are listed to the left of the micrographs. Scale bar: 5 µm. (D) Pie charts showing the proportion of specific phenotype mutants (SPMs) that have one or more distinct aberrant phenotypes, and non-phenotype-specific mutants for each of the compartments screened.


Representative images of wild - type and mutant cells organized by marker and phenotype ( labels on the left of each panel ) . For each phenotype , three cells labelled for the training set ( labelled single cells ) and cells identified by the 2NN classifier ( identified single cells ) are shown . The table to the right of the images shows ( from left to right ) : [ 1 ] the occurrence of each phenotype in a wild - type population ( % in WT ) ; [ 2 ] the number of specific phenotype mutants ( all ) and stringent specific phenotype mutants ( str ) for each of the 17 mutant morphologies ; [ 3 ] the most significantly enriched GO Slim biological process and ; [ 4 ] the most significantly enriched protein complex . # denotes term below statistical significance .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of wild-type and mutant cells organized by marker and phenotype (labels on the left of each panel). For each phenotype, three cells labelled for the training set (labelled single cells) and cells identified by the 2NN classifier (identified single cells) are shown. The table to the right of the images shows (from left to right): [1] the occurrence of each phenotype in a wild-type population (% in WT); [2] the number of specific phenotype mutants (all) and stringent specific phenotype mutants (str) for each of the 17 mutant morphologies; [3] the most significantly enriched GO Slim biological process and; [4] the most significantly enriched protein complex. # denotes term below statistical significance.


Endocytic patch formation dynamics in the ydl176wΔ ( ipf1Δ ) strain . Patch dynamics were examined using time - lapse fluorescence microscopy of wild - type ( WT ) and ipf1Δ deletion strains carrying reporters for the coat ( Sla1 - GFP ; green ) , and actin ( Sac6 - tdTomato ; red ) modules . Left : Representative kymographs for the WT and ipf1Δ strains . Right : Box plot illustrating the distribution of lifetimes of Sla1 - GFP and Sac6 - tdTomato patches . Significance was determined using unpaired t - tests . Scale bar : 10 seconds .

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

Endocytic patch formation dynamics in the ydl176wΔ (ipf1Δ) strain. Patch dynamics were examined using time-lapse fluorescence microscopy of wild-type (WT) and ipf1Δ deletion strains carrying reporters for the coat (Sla1-GFP; green), and actin (Sac6-tdTomato; red) modules. Left: Representative kymographs for the WT and ipf1Δ strains. Right: Box plot illustrating the distribution of lifetimes of Sla1-GFP and Sac6-tdTomato patches. Significance was determined using unpaired t-tests. Scale bar: 10 seconds.


Penetrance as a function of replicative age . Top : Bar graph showing the fraction of outliers in populations of increasing replicative age ( # of divisions ) for wild - type ( WT ) , and 5 mutant strains ( rrd2Δ , rpl20bΔ , cka2Δ , vac8Δ and vac17Δ ) . Data are presented as mean of 3 biological replicates + / - SD . Bottom : Box plot with the distribution of cell sizes for the same populations of cells . Whiskers extend to the 5th and 95th percentile .

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

Penetrance as a function of replicative age. Top: Bar graph showing the fraction of outliers in populations of increasing replicative age (# of divisions) for wild-type (WT), and 5 mutant strains (rrd2Δ, rpl20bΔ, cka2Δ, vac8Δ and vac17Δ). Data are presented as mean of 3 biological replicates +/- SD. Bottom: Box plot with the distribution of cell sizes for the same populations of cells. Whiskers extend to the 5th and 95th percentile.


Micrographs of young ( top row of images ) and older ( bottom row of images ) wild - type ( WT ) cells expressing Vph1 - EGFP ( green vacuole ) and stained with CF640R WGA ( magenta bud scars ) . Scale bar : 5 µm .

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

Micrographs of young (top row of images) and older (bottom row of images) wild-type (WT) cells expressing Vph1-EGFP (green vacuole) and stained with CF640R WGA (magenta bud scars). Scale bar: 5 µm.


Combined effect of replicative age and a vacuole inheritance defect on penetrance . Micrographs of wild - type and vac17Δ cells expressing Vph1 - EGFP ( green vacuole ) and Hta2 - mCherry ( red nucleus ) , stained with CF640R WGA ( magenta bud scars ) . Cells with increasing bud - scar staining ( replicative age ) are shown from left to right . Scale bar : 5 µm .

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

Combined effect of replicative age and a vacuole inheritance defect on penetrance. Micrographs of wild-type and vac17Δ cells expressing Vph1-EGFP (green vacuole) and Hta2-mCherry (red nucleus), stained with CF640R WGA (magenta bud scars). Cells with increasing bud-scar staining (replicative age) are shown from left to right. Scale bar: 5 µm.


Relationship between stress response and penetrance . Single - cell UPRE - RFP levels were measured in ~ 60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology . Cells were binned into equal - sized bins , from low to high stress response , assessed as outlier or inlier , and clustered based on their penetrance profile ( composed from the fraction of outliers in each stress - response bin ) . Each line plot represents a penetrance profile . * denotes the cluster with a profile most similar to wild - type . Insert pie charts show the proportion of mutant strains in each cluster .

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

Relationship between stress response and penetrance. Single-cell UPRE-RFP levels were measured in ~60 different mutant strains that we had identified as penetrance mutants with intermediate penetrance with defects in actin or vacuole morphology. Cells were binned into equal-sized bins, from low to high stress response, assessed as outlier or inlier, and clustered based on their penetrance profile (composed from the fraction of outliers in each stress-response bin). Each line plot represents a penetrance profile. * denotes the cluster with a profile most similar to wild-type. Insert pie charts show the proportion of mutant strains in each cluster.


( A ) C - circle assay for tumor samples . The presence of C - circles was tested in 22 osteosarcomas by Rolling Circle Amplification ( RCA ) assay using Φ29 DNA polymerase . Φ29 DNA polymerase negative controls and Φ29 DNA polymerase - based reactions starting respectively with 75 , 37 . 5 , and 18 . 75 ng of DNA were applied to dot blots and C - circles were detected by hybridization with a 32P - ( CCCTAA ) 4 telomeric probe . U2OS and HeLa cells correspond to positive and negative control samples , 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 O O O O O O O O O O O O O O O O O O O

(A) C-circle assay for tumor samples. The presence of C-circles was tested in 22 osteosarcomas by Rolling Circle Amplification (RCA) assay using Φ29 DNA polymerase. Φ29 DNA polymerase negative controls and Φ29 DNA polymerase-based reactions starting respectively with 75, 37.5, and 18.75 ng of DNA were applied to dot blots and C-circles were detected by hybridization with a 32P-(CCCTAA)4 telomeric probe. U2OS and HeLa cells correspond to positive and negative control samples, respectively.


( B ) Representative images of telomere FISH ( red ) and PML IF ( green ) colocalizations ( APBs ) in osteosarcoma tumor samples . DNA is counterstained blue with DAPI . APBs are indicated by white arrows . Scale bars are 5 μm .

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

(B) Representative images of telomere FISH (red) and PML IF (green) colocalizations (APBs) in osteosarcoma tumor samples. DNA is counterstained blue with DAPI. APBs are indicated by white arrows. Scale bars are 5 μm.


