Figure 3 ( A ) Growth analysis of strains expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Growth curves and calculated doubling times from the exponential phase ( insert ) are presented . Cells were cultivated in media containing either glucose or glycerol as carbon source . ( B ) Determination of chronological lifespan via propidium iodide ( PI ) staining of cells described above . ( D , E ) Analysis of competitive fitness as described in ( C ) . Cor1WT strains harbored a chromosomally - integrated hygromycin selection cassette , whereas Cor1 * * contained a clonNAT selection cassette ( D ) . Subsequently , selection markers were switched as a control ( E ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(A) Growth analysis of strains expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Growth curves and calculated doubling times from the exponential phase (insert) are presented. Cells were cultivated in media containing either glucose or glycerol as carbon source.(B) Determination of chronological lifespan via propidium iodide (PI) staining of cells described above.(D,E) Analysis of competitive fitness as described in (C). Cor1WT strains harbored a chromosomally-integrated hygromycin selection cassette, whereas Cor1** contained a clonNAT selection cassette (D). Subsequently, selection markers were switched as a control (E).


Figure 4 ( A , B ) Spectrophotometric measurement of Cyt c oxidase ( CIV ; A ) and NADH Cyt c reductase ( CIII ; B ) activities in solubilized mitochondrial extracts . Mitochondria were isolated from cells expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . ( C , D ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH ( C ) or succinate + glycerol - 3 - phosphate ( G3P ; D ) in isolated coupled mitochondria of indicated strains . Measurement was performed in the absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP . ( E ) Respiratory control ratio calculated from polarographic measurements of KCN - sensitive oxygen consumption driven by NADH and succinate + glycerol - 3 - phosphate ( G3P ) in isolated coupled mitochondria of indicated strains . ( F , G ) Polarographic measurement of KCN - sensitive oxygen consumption driven by NADH in mitoplasts in the absence ( endog . Cyt c ) or presence ( + exog . Cyt c ) of exogenous oxidized Cyt c . NADH oxidation ( E ) and the calculated ratio + Cyt c / - Cyt c of substrate oxidation ( F ) are visualized .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(A,B) Spectrophotometric measurement of Cyt c oxidase (CIV; A) and NADH Cyt c reductase (CIII; B) activities in solubilized mitochondrial extracts. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**).(C,D) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH (C) or succinate + glycerol-3-phosphate (G3P; D) in isolated coupled mitochondria of indicated strains. Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.(E) Respiratory control ratio calculated from polarographic measurements of KCN-sensitive oxygen consumption driven by NADH and succinate + glycerol-3-phosphate (G3P) in isolated coupled mitochondria of indicated strains.(F,G) Polarographic measurement of KCN-sensitive oxygen consumption driven by NADH in mitoplasts in the absence (endog. Cyt c) or presence (+ exog. Cyt c) of exogenous oxidized Cyt c. NADH oxidation (E) and the calculated ratio +Cyt c/- Cyt c of substrate oxidation (F) are visualized.


Figure 5 ( A ) Polarographic measurement of KCN - sensitive oxygen consumption , driven by NADH in isolated coupled mitochondria . Mitochondria were isolated from cells expressing the wild type form of Cor1 ( Cor1WT ) or the mutant Cor1N63A , N187A , D192A , V189A , Y65A , L238A , K240A ( Cor1 * * ) . Thereby , strains either overexpressed cytochrome c ( Cyt cOE ) or harbored the empty plasmid as a control ( Empty ) . Measurement was performed in the absence ( basal respiration ) or presence ( phosphorylating condition ) of ADP . Analysis of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing glucose ( B ) as carbon source . Analysis of competitive fitness of strains described in ( A ) . Cor1WT strains harbored a chromosomally - integrated clonNAT selection cassette , whereas Cor1 * * contained a hygromycin selection cassette . Strains were cultivated in CM media containing galactose ( C ) or glycerol ( D ) as carbon source .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(A) Polarographic measurement of KCN-sensitive oxygen consumption, driven by NADH in isolated coupled mitochondria. Mitochondria were isolated from cells expressing the wild type form of Cor1 (Cor1WT) or the mutant Cor1N63A, N187A, D192A, V189A, Y65A, L238A, K240A (Cor1**). Thereby, strains either overexpressed cytochrome c (Cyt cOE) or harbored the empty plasmid as a control (Empty). Measurement was performed in the absence (basal respiration) or presence (phosphorylating condition) of ADP.Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing glucose (B) as carbon source.Analysis of competitive fitness of strains described in (A). Cor1WT strains harbored a chromosomally-integrated clonNAT selection cassette, whereas Cor1** contained a hygromycin selection cassette. Strains were cultivated in CM media containing galactose (C) or glycerol (D) as carbon source.