( C ) Representative image of TeSLA Southern Blot of two representative samples of ALT - positive ( left ) and telomerase - positive ( right ) osteosarcomas . Nine TeSLA PCRs ( 30 pg each reaction ) were done for each DNA sample . DIG - labelled MW ladder has been added a posteriori . ( D ) Plot of cumulative TeSLA fragment sizes ( in kb ) in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Points and error bars ( ± SEM ) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs ( n = 9 per tumor sample ) from all the patients in both groups ( n = 16 in ALT - positive group , and n = 6 in ALT - negative group ) . The Kolmogorov - Smirnov test was applied to identify statistical differences in TeSLA fragments distributions ( p = 0 . 0016 ) . ( E ) Individual TeSLa fragment lengths in ALT - positive ( n = 16 ) and ALT - negative ( n = 6 ) osteosarcomas . Each dot represents a TeSLA fragment , bars represent the median value for individual patient ' s 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative image of TeSLA Southern Blot of two representative samples of ALT-positive (left) and telomerase-positive (right) osteosarcomas. Nine TeSLA PCRs (30 pg each reaction) were done for each DNA sample. DIG-labelled MW ladder has been added a posteriori. (D) Plot of cumulative TeSLA fragment sizes (in kb) in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Points and error bars (± SEM) represent cumulative percentage of TeSLA fragments from all the TeSLA PCRs (n=9 per tumor sample) from all the patients in both groups (n=16 in ALT-positive group, and n=6 in ALT-negative group). The Kolmogorov-Smirnov test was applied to identify statistical differences in TeSLA fragments distributions (p=0.0016). (E) Individual TeSLa fragment lengths in ALT-positive (n=16) and ALT-negative (n=6) osteosarcomas. Each dot represents a TeSLA fragment, bars represent the median value for individual patient's samples.


( A ) Oncoprint graph showing the distribution of mutations ( green rectangles ) and copy number variations ( red rectangle , amplification ; blue rectangle , copy loss ) in all samples . Tumors are distributed according to ALT - positivity ( ALT + ) or - negativity ( ALT - ) and ATRX status ( ATRX - mutated or ATRX - wt ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Oncoprint graph showing the distribution of mutations (green rectangles) and copy number variations (red rectangle, amplification; blue rectangle, copy loss) in all samples. Tumors are distributed according to ALT-positivity (ALT+) or -negativity (ALT-) and ATRX status (ATRX-mutated or ATRX-wt).


( B ) Tissue sections of two representative high - grade osteosarcomas with HES ( hematoxylin eosin saffron ) staining ( left panel ) , anti - ATRX ( HPA064684 ) immunochemistry ( middle panel ) , and anti - DAXX ( HPA008736 ) immunochemistry ( right panel ) , showing ATRX protein expression in two ALT - positive samples . In the top panel , intratumoral ATRX expression is negative ( positive control osteoclasts are shown ) . In the bottom panel , intratumoral ATRX expression is high . Scale bars are 250 μm .

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

(B) Tissue sections of two representative high-grade osteosarcomas with HES (hematoxylin eosin saffron) staining (left panel), anti-ATRX (HPA064684) immunochemistry (middle panel), and anti-DAXX (HPA008736) immunochemistry (right panel), showing ATRX protein expression in two ALT-positive samples. In the top panel, intratumoral ATRX expression is negative (positive control osteoclasts are shown). In the bottom panel, intratumoral ATRX expression is high. Scale bars are 250 μm.


( A ) Copy - number - alteration ( CNA ) profiles of osteosarcomas . Representation of the GISTIC analysis with false discovery rate ( FDR ( q - value ) ) for all cases ( n = 22 ; left panel ) , ALT - positive / ATRX - wt cases ( n = 11 ; middle panel ) , and ALT - positive / ATRX - mutated cases ( n = 5 ; right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Copy-number-alteration (CNA) profiles of osteosarcomas. Representation of the GISTIC analysis with false discovery rate (FDR (q-value)) for all cases (n=22; left panel), ALT-positive/ATRX-wt cases (n=11; middle panel), and ALT-positive/ATRX-mutated cases (n=5; right panel).


( B ) Gain / amplification of TOP3A gene region ( 17p11 ) is a genomic signature of ATRX - wt osteosarcomas . For each ATRX - mutated ( top part ) and ATRX - wt ( bottom part ) case , from the left to the right , the chromosome 17 and regional genomic profiles were established , with CGH Analytics software . A focus was made within the genomic interval ( 17 . 25 - 19 . 11 Mb ) of the short arm of chromosome 17 ( hg38 human genome mapping ; Build 38 from NCBI , December 2013 version ) including TOP3 . Color profiles corresponding to the different tumors are defined at the top of each group . Most ATRX - wt cases show gain or amplification of this region ( bottom part ) , whereas no ATRX - mutated cases display this alteration ( top part ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gain/amplification of TOP3A gene region (17p11) is a genomic signature of ATRX-wt osteosarcomas. For each ATRX-mutated (top part) and ATRX-wt (bottom part) case, from the left to the right, the chromosome 17 and regional genomic profiles were established, with CGH Analytics software. A focus was made within the genomic interval (17.25-19.11 Mb) of the short arm of chromosome 17 (hg38 human genome mapping; Build 38 from NCBI, December 2013 version) including TOP3. Color profiles corresponding to the different tumors are defined at the top of each group. Most ATRX-wt cases show gain or amplification of this region (bottom part), whereas no ATRX-mutated cases display this alteration (top part).


( C ) Chromosome 17 copy number variation analysis showing amplification / gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX - wt tumors ( left panel ) , and amplification of the TOP3A region for which TP53 region is at the edge of the amplification / gain in ALT - positive ATRX - wt tumors ( right panel ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Chromosome 17 copy number variation analysis showing amplification/gain of the TOP3A region that do not extend into the TP53 gene in ALT ATRX-wt tumors (left panel), and amplification of the TOP3A region for which TP53 region is at the edge of the amplification/gain in ALT-positive ATRX-wt tumors (right panel).


( A ) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT - positive vs . ALT - negative samples , showing upregulated ( in red ) and downregulated ( in green ) genes . Twelve tumors had sufficient RNA quality to be analyzed : seven ALT - positive ATRX - wt , two ALT - positive / ATRX - mutated and three ALT - negative .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Volcano plot of the supervised analysis of mRNA expression profiles of osteosarcomas according to ALT-positive vs. ALT-negative samples, showing upregulated (in red) and downregulated (in green) genes. Twelve tumors had sufficient RNA quality to be analyzed: seven ALT-positive ATRX-wt, two ALT-positive/ATRX-mutated and three ALT-negative.


( B ) TOP3A gene expression according to ALT and ATRX status : ALT + / ATRX - wt ( n = 7 ) , ALT + / ATRX - mut ( n = 2 ) and ALT - ( n = 3 ) . Box - and - whisker plot were defined with default parameters by median value ( central band at the 50th percentile ) , interquartile ranges ( IQR , box limited by 25th and 75th percentile ) and whisker boundaries defined by minimum and maximum value . An ANOVA statistical test , and Tukey ' s range test were used to compare modalities .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) TOP3A gene expression according to ALT and ATRX status: ALT+/ATRX-wt (n=7), ALT+/ATRX-mut (n=2) and ALT- (n=3). Box-and-whisker plot were defined with default parameters by median value (central band at the 50th percentile), interquartile ranges (IQR, box limited by 25th and 75th percentile) and whisker boundaries defined by minimum and maximum value. An ANOVA statistical test, and Tukey's range test were used to compare modalities.


( A ) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control .

(A) Western blot showing ATRX protein expression in nine osteosarcoma cell lines and HTC116 as positive control.


( B ) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control .

(B) Western blot showing TOP3A protein expression in nine osteosarcoma cell lines and NY siTOP3A as control.


( D ) C - circle assay showing inverse effect of ATRX ectopic expression on C - circle levels in ATRX - mutated and ATRX - wt cell lines . Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test .

(D) C-circle assay showing inverse effect of ATRX ectopic expression on C-circle levels in ATRX-mutated and ATRX-wt cell lines. Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test.


( E ) C - circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments , n . s . = non - significant , * p < 0 . 05 , Mann - Whitney test . Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) C-circle assay showing rescue of ATRX ectopic expression by TOP3A overexpression in U2OS osteosarcoma cells Error bars represent the mean ± SEM from n = 2 experiments, n.s. = non-significant, *p < 0.05, Mann-Whitney test. Western blot and quantification of TOP3A expression is shown for U2OS cells overexpressing TOP3A.


( B ) Representative images of telomeric DNA ( green ) and PML protein ( red ) colocalizations ( APBs ) in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD . Scale bars are 5 μm . ( C ) Quantification of APB frequency in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . , non - significant ; Mann - Whitney test . ( D ) Effect of TOP3A KD on Telomere Dysfunction - Induced Foci ( TIFs ) in osteosarcoma and in vitro - immortalized cell lines ; Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , * p < 0 . 05 , * * p < 0 . 005 ; Mann - Whitney 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 images of telomeric DNA (green) and PML protein (red) colocalizations (APBs) in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD. Scale bars are 5 μm. (C) Quantification of APB frequency in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s., non-significant; Mann-Whitney test. (D) Effect of TOP3A KD on Telomere Dysfunction-Induced Foci (TIFs) in osteosarcoma and in vitro-immortalized cell lines; Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, *p < 0.05, **p < 0.005; Mann-Whitney test.


( E ) Distributions of TeSLA fragments in osteosarcoma and in vitro - immortalized ATRX - mutated or ATRX - wt cell lines according to TOP3A KD ( left panel ; each dot represents a TeSLA fragment ) , and representative TeSLA Southern Blot image for NY ( right panel ) .

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

(E) Distributions of TeSLA fragments in osteosarcoma and in vitro-immortalized ATRX-mutated or ATRX-wt cell lines according to TOP3A KD (left panel; each dot represents a TeSLA fragment), and representative TeSLA Southern Blot image for NY (right panel).


( A ) FISH - IF of telomeres ( in red ) and BLM ( in green ) in TOP3A - depleted cells BLM foci at telomeres are indicated by white arrows . Error bars represent the mean ± SEM from n = 3 experiments , n = 150 cells scored per treatment , n . s . = non - significant , * * p < 0 . 01 , Mann - Whitney test . Scale bars are 5 μm .

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

(A) FISH-IF of telomeres (in red) and BLM (in green) in TOP3A-depleted cells BLM foci at telomeres are indicated by white arrows. Error bars represent the mean ± SEM from n = 3 experiments, n = 150 cells scored per treatment, n.s. = non-significant, **p < 0.01, Mann-Whitney test. Scale bars are 5 μm.


( B ) ATSA assay assessing ALT - mediated telomeric DNA synthesis ( telomere in red , EdU in purple ) in PML ( in green ) according to TOP3A KD . EdU signal at APB ( telomere / PML ) are indicated by white arrows . Approximately 150 cells were divided into five groups ( 0 , 1 - 2 , 3 - 4 , 5 - 6n and ≥ 7 ) based on the number of EdU + APBs . Scale bars are 5 μm .

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

(B) ATSA assay assessing ALT-mediated telomeric DNA synthesis (telomere in red, EdU in purple) in PML (in green) according to TOP3A KD. EdU signal at APB (telomere/PML) are indicated by white arrows. Approximately 150 cells were divided into five groups (0,1-2, 3-4, 5-6n and ≥7) based on the number of EdU+APBs. Scale bars are 5 μm.


( A - D ) Primary cultures from D . melanogaster embryos expressing GFP - Atg8a under the control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted line . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout the muscle cytoplasm and nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in the formation of small GFP - Atg8a labeled punctae . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in the accumulation of large GFP - Atg8a labeled vesicles that localize around the nucleus and between the myofibers . ( D ) Addition of both CQ and Rap for 12hrs , triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone . ( E ) Knockdown of the core autophagy gene , Atg1 , prevents the formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) Quantification of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . Error bars indicate SEM with * * p < . 01 ; * * * p < . 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the muscle cytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the muscles than either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.


( A - D ) Primary cultures from D . melanogaster embryos expressing GFP - Atg8a under the control of Dmef2 - Gal4 , stained with Phalloidin ( actin ) in red . Inset are magnifications of areas denoted by dotted line . ( A ) In untreated control cultures , GFP - Atg8a localizes throughout the musclecytoplasm and nuclei . ( B ) Addition of rapamycin ( Rap ) for 12 hrs results in the formation of small GFP - Atg8a labeled punctae . ( C ) Addition of chloroquine ( CQ ) for 12 hrs results in the accumulation of large GFP - Atg8a labeled vesicles that localize around the nucleus and between the myofibers . ( D ) Addition of both CQ and Rap for 12hrs , triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone . ( E ) Knockdown of the core autophagy gene , Atg1 , prevents the formation of GFP - Atg8a vesicles in CQ + Rap treated cultures . ( F ) Quantification of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . Error bars indicate SEM with * * p < . 01 ; * * * p < . 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 B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-D) Primary cultures from D. melanogaster embryos expressing GFP-Atg8a under the control of Dmef2-Gal4, stained with Phalloidin (actin) in red. Inset are magnifications of areas denoted by dotted line. (A) In untreated control cultures, GFP-Atg8a localizes throughout the musclecytoplasm and nuclei. (B) Addition of rapamycin (Rap) for 12 hrs results in the formation of small GFP-Atg8a labeled punctae. (C) Addition of chloroquine (CQ) for 12 hrs results in the accumulation of large GFP-Atg8a labeled vesicles that localize around the nucleus and between the myofibers. (D) Addition of both CQ and Rap for 12hrs, triggers the formation of greater numbers of enlarged vesicles in the musclesthan either drug alone. (E) Knockdown of the core autophagy gene, Atg1, prevents the formation of GFP-Atg8a vesicles in CQ+Rap treated cultures. (F) Quantification of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). Error bars indicate SEM with **p<.01; ***p<.001.


( E ) Control white RNAi muscles starved and treated with CQ accumulate GFP - Atg8a punctae , but very few colocalize with ubiquitin ( Ubi ) . ( F ) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP - Atg8a punctae . The remainder colocalize with ubiquitin . ( G ) Quantification of the mean GFP - Atg8a punctae area and Mander ' s overlap coefficient for GFP - Atg8a and Ubi for genotypes shown in E and F . Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals , with p - values for Rpn1 RNAi relative to white RNAi control ( * * * p < . 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

(E) Control white RNAi muscles starved and treated with CQ accumulate GFP-Atg8a punctae, but very few colocalize with ubiquitin (Ubi). (F) Knockdown of Rpn1 in starved + CQ muscle strongly inhibits the formation of GFP-Atg8a punctae. The remainder colocalize with ubiquitin. (G) Quantification of the mean GFP-Atg8a punctae area and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in E and F. Error bars indicate SEM for n = 8 ventral longitudinal muscles from individual animals, with p-values for Rpn1 RNAi relative to white RNAi control (***p<.001).