Figure 4D ) Proline biosynthesis protein levels in breast cancer subtypes ( Log2 ratios of light vs . SILAC standard ) . The upper and lower limits of the box show values from the first to the third quartile , with the horizontal line indicating the median . The whiskers extend 1 . 5 times the interquartile range from the edges of the box . Outlier values were plotted outside the whiskers . Significance was determined using paired Student ' s t - test and p values were corrected using permutation based FDR . * p < 0 . 05 and * * * p < 0 . 001 . E ) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study ( n = 3 ) ; Scale bar : 200 μm . F ) Quantification of IHC shows percentage of cells with 2 + and 3 + PYCR1 staining intensity . Data is expressed as mean ± SD of three patient samples . Paired samples were compared by a Student ' s t - test . * p < 0 . 05 and * * * p < 0 . 001 . G ) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data . Cox univariate p value , risk table and hazard ratio with 95 % CI indicated .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4D) Proline biosynthesis protein levels in breast cancer subtypes (Log2 ratios of light vs. SILAC standard). The upper and lower limits of the box show values from the first to the third quartile, with the horizontal line indicating the median. The whiskers extend 1.5 times the interquartile range from the edges of the box. Outlier values were plotted outside the whiskers. Significance was determined using paired Student's t-test and p values were corrected using permutation based FDR . *p < 0.05 and *** p < 0.001.E) High abundance of PYCR1 in the cancer tissue was validated by immunohistochemistry on matched tissue specimens from patients in this study (n=3); Scale bar: 200 μm.F) Quantification of IHC shows percentage of cells with 2+ and 3+ PYCR1 staining intensity. Data is expressed as mean ± SD of three patient samples. Paired samples were compared by a Student's t-test. *p < 0.05 and *** p < 0.001.G) Kaplan Meier survival curve for PYCR1 levels in residual cancer in the current proteomics data. Cox univariate p value, risk table and hazard ratio with 95% CI indicated.


Figure 5A ) Seahorse experiment measuring oxygen consumption rate ( OCR ) in MCF7 cells upon addition of oligomycin , FCCP and a mixture of antimycin and rotenone . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values were indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . B ) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . C ) 13C5 - Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate , malate and citrate in MCF7 cells . Data are presented as mean ± SD of triplicate samples . Isotopologues are represented as M + n where M indicates the mass and n equals number of 13C incorporatedD ) Seahorse experiment measuring extracellular acidification rate ( ECAR ) in MCF7 cells upon addition of glucose , oligomycin and 2 - deoxyglucose ( 2DG ) . Arrows indicate the points of addition of each inhibitor . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . E ) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements . Data are presented as mean ± SD of one representative experiment ( n = 4 wells ) . Similar results were obtained in three biological experiments . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * * p < 0 . 01 and * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . F ) Normalized lactate concentration upon PYCR1 KO . Data represents mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A) Seahorse experiment measuring oxygen consumption rate (OCR) in MCF7 cells upon addition of oligomycin, FCCP and a mixture of antimycin and rotenone. Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values were indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.B) Bar plot indicates average basal OCR and spare respiratory capacity in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.C) 13C5 -Glutamine derived carbon labeling patterns of TCA cycle intermediates fumarate, malate and citrate in MCF7 cells. Data are presented as mean ± SD of triplicate samples. Isotopologues are represented as M+n where M indicates the mass and n equals number of 13C incorporatedD) Seahorse experiment measuring extracellular acidification rate (ECAR) in MCF7 cells upon addition of glucose, oligomycin and 2-deoxyglucose (2DG). Arrows indicate the points of addition of each inhibitor. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.E) Bar plot indicates average glycolytic capacity and glycolytic reserve in seahorse measurements. Data are presented as mean ± SD of one representative experiment (n = 4 wells). Similar results were obtained in three biological experiments.Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows ** p < 0.01 and *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.F) Normalized lactate concentration upon PYCR1 KO. Data represents mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