( H , I ) Autophagosome number is reduced , but autophagic flux is not significantly altered in starved Rpn1 vs . white RNAi muscles expressing GFP - mCherry - Atg8 . ( J ) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP - mCherry ( n = 6 ) . SEM is indicated .

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

(H, I) Autophagosome number is reduced, but autophagic flux is not significantly altered in starved Rpn1 vs. white RNAi muscles expressing GFP-mCherry-Atg8. (J) Flux measurement is shown on the right as the ratio of mCherry punctae to GFP-mCherry (n = 6). SEM is indicated.


( K ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 ; UAS - RNAi animals . In fed white RNAi control , endogenous Atg8a appears as a single band ( Atg8a - I ) . 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band ( Atg8a - II ) . Knockdown of Atg1 or Rpn1 inhibits Atg8a modification , indicating reduced autophagy levels .

(K) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4; UAS-RNAi animals. In fed white RNAi control, endogenous Atg8a appears as a single band (Atg8a-I). 6 hrs starvation induces lipid modification of Atg8a and the appearance of a second band (Atg8a-II). Knockdown of Atg1 or Rpn1 inhibits Atg8a modification, indicating reduced autophagy levels.


( A - D ) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant ( DTS ) . Dmef2 - Gal4 , UAS - GFP - Atg8a control and UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection . 18 hrs post - shift ( PS ) , both control ( A ) and DTS muscles ( B ) accumulate GFP - Atg8a punctae , but the latter also accumulate ubiquitin ( Ubi ) aggregates . 48 hrs PS , control muscles ( C ) maintain strong autophagy induction , but DTS mutant muscles ( D ) have reduced GFP - Atg8a punctae accumulation , similar to the Rpn1 knockdown phenotype . ( E ) Quantification of the mean GFP - Atg8a punctae and Mander ' s overlap coefficient for GFP - Atg8a and Ubi for genotypes shown in A - D ( SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p - values of DTS relative to control ) . Dark green = Dmef2 - Gal4 , UAS - GFP - Atg8a ; Light green = UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a .

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

(A-D) Inhibition of the proteasome by overexpression of a temperature sensitive proteasome mutant (DTS). Dmef2-Gal4, UAS-GFP-Atg8a control and UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and animals were shifted to the restrictive temperature then starved and CQ treated 6 hrs prior to dissection. 18 hrs post-shift (PS), both control (A) and DTS muscles (B) accumulate GFP-Atg8a punctae, but the latter also accumulate ubiquitin (Ubi) aggregates. 48 hrs PS, control muscles (C) maintain strong autophagy induction, but DTS mutant muscles (D) have reduced GFP-Atg8a punctae accumulation, similar to the Rpn1 knockdown phenotype. (E) Quantification of the mean GFP-Atg8a punctae and Mander's overlap coefficient for GFP-Atg8a and Ubi for genotypes shown in A-D (SEM is indicated for n = 8 ventral longitudinal muscles from individual animals with p-values of DTS relative to control). Dark green = Dmef2-Gal4, UAS-GFP-Atg8a; Light green = UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a.


( F ) Western blot analysis of endogenous Atg8a from third instar muscle lysate . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - Gal4 control at both 18 hrs and 48 hrs PS , but not in DTS at 48 hrs PS .

O O O O O O O O O O O O O O O O O O O O O O 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) Western blot analysis of endogenous Atg8a from third instar muscle lysate. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-Gal4 control at both 18 hrs and 48 hrs PS, but not in DTS at 48 hrs PS.


( A - C ) Time course of effect of temperature shift on aggregates , autophagosomes , and larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a animals starved 6 hrs prior to analysis . ( A ) The number of ubiquitin aggregates peaks at 18 - 24 hrs PS , and decreases significantly from 18 to 48 hrs PS ( n = 10 and p - values shown relative to 18 hrs PS ) . ( B ) The number of GFP - Atg8a punctae peaks at 12 - 24 hrs PS and decreases significantly from 18 to 48 hrs PS ( n = 10 and p - values shown relative to 18 hrs PS ) . ( C ) Larval locomotion , measured as the time to crawl 3cm , gradually and significantly worsens from 18 to 48 hrs PS ( n = 6 and p - values shown relative to 0 hrs PS .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-C) Time course of effect of temperature shift on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals starved 6 hrs prior to analysis. (A) The number of ubiquitin aggregates peaks at 18-24 hrs PS, and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (B) The number of GFP-Atg8a punctae peaks at 12-24 hrs PS and decreases significantly from 18 to 48 hrs PS (n = 10 and p-values shown relative to 18 hrs PS). (C) Larval locomotion, measured as the time to crawl 3cm, gradually and significantly worsens from 18 to 48 hrs PS (n = 6 and p-values shown relative to 0 hrs PS.


( D - E ) tyramine ( Tyr ) treatment reduces GFP - Atg8a punctae in larval muscle . UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a animals were placed on 100mg / ml Tyr ( D ) or control food ( E ) , shifted to restrictive temperature for 18hrs , and starved 6 hrs prior to dissection .

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

(D-E) tyramine (Tyr) treatment reduces GFP-Atg8a punctae in larval muscle. UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a animals were placed on 100mg/ml Tyr (D) or control food (E), shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection.


( F - H ) Effect of Tyr treatment on aggregates , autophagosomes , and larval locomotion in UAS - DTS ; Dmef2 - Gal4 , UAS - GFP - Atg8a and Dmef2 - Gal4 , UAS - GFP - Atg8a animals , shifted to restrictive temperature for 18hrs , and starved 6 hrs prior to dissection . Aggregate ( F ) and GFP - Atg8a punctae ( G ) numbers in muscles are significantly reduced with 100 mg / ml Tyr treatment in DTS but not control animals 18 hrs post - shift ( PS ) . ( H ) 100 mg / ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS . n = 12 and p - values shown relative to non - Tyr treated animals . For all measurements , SEM is shown with * p < . 05 ; * * p < . 01 ; * * * p < . 001 .

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

(F-H) Effect of Tyr treatment on aggregates, autophagosomes, and larval locomotion in UAS-DTS; Dmef2-Gal4, UAS-GFP-Atg8a and Dmef2-Gal4, UAS-GFP-Atg8a animals, shifted to restrictive temperature for 18hrs, and starved 6 hrs prior to dissection. Aggregate (F) and GFP-Atg8a punctae (G) numbers in muscles are significantly reduced with 100 mg/ml Tyr treatment in DTS but not control animals 18 hrs post-shift (PS). (H) 100 mg/ml Tyr treatment significantly worsens larval locomotion in both control and DTS animals 18 hrs PS. n = 12 and p-values shown relative to non-Tyr treated animals. For all measurements, SEM is shown with *p<.05; **p<.01; ***p<.001.


( I ) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS . 6 hrs starvation induces lipidated Atg8a - II in Dmef2 - G4 control with or without 100 mg / ml Tyr . After 6hrs starvation , Atg8a - II levels are reduced in DTS + 100 mg / ml Tyr relative to untreated DTS .

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

(I) Western blot analysis of endogenous Atg8a from third instar muscle lysate 18 hrs PS. 6 hrs starvation induces lipidated Atg8a-II in Dmef2-G4 control with or without 100 mg/ml Tyr. After 6hrs starvation, Atg8a-II levels are reduced in DTS + 100 mg/ml Tyr relative to untreated DTS.


( D - H ) Dmef2 - Gal4 drives expression of UAS - GFP - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . RNAi knockdown of CG41099 ( E ) , CG7112 ( F ) , CG31935 ( G ) , and Rab3 - GAP ( H ) , blocks the formation of GFP - Atg8a labeled autophagosomes compared to knockdown of white ( 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 O O O O O O O O O O O O O O O O O O O O O O O O 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-H)Dmef2-Gal4 drives expression of UAS-GFP-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. RNAi knockdown of CG41099 (E), CG7112 (F), CG31935 (G), and Rab3-GAP (H), blocks the formation of GFP-Atg8a labeled autophagosomes compared to knockdown of white (D).


( I - M ) Dmef2 - Gal4 drives expression of UAS - myc - Atg8a in animals starved on low nutrient food for 6 hrs + 2 . 5 mg / ml CQ for 6 hrs . The effect of the RAB3GAPs is not via Rab3 or Rab5 function , as a Rab3 null mutation ( J ) , expression of a constitutive active form of Rab3 ( Q80L ) ( K ) or Rab5 ( Q88L ) ( L ) has no effect on muscle autophagy compared to control ( I ) . Expression of a constitutive active form of Rab39 ( Q69L ) ( M ) reduces myc - Atg8a labeled vesicles . ( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 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

(I-M) Dmef2-Gal4 drives expression of UAS-myc-Atg8a in animals starved on low nutrient food for 6 hrs + 2.5 mg/ml CQ for 6 hrs. The effect of the RAB3GAPs is not via Rab3 or Rab5 function, as a Rab3 null mutation (J), expression of a constitutive active form of Rab3 (Q80L) (K) or Rab5 (Q88L) (L) has no effect on muscle autophagy compared to control (I). Expression of a constitutive active form of Rab39 (Q69L) (M) reduces myc-Atg8a labeled vesicles. (N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.


( N ) Quantification of autophagy phenotypes from panels D - M . SEM is indicated , with n = 8 ventral longitudinal muscles from individual animals and * * p < . 01 ; * * * p < . 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

(N) Quantification of autophagy phenotypes from panels D-M. SEM is indicated, with n = 8 ventral longitudinal muscles from individual animals and **p<.01; ***p<.001.


( O ) Atg8a and Rab3 - GAP colocalize to autophagosomes . Dmef2 - Gal4 drives expression of UAS - myc - Atg8a and UAS - CFP - Rab3 - GAP in animals starved and treated with CQ for 6 hrs . In all panels green = GFP - Atg8a and red = myc - Atg8a , and blue = CFP - Rab3Gap2 .

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

(O) Atg8a and Rab3-GAP colocalize to autophagosomes. Dmef2-Gal4 drives expression of UAS-myc-Atg8a and UAS-CFP-Rab3-GAP in animals starved and treated with CQ for 6 hrs. In all panels green = GFP-Atg8a and red = myc-Atg8a, and blue = CFP-Rab3Gap2.


( P ) Anti - Atg8a Western blot of third instar muscle lysate from Dmef2 - Gal4 driving the RNAi or overexpression constructs shown in D - N . Total levels of endogenous Atg8a are variable , but only knockdown of CG41099 , CG7112 , CG31935 , Rab3 - GAP , or overexpression of Rab39 ( Q69L ) block induction of lipidated Atg8a - II following 6 hrs starvation .

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

(P) Anti-Atg8a Western blot of third instar muscle lysate from Dmef2-Gal4 driving the RNAi or overexpression constructs shown in D-N. Total levels of endogenous Atg8a are variable, but only knockdown of CG41099, CG7112, CG31935, Rab3-GAP, or overexpression of Rab39 (Q69L) block induction of lipidated Atg8a-II following 6 hrs starvation.


( A ) Primary cultured muscles with control lacZ RNAi , treated for 12 hrs with Rap + CQ . Note the formation of large GFP - Atg8a vesicles throughout the muscles . ( B ) GFP - Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi , treated for 12 hrs with Rap + CQ . Knockdown of Pfk ( C ) or CG9485 ( D ) causes increased accumulation of GFP - Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap + CQ . ( E ) Quantification for A - D of the mean ratio of GFP - Atg8a punctae area to total muscle area per well ( n = 4 ) . SEM is indicated with * p < . 05 ; * * p < . 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

(A) Primary cultured muscles with control lacZ RNAi, treated for 12 hrs with Rap+CQ. Note the formation of large GFP-Atg8a vesicles throughout the muscles. (B) GFP-Atg8a vesicles are reduced in primary cultured muscles with GlyS RNAi, treated for 12 hrs with Rap+CQ. Knockdown of Pfk (C) or CG9485 (D) causes increased accumulation of GFP-Atg8a vesicles in primary cultured muscles treated for 12 hrs with Rap+CQ. (E) Quantification for A-D of the mean ratio of GFP-Atg8a punctae area to total muscle area per well (n = 4). SEM is indicated with *p<.05; **p<.01.


( F ) Control white RNAi muscles accumulate large autophagosomes at 1 mg / ml CQ . ( G ) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle . ( H ) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle . ( I ) Quantification of the GFP - Atg8a punctae area in muscles from white , CG9485 , and AGBE knockdowns . n = 6 larval muscles with SEM indicated and * p < . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Control white RNAi muscles accumulate large autophagosomes at 1 mg/ml CQ. (G) RNAi knockdown of CG9485 increases autophagosome accumulation in the muscle. (H) RNAi knockdown of AGBE reduces autophagic vesicle accumulation in the muscle. (I) Quantification of the GFP-Atg8a punctae area in muscles from white, CG9485, and AGBE knockdowns.n = 6 larval muscles with SEM indicated and *p<.05.


( J ) Western blot analysis of endogenous Atg8a from third instar muscle lysate . Dmef2 - Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation , indicating reduced autophagy levels .

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

(J) Western blot analysis of endogenous Atg8a from third instar muscle lysate. Dmef2-Gal4 driven knockdown of AGBE but not CG9485 inhibits Atg8a lipidation, indicating reduced autophagy levels.


A H & E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID - 19 patients who presented with gastrointestinal ( GI ) symptoms . The inflammatory infiltrates were indicated by a yellow arrow . The edema area was indicated by a red star . Scale bars , 100 µm . B The bar chart shows the degrees of intestinal inflammation in patients with COVID - 19 . Number of samples for each group as indicated . Data information : All data are shown as 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 O O O O O O O O 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 H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm. B The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated. Data information: All data are shown as mean ± SD.


C Cytokine levels in the plasma of COVID - 19 patients with ( n = 12 ) or without ( n = 6 ) GI symptoms by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test .

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

C Cytokine levels in the plasma of COVID-19 patients with (n=12) or without (n=6) GI symptoms by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


D Levels of plasma VEGF in COVID - 19 patients with ( n = 8 ) or without ( n = 10 ) disease progression by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test .

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

D Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=10) disease progression by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


E Temporal course of plasma VEGF at the early and late stage of COVID - 19 infection by cytokine assay . Data shown are the levels of plasma VEGF in patients with ( n = 8 ) and without ( n = 10 ) disease progression at the early stage ( one to three days after laboratory - confirmed for COVID - 19 ) and late stage ( more than three days after laboratory - confirmed for COVID - 19 ) .

E Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n=8) and without (n=10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19).


F Levels of plasma VEGF in COVID - 19 patients with ( n = 7 ) or without ( n = 6 ) interstitial vasodilation by cytokine assay . G Levels of plasma VEGF in COVID - 19 patients with ( n = 8 ) or without ( n = 5 ) interstitial edema by cytokine assay . Data information : All data are shown as mean ± SD . P values are determined by Student ' s t - test .