Figure 6A ) Representative pictures of transwell migration and invasion after PYCR1 knockout . Scale bar : 100 μm . B ) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion . Samples were compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . C ) Representative images of colony formation in soft agar upon PYCR1 KO . Scale bar : 100 μm . D ) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours . Data represents mean ± SE of three biological experiments . Samples were compared using paired Student ' s t - test . E - H ) Bar plots show percentage of viable cells after treatment with 0 . 75 mM H2O2 ( E ) , 0 . 06 µM paclitaxel ( F ) , 0 . 15 µM doxorubicin ( G ) , 2 . 5 mM cyclophosphamide ( H ) . Data represent mean ± SE of three biological experiments . Samples are compared using Kruskal Wallis test followed by Dunnett ' s test for multiple pairwise comparisons . Corrected p values are indicated as follows * p < 0 . 05 . I ) Tumor volume measurements for 26 days in MCF7 injected NSG mice . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) are shown . Data represents mean ± SE . Pax : Paclitaxel , Dox : Doxorubicin . Groups were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * p < 0 . 05 , * * p < 0 . 01 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances . J ) Bar plot indicates mean ± SE of tumor volume measurements for day 26 . CRISPR control and PYCR1 KO tumors with ( n = 8 ) and without treatment ( n = 5 ) were compared by one way ANOVA followed by Tukey ' s multiple comparisons test for pairwise group comparisons . Corrected p - values are reported as follows * * * p < 0 . 001 . Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A) Representative pictures of transwell migration and invasion after PYCR1 knockout. Scale bar :100 μm. B) Bar plot represents mean ± SD of three biological replicates for transwell migration and invasion. Samples were compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p-values are reported as follows *** p < 0.001. C) Representative images of colony formation in soft agar upon PYCR1 KO. Scale bar: 100 μm.D) Growth measurements of MCF7 wild type and PYCR1 KO cells over 96 hours. Data represents mean ± SE of three biological experiments. Samples were compared using paired Student's t-test.E-H) Bar plots show percentage of viable cells after treatment with 0.75 mM H2O2 (E), 0.06 µM paclitaxel (F), 0.15 µM doxorubicin (G), 2.5 mM cyclophosphamide (H). Data represent mean ± SE of three biological experiments. Samples are compared using Kruskal Wallis test followed by Dunnett's test for multiple pairwise comparisons. Corrected p values are indicated as follows *p < 0.05.I) Tumor volume measurements for 26 days in MCF7 injected NSG mice. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) are shown. Data represents mean ± SE. Pax: Paclitaxel, Dox: Doxorubicin. Groups were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *p < 0.05, ** p < 0.01. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.J) Bar plot indicates mean ± SE of tumor volume measurements for day 26. CRISPR control and PYCR1 KO tumors with (n=8) and without treatment (n=5) were compared by one way ANOVA followed by Tukey's multiple comparisons test for pairwise group comparisons. Corrected p-values are reported as follows *** p < 0.001. Shapiro Wilk test was used to check data normality and Bartlett test was used to examine homogeneity of variances.


Figure 1 ( b ) Representative electron micrograph of a viroplasm ( V ) formed in NSP5 - EGFP cells infected with rotaviruses 8 hours post infection . Inset - double - layered particles emerging from the surface of a viroplasm . Scale bar = 300 nm .

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Figure 1(b) Representative electron micrograph of a viroplasm (V) formed in NSP5-EGFP cells infected with rotaviruses 8 hours post infection. Inset - double-layered particles emerging from the surface of a viroplasm. Scale bar = 300 nm.


Figure 2 ( a ) Fluorescence recovery after photobleaching ( FRAP ) of EGFP - tagged replication factories after 4 HPI ( early infection ) and 12 HPI ( late infection ) . Scale bar , 10 μm .

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Figure 2(a) Fluorescence recovery after photobleaching (FRAP) of EGFP-tagged replication factories after 4 HPI (early infection) and 12 HPI (late infection). Scale bar, 10 μm.


Figure 3 ( b ) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI . Left to right - 1 , 6 - hexanediol ( 1 , 6HD ) ; 1 , 3 - propylene diol ( 1 , 3PD ) ; 1 , 2 - propylene diol , or propylene glycol ( PG ) ; glycerol ( Gly ) ; polyethylene glycol 200 ( PEG200 ) . Top panels - before application and bottom panels - 1 min after application of these compounds ( 4 . 5 % v / v ) . Scale bar , 30 µm .

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Figure 3(b) Sensitivity of RV replication factories to aliphatic alcohols at 4 HPI. Left to right -1,6-hexanediol (1,6HD); 1,3-propylene diol (1,3PD); 1,2-propylene diol, or propylene glycol (PG); glycerol (Gly); polyethylene glycol 200 (PEG200). Top panels - before application and bottom panels - 1 min after application of these compounds (4.5% v/v). Scale bar, 30 µm.


Figure 4 ( a ) Recombinantly expressed , Atto647 - dye labelled NSP5 , Atto488 - dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase - separation properties . All labelled protein samples were mixed with the unlabeled NSP5 ( 1 : 10 molar ratio ) to minimise the effect of labelling on LLPS . Top panel , left to right : NSP5 - 647 / NSP2 - 488 droplets formed upon mixing of both proteins ( 10 μM each ) . Both channels are shown , along with an image of both channels overlaid . 4 % ( v / v ) 1 , 6 - hexanediol ( 1 , 6HD ) dissolves these condensates . Scale bar , 10 µm . Middle panel , left to right : NSP5 - 647 ( 50 μM ) + poly - arginine ( polyArg , 5 μΜ ) ; NSP5 - polyArg condensate + 1 , 6 - HD ; NSP5 - polyArg condensates + 0 . 5 M NaCl ; NSP5 + R9 peptide ( Arg - 9 , 5 μΜ ) . Bottom row , left to right : NSP5 - 647 sample ( 35 μM ) alone ; after addition of 10 % v / v PEG - 20K ; NSP2 - 488 ( 25 μM ) alone ; after addition of 10 % v / v PEG - 20K .

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Figure 4(a) Recombinantly expressed, Atto647-dye labelled NSP5, Atto488-dye labelled NSP2 and unlabeled NSP5 protein samples were used for investigating their phase-separation properties. All labelled protein samples were mixed with the unlabeled NSP5 (1:10 molar ratio) to minimise the effect of labelling on LLPS. Top panel, left to right: NSP5-647/NSP2-488 droplets formed upon mixing of both proteins (10 μM each). Both channels are shown, along with an image of both channels overlaid. 4% (v/v) 1,6-hexanediol (1,6HD) dissolves these condensates. Scale bar, 10 µm. Middle panel, left to right: NSP5-647 (50 μM) + poly-arginine (polyArg, 5 μΜ); NSP5-polyArg condensate + 1,6-HD; NSP5-polyArg condensates + 0.5 M NaCl; NSP5 + R9 peptide (Arg-9, 5 μΜ). Bottom row, left to right: NSP5-647 sample (35 μM) alone; after addition of 10% v/v PEG-20K; NSP2-488 (25 μM) alone; after addition of 10% v/v PEG-20K.


Figure 5Top panel , left to right : NSP5 - ∆ CTR labelled with Atto - 647 ( 25 μM ) incubated with unlabeled NSP2 ( 10 μΜ ) ; NSP5 - ∆ CTR in the presence of 10 % v / v PEG - 20K ; and with 5 μΜ of poly - arginine ( polyArg ) . Bottom panel : Atto - 647 - labelled NSP5 - ∆ CTR ( 5 μΜ ) incubated with unlabeled NSP5 ( 25 μΜ ) and Atto - 488 labelled NSP2 ( 10 μΜ ) . NSP5 / NSP2 droplets containing labelled NSP2 ( green ) also contain NSP5 - ∆ CTR - Atto647 ( magenta ) , shown along with an image of both 488 / 647 channels overlaid . Scale bar , 10 µm .

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Figure 5Top panel, left to right: NSP5-∆CTR labelled with Atto-647 (25 μM) incubated with unlabeled NSP2 (10 μΜ); NSP5-∆CTR in the presence of 10% v/v PEG-20K; and with 5 μΜ of poly-arginine (polyArg). Bottom panel: Atto-647-labelled NSP5-∆CTR (5 μΜ) incubated with unlabeled NSP5 (25 μΜ) and Atto-488 labelled NSP2 (10 μΜ). NSP5/NSP2 droplets containing labelled NSP2 (green) also contain NSP5-∆CTR-Atto647 (magenta), shown along with an image of both 488/647 channels overlaid. Scale bar, 10 µm.