F Levels of plasma VEGF in COVID-19 patients with (n=7) or without (n=6) interstitial vasodilation by cytokine assay. G Levels of plasma VEGF in COVID-19 patients with (n=8) or without (n=5) interstitial edema by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student's t-test.


A Levels of VEGF - A mRNA in the intestinal tissues from COVID - 19 patients ( n = 5 ) or healthy controls ( n = 5 ) by RNA - seq . Data information : All data are shown as mean ± SD . For ( A ) , P values are determined by Student ' s t -

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

A Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n=5) or healthy controls (n=5) by RNA-seq. Data information: All data are shown as mean ± SD. For (A), P values are determined by Student's t-test


C , D Representative H & E images ( C ) and the quantitative analysis of inflammation ( D ) of the intestinal tissues from animals treated by the protocol shown in ( B ) . The inflammatory infiltrates were indicated by a yellow arrow . Scale bar , 100 µm . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) .

C, D Representative H&E images (C) and the quantitative analysis of inflammation (D) of the intestinal tissues from animals treated by the protocol shown in (B). The inflammatory infiltrates were indicated by a yellow arrow. Scale bar, 100 µm. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).


E qRT - PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 5 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney 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

E qRT-PCR analysis of levels of inflammatory factors in duodenum tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 5; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test.


F ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control - Fc or Spike RBD - Fc . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . For ( F ) P values are determined by Student ' s t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ELISA analysis of VEGF concentration in the intestinal tissues of mice treated with Control-Fc or Spike RBD-Fc. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (F) P values are determined by Student's t-test


G ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily . Control - Fc , n = 4 ; Spike RBD - Fc , n = 6 . n , biologically independent samples ( mice ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 ELISA analysis of VEGF concentration in the plasma of mice after six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily. Control-Fc, n = 4; Spike RBD-Fc, n = 6. n, biologically independent samples (mice).


H Evaluation of murine peritoneal permeability six hours after treatment with Control - Fc or Spike RBD - Fc , or seven days after treated with Control - Fc or Spike RBD - Fc daily by TRITC - dextran dye extravasation assay . For each group , n = 8 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . For ( H ) , P values are determined by Student ' s t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Evaluation of murine peritoneal permeability six hours after treatment with Control-Fc or Spike RBD-Fc, or seven days after treated with Control-Fc or Spike RBD-Fc daily by TRITC-dextran dye extravasation assay. For each group, n = 8. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. For (H), P values are determined by Student's t-test


A qRT - PCR analysis of levels of VEGF mRNA in HUVEC treated with Control - Fc or Spike RBD - Fc . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t -

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

A qRT-PCR analysis of levels of VEGF mRNA in HUVEC treated with Control-Fc or Spike RBD-Fc. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test


B ELISA analysis of VEGF concentration in the supernatant from Caco - 2 cells treated with Control - Fc or Spike - Fc . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : All data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t - test ;

B ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc or Spike-Fc. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test;


C Immunofluorescence analysis of VEGF , cytokeratin ( CK ) and CD34 protein in the intestinal tissues of COVID - 19 patients . CK and CD34 staining in green , VEGF staining in red and nuclear staining in blue . Scale bars , 50 µm .

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

C Immunofluorescence analysis of VEGF, cytokeratin (CK) and CD34 protein in the intestinal tissues of COVID-19 patients. CK and CD34 staining in green, VEGF staining in red and nuclear staining in blue. Scale bars, 50 µm.


D Levels of ERK and pERK in Caco - 2 cells treated with Control - Fc or Spike RBD - Fc by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( D ) , P values are determined by Paired Student ' s t -

D Levels of ERK and pERK in Caco-2 cells treated with Control-Fc or Spike RBD-Fc by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (D), P values are determined by Paired Student's t-test


E ELISA analysis of VEGF concentration in the supernatant from Caco - 2 cells treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 . For each group , n = 4 . Data are from 4 independent biological experiments . Data information : All data are shown as mean ± SD . ; for ( E ) , P values are determined by Mann - Whitney 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 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

E ELISA analysis of VEGF concentration in the supernatant from Caco-2 cells treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984. For each group, n=4. Data are from 4 independent biological experiments. Data information: All data are shown as mean ± SD. ; for (E), P values are determined by Mann-Whitney test;


F , G Immunohistochemical staining shows ERK ( E ) and pERK ( F ) expression in the duodenum of COVID - 19 patients and healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : All data are shown as mean ± SD . For ( F ) and ( G ) , P values are determined by Student ' s t -

F, G Immunohistochemical staining shows ERK (E) and pERK (F) expression in the duodenum of COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. For (F) and (G), P values are determined by Student's t-test


H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 . Scale bar , 100 µm . For each group , n = 4 . Data information : All data are shown as mean ± SD . for ( H ) P values are determined by one - way ANOVA .

O O O O O O O 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

H Immunohistochemical staining shows ERK and pERK expression in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984. Scale bar, 100 µm. For each group, n = 4. Data information: All data are shown as mean ± SD. for (H) P values are determined by one-way ANOVA.


I The levels of Ras , c - Raf , pMEK , ERK , pERK and p - P90RSK in Caco - 2 cells treated with Control - Fc , Spike RBD - Fc or Spike RBD - Fc combined with SCH772984 by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . for ( I ) , P values are determined by one - way ANOVA .

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

I The levels of Ras, c-Raf, pMEK, ERK, pERK and p-P90RSK in Caco-2 cells treated with Control-Fc, Spike RBD-Fc or Spike RBD-Fc combined with SCH772984 by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. for (I), P values are determined by one-way ANOVA.


A The normalized fluorescence intensity of TRITC - Dextran dye from coculture systems in which Caco - 2 cells were incubated with Control - Fc or Spike RBD - Fc after 24 hours . For each group , n = 3 . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( A ) P values are determined by Paired Student ' s t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 normalized fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc or Spike RBD-Fc after 24 hours. For each group, n=3. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (A) P values are determined by Paired Student's t-test


B The expression of junctional proteins ZO - 1 , VE - cadherin ( VE - cad ) , phosphorylated VE - cad ( pVE - cad ; 658 ) and pVE - cad ( 731 ) in HUVECs co - cultured with the supernatant of Caco - 2 cells that were treated with either Control - Fc or Spike RBD - Fc detected by Western blot . Protein expression was normalized to β - actin . Data are from 3 independent biological experiments . Data information : All data are shown as mean ± SD . For ( B ) , P values are determined by Paired Student ' s t -

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 expression of junctional proteins ZO-1, VE-cadherin (VE-cad), phosphorylated VE-cad (pVE-cad; 658) and pVE-cad (731) in HUVECs co-cultured with the supernatant of Caco-2 cells that were treated with either Control-Fc or Spike RBD-Fc detected by Western blot. Protein expression was normalized to β-actin. Data are from 3 independent biological experiments. Data information: All data are shown as mean ± SD. For (B), P values are determined by Paired Student's t-test


C , D Immunohistochemical staining shows levels of VE - cad ( C ) and pVE - Cad ( 731 ) ( D ) in the duodenum tissue from COVID - 19 patients and healthy controls . Scale bars , 100 μm . For each group , n = 7 . n , biologically independent samples ( human specimen ) . Data information : All data are shown as mean ± SD . for ( C ) and ( D ) , P values are determined by Student ' s t -

C, D Immunohistochemical staining shows levels of VE-cad (C) and pVE-Cad (731) (D) in the duodenum tissue from COVID-19 patients and healthy controls. Scale bars, 100 μm. For each group, n=7. n, biologically independent samples (human specimen). Data information: All data are shown as mean ± SD. for (C) and (D), P values are determined by Student's t-test


E Immunofluorescence analysis of VE - cad expression and subcellular localization in Caco - 2 cells that were treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab by confocal microscopy . VE - cad staining in green and nuclear staining in blue . Scale bar , 20 µm .

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

E Immunofluorescence analysis of VE-cad expression and subcellular localization in Caco-2 cells that were treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab by confocal microscopy. VE-cad staining in green and nuclear staining in blue. Scale bar, 20 µm.


F Fluorescence intensity of TRITC - Dextran dye from coculture systems in which Caco - 2 cells were incubated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Data are from five technical replicates with similar results from three biological replicates . Data information : All data are shown as mean ± SD . ; for ( F ) , P values are determined by one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O 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 Fluorescence intensity of TRITC-Dextran dye from coculture systems in which Caco-2 cells were incubated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Data are from five technical replicates with similar results from three biological replicates. Data information: All data are shown as mean ± SD. ; for (F) , P values are determined by one-way ANOVA.


G Immunohistochemical staining shows levels of VE - cad and pVE - Cad ( 731 ) in the intestinal tissues of mice treated with Control - Fc , Spike RBD - Fc , or Spike RBD - Fc combined with SCH772984 or Bevacizumab . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . for ( G ) , P values are determined by one - way ANOVA .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunohistochemical staining shows levels of VE-cad and pVE-Cad (731) in the intestinal tissues of mice treated with Control-Fc, Spike RBD-Fc, or Spike RBD-Fc combined with SCH772984 or Bevacizumab. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. for (G), P values are determined by one-way ANOVA.


A ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti - VEGF antibody . SCH772984 , ERK inhibitor ; Bevacizumab , anti - VEGF antibody . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA .

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 O O O O O O O O O O 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 ELISA analysis of VEGF concentration in intestinal tissues of mice treated with ERK inhibitor or anti-VEGF antibody. SCH772984, ERK inhibitor; Bevacizumab, anti-VEGF antibody. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay . For each group , n = 6 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA .

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

B Evaluation of intestinal permeability in animals treated with SCH772984 or Bevacizumab by Evans Blue dye extravasation assay. For each group, n = 6. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


C , D Representative H & E images ( C ) and the degrees of inflammation ( D ) of intestinal tissues from animals treated with SCH772984 or Bevacizumab . The inflammatory infiltrates were indicated by a yellow arrow . The edema area was indicated by a red star . Scale bar , 100 µm . For each group , n = 4 . n , biologically independent samples ( mice ) . Data information : All data are shown as mean ± SD . P values are determined by one - way ANOVA .

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

C, D Representative H&E images (C) and the degrees of inflammation (D) of intestinal tissues from animals treated with SCH772984 or Bevacizumab. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bar, 100 µm. For each group, n = 4. n, biologically independent samples (mice). Data information: All data are shown as mean ± SD. P values are determined by one-way ANOVA.


( A ) Electron micrograph of an area with abundant double - membrane spherules ( DMSs ) . DMVs ( asterisks ) are interspersed and surrounding the DMS cluster . ( B ) Slice through a tomogram ( left ) and corresponding surface - rendered model ( right ) of a representative area containing the different types of MERS - CoV - induced membrane modifications : DMSs ( orange ) , convoluted membranes ( CM , blue ) and DMVs ( yellow and lilac , outer and inner membranes ) . The model also highlights ER membranes ( green ) and a vesicle ( silver ) containing new virions ( pink ) . ( See also S1 Video ) . ( C ) Comparison of DMSs and virions ( arrowheads in left and right panels , respectively ) in enlarged views of tomographic slices from the regions boxed in ( B ) . The DMSs are similar in size but distinct in appearance from newly - formed MERS - CoV particles . ( D ) Whisker plots of the size distribution of DMSs ( n = 58 ) , virions ( n = 28 ) and DMVs ( n = 109 ) , as measured from the tomograms . DMSs and virions have a comparable size ( median diameter , 80 nm ) , while the median diameter of the DMVs is 247 nm . ( E ) Models and tomographic slices through an open ( left ) and closed ( right ) DMS . Both types of DMSs are connected with the CM . In open DMSs , both the inner and outer membranes ( dark blue and orange , respectively ) are continuous with CM . Two ~ 8 - nm apart slices through the reconstruction are shown . For closed DMSs only the outer membrane is connected to CM , while the inner membrane seems to define a closed compartment .

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

(A) Electron micrograph of an area with abundant double-membrane spherules (DMSs). DMVs (asterisks) are interspersed and surrounding the DMS cluster. (B) Slice through a tomogram (left) and corresponding surface-rendered model (right) of a representative area containing the different types of MERS-CoV-induced membrane modifications: DMSs (orange), convoluted membranes (CM, blue) and DMVs (yellow and lilac, outer and inner membranes). The model also highlights ER membranes (green) and a vesicle (silver) containing new virions (pink). (See also S1 Video). (C) Comparison of DMSs and virions (arrowheads in left and right panels, respectively) in enlarged views of tomographic slices from the regions boxed in (B). The DMSs are similar in size but distinct in appearance from newly-formed MERS-CoV particles. (D) Whisker plots of the size distribution of DMSs (n=58), virions (n=28) and DMVs (n=109), as measured from the tomograms. DMSs and virions have a comparable size (median diameter, 80 nm), while the median diameter of the DMVs is 247 nm. (E) Models and tomographic slices through an open (left) and closed (right) DMS. Both types of DMSs are connected with the CM. In open DMSs, both the inner and outer membranes (dark blue and orange, respectively) are continuous with CM. Two ~8-nm apart slices through the reconstruction are shown. For closed DMSs only the outer membrane is connected to CM, while the inner membrane seems to define a closed compartment.


( F ) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS - CoV RO , and of these with the ER . These include CM - ER ( black arrowheads ) , DMV - ER ( white arrowheads ) , CM - DMV ( dark grey arrowheads ) , and CM - DMS ( light grey arrowhead ) connections . Scale bars , 250 nm ( A , B ) , and 100 nm ( C - F ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Gallery of tomographic slices highlighting membrane connections between different elements of the MERS-CoV RO, and of these with the ER. These include CM-ER (black arrowheads), DMV-ER (white arrowheads), CM-DMV (dark grey arrowheads), and CM-DMS (light grey arrowhead) connections. Scale bars, 250 nm (A, B), and 100 nm (C -F).


alpha - CoVs . 2D - EM images from 100 - nm thick sections of different mammal cells infected with ( A ) SARS - CoV ( MOI 10 , 9 hpi ) , ( B ) MHV ( MOI 10 , 8 hpi ) and ( C ) HCoV - 229E ( MOI 5 , 24 hpi ) . Both beta - CoVs ( A , B ) and the alpha - CoV ( C ) induce membrane modifications that include not only DMVs ( asterisks ) and CM , but also DMSs ( white arrowheads ) . Scale bars , 250 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

alpha-CoVs.2D-EM images from 100-nm thick sections of different mammal cells infected with (A) SARS-CoV (MOI 10, 9 hpi), (B) MHV (MOI 10, 8 hpi) and (C) HCoV-229E (MOI 5, 24 hpi). Both beta-CoVs (A,B) and the alpha-CoV (C) induce membrane modifications that include not only DMVs (asterisks) and CM, but also DMSs (white arrowheads). Scale bars, 250 nm.


( A ) Overview of an infected cell in which regions with different virus - induced modifications are annotated in yellow ( DMVs ) , blue ( CM ) and orange ( DMSs ) . Several densely - labelled regions containing DMVs , but not the other virus - induced structures are apparent . A close - up of one of these regions ( boxed area ) is shown in ( B ) .

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

(A) Overview of an infected cell in which regions with different virus-induced modifications are annotated in yellow (DMVs), blue (CM) and orange (DMSs). Several densely-labelled regions containing DMVs, but not the other virus-induced structures are apparent. A close-up of one of these regions (boxed area) is shown in (B).


( C , D ) Distribution of the autoradiography signal around DMVs ( nDMVs = 36 , see Material and Methods for selection criteria and details ) . The data is plotted ( C ) as a histogram , or ( D ) normalized by the radius to the DMV centre to account for the increase in the screened area . Scale bars , ( A ) 5 μm , ( B ) 500 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

(C, D) Distribution of the autoradiography signal around DMVs (nDMVs = 36, see Material and Methods for selection criteria and details). The data is plotted (C) as a histogram, or (D) normalized by the radius to the DMV centre to account for the increase in the screened area. Scale bars, (A) 5 μm, (B) 500 nm.


( A ) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS - CoV - infected Vero E6 cells ( MOI 10 ) , as measured by scintillation counting on the RNA isolated from the cells . The label was provided simultaneously to all the samples at 6 hpi .

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

(A) Analysis of the amount of radioactive label incorporated into RNA as a function of the labelling time in SARS-CoV-infected Vero E6 cells (MOI 10), as measured by scintillation counting on the RNA isolated from the cells. The label was provided simultaneously to all the samples at 6 hpi.


( B - D ) EM detection by autoradiography . ( B ) Overview of a SARS - CoV - infected Vero E6 cell ( MOI 10 ) were labelled for 20 min just before fixation at 7 hpi , and then processed for EM autoradiography . Autoradiography grains accumulate in the RO regions . N , nucleus ; m , mitochondria ; VCR , virion - containing regions ; LD , lipid droplets . Scale bar , 1 μm .

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

(B-D) EM detection by autoradiography. (B) Overview of a SARS-CoV-infected Vero E6 cell (MOI 10) were labelled for 20 min just before fixation at 7 hpi, and then processed for EM autoradiography. Autoradiography grains accumulate in the RO regions. N, nucleus; m, mitochondria; VCR, virion-containing regions; LD, lipid droplets. Scale bar, 1 μm.


( C , D ) Quantification of the autoradiography signal per subcellular structure ( see also S1 Table ) . Labelling densities and relative labelling indexes ( RLI ) in different subcellular regions of ( C ) Vero E6 cells infected with SARS - CoV ( MOI 10 ) or ( D ) Huh7 cells infected with MERS - CoV ( MOI 5 ) . Radioactively - labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi , respectively . Control mock - infected cells are excluded from the RLI plots , as RLI comparisons between conditions require the same number of classes ( subcellular regions ) and these cells lack ROs and virions .

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

(C, D) Quantification of the autoradiography signal per subcellular structure (see also S1 Table). Labelling densities and relative labelling indexes (RLI) in different subcellular regions of (C) Vero E6 cells infected with SARS-CoV (MOI 10) or (D) Huh7 cells infected with MERS-CoV (MOI 5). Radioactively-labelled uridine was provided for the indicated periods of time immediately before fixation at 7 hpi and 12 hpi, respectively. Control mock-infected cells are excluded from the RLI plots, as RLI comparisons between conditions require the same number of classes (subcellular regions) and these cells lack ROs and virions.


( A ) Overview of a cluster of MERS - CoV - induced membrane modifications in Huh7 cells prepared as described in Fig . 4 . Some DMSs are boxed in orange while regions with CM are encircled in blue . In comparison with the densely - labelled surrounding DMVs , these regions are relatively devoid of autoradiography signal .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Overview of a cluster of MERS-CoV-induced membrane modifications in Huh7 cells prepared as described in Fig. 4. Some DMSs are boxed in orange while regions with CM are encircled in blue. In comparison with the densely-labelled surrounding DMVs, these regions are relatively devoid of autoradiography signal.


( B ) The distribution of autoradiography grains on CM was not homogeneous ( nCM = 9 ) , and label was predominantly found close to the boundaries of the CM , as expected if the signal arises from the surrounding DMVs .

O O O O O O O O O O O O O O O O O O O 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 distribution of autoradiography grains on CM was not homogeneous (nCM=9), and label was predominantly found close to the boundaries of the CM, as expected if the signal arises from the surrounding DMVs.


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( C ) Enlargements of the DMS areas boxed in ( A ) .

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

(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (C) Enlargements of the DMS areas boxed in (A).


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . Most DMSs were devoid of signal and those who contained label were close to labelled DMVs ( D ) ( nDMS = 105 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). Most DMSs were devoid of signal and those who contained label were close to labelled DMVs (D) (nDMS = 105).


( C - E ) Analysis of the label around / on the DMSs ( see Materials and Methods for selection criteria and details ) . ( E ) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre , as expected from a random distribution ( nDMSs = 54 ) . Scale bars , ( A ) 500 nm , ( C ) 100 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

(C-E) Analysis of the label around/on the DMSs (see Materials and Methods for selection criteria and details). (E) The distribution of signal around DMSs shows an increase in the amount of autoradiography grains with the distance from the DMS centre, as expected from a random distribution (nDMSs = 54). Scale bars, (A) 500 nm, (C) 100 nm.


IEM detection of viral markers in MERS - CoV - infected cells . ( A - G ) Immunogold labeling of thawed cryo - sections of MERS - CoV - infected Huh7 cells ( 12 hpi ) for the detection of the indicated viral proteins . ( A - C ) Structural proteins were detected on virions ( black arrowheads ) and , for the M and S proteins , also on Golgi cisterna . While regions containing DMS ( white arrowheads ) and CM labelled for the N protein ( D ) and nsp3 ( G ) , the M and S protein were not detected in these areas . ( H - I ) Immunogold labeling of dsRNA in HPF - FS samples of MERS - CoV - infected Huh7 cells ( 13 hpi ) . The label accumulated on DMVs , which could be easily detected in this type of samples ( grey arrowheads ) , while the regions with CM and DMSs , which appeared as dark areas among the DMV clusters , where devoid of dsRNA signal . G , Golgi complex ; m , mitochondria . Scale bars , 250 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 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 B-MEASURED_VAR O O O O O O O O O O O O O O O O O

IEM detection of viral markers in MERS-CoV-infected cells.(A-G) Immunogold labeling of thawed cryo-sections of MERS-CoV-infected Huh7 cells (12 hpi) for the detection of the indicated viral proteins. (A-C) Structural proteins were detected on virions (black arrowheads) and, for the M and S proteins, also on Golgi cisterna. While regions containing DMS (white arrowheads) and CM labelled for the N protein (D) and nsp3 (G), the M and S protein were not detected in these areas. (H-I) Immunogold labeling of dsRNA in HPF-FS samples of MERS-CoV-infected Huh7 cells (13 hpi). The label accumulated on DMVs, which could be easily detected in this type of samples (grey arrowheads), while the regions with CM and DMSs, which appeared as dark areas among the DMV clusters, where devoid of dsRNA signal. G, Golgi complex; m, mitochondria. Scale bars, 250 nm.