Figure 6 ( a ) RV - infected MA - NSP5 - EGFP cells at 6 HPI . NSP5 - EGFP - tagged viral factories ( green ) are dissolved in the presence of 4 . 7 % ( v / v ) propylene glycol ( PG , middle ) . Viral RNA - protein condensates rapidly reform ( < 10 min ) after replacing the PG - containing cell culture medium ( PG removed , right ) . Seg3 ( magenta ) and Seg4 ( cyan ) transcripts are detected by smFISH , and colocalising Seg3 and Seg4 RNA signals ( white ) . Scale bars : 50 µm , zoomed - in regions : 10 µm . Effects of PG treatments on the viral production . PG - containing medium ( 2 % or 4 . 7 % PG ) , or standard cell culture medium , were applied to RV - infected cells ( MOI of 10 ) 2 HPI ( 1 hour after virus absorption ) . ( e ) Effects of PG treatments on virus production at different infection points . 4 . 7 % ( v / v ) PG was applied between 2 - 7 HPI ( ' early infection ' ) , and then diluted to 2 % between 7 - 11 HPI , or 4 . 7 % PG was applied between 7 - 11 HPI ( ' late infection ' ) , and diluted prior to harvesting the virus . Each group was compared to the control group ( 10 h treatment with 2 % PG ) . Each group was compared to the control group ( 10 h treatment with 2 % PG ) . Application of 4 . 7 % PG for 5 h significantly reduced virus replication ( P = 0 . 0016 ) between 2 - 7 HPI compared to the control group . PG treatment between 7 - 12 HPI did not significantly reduce the viral titre compared to the control group ( P = 0 . 1141 ) . Infectious titres represent mean ± SD values estimated for four independent biological replicates . Statistical analyses were performed using a Kruskal - Wallis test , followed by uncorrected Dunn ' s multiple comparisons test ( * * P < 0 . 002 ) . ( f , g ) 4 . 7 % PG treatment of RV - infected cells results in NSP5 dephosphorylation . ( f ) Western blot analysis of RV - infected cells harvested at 7 and 12 HPI , treated with 2 % or 4 . 7 % PG at different infection time points indicated in the figure . Multiple phosphorylated forms of NSP5 can be seen as higher MW bands . Treatment with 4 . 7 % PG reduces phosphorylation , which is not perturbed in the presence of 2 % PG . Treatments administered between 2 - 7 and 7 - 12 hpi reduce NSP5 phosphorylation , albeit to a considerably lower degree compared to the 2 - 12 hpi treatment . ( g ) Okadaic acid ( 5 μM ) applied to 4 . 7 % PG - treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6(a) RV-infected MA-NSP5-EGFP cells at 6 HPI. NSP5-EGFP-tagged viral factories (green) are dissolved in the presence of 4.7% (v/v) propylene glycol (PG, middle). Viral RNA-protein condensates rapidly reform (<10 min) after replacing the PG-containing cell culture medium (PG removed, right). Seg3 (magenta) and Seg4 (cyan) transcripts are detected by smFISH, and colocalising Seg3 and Seg4 RNA signals (white). Scale bars: 50 µm, zoomed-in regions: 10 µm.Effects of PG treatments on the viral production. PG-containing medium (2% or 4.7% PG), or standard cell culture medium, were applied to RV-infected cells (MOI of 10) 2 HPI (1 hour after virus absorption). (e) Effects of PG treatments on virus production at different infection points. 4.7% (v/v) PG was applied between 2-7 HPI ('early infection'), and then diluted to 2% between 7-11 HPI, or 4.7% PG was applied between 7-11 HPI ('late infection'), and diluted prior to harvesting the virus. Each group was compared to the control group (10 h treatment with 2% PG). Each group was compared to the control group (10 h treatment with 2% PG). Application of 4.7% PG for 5 h significantly reduced virus replication (P = 0.0016) between 2-7 HPI compared to the control group. PG treatment between 7-12 HPI did not significantly reduce the viral titre compared to the control group (P = 0.1141). Infectious titres represent mean±SD values estimated for four independent biological replicates. Statistical analyses were performed using a Kruskal-Wallis test, followed by uncorrected Dunn's multiple comparisons test (**P < 0.002).(f, g) 4.7% PG treatment of RV-infected cells results in NSP5 dephosphorylation. (f) Western blot analysis of RV-infected cells harvested at 7 and 12 HPI, treated with 2% or 4.7% PG at different infection time points indicated in the figure. Multiple phosphorylated forms of NSP5 can be seen as higher MW bands. Treatment with 4.7% PG reduces phosphorylation, which is not perturbed in the presence of 2% PG. Treatments administered between 2-7 and 7-12 hpi reduce NSP5 phosphorylation, albeit to a considerably lower degree compared to the 2-12 hpi treatment. (g) Okadaic acid (5 μM) applied to 4.7% PG-treated infected cells blocks NSP5 dephosphorylation restoring its phosphorylation pattern.


Figure 7 ( e ) 3D DNA - PAINT analysis of Seg3 RNA foci in RV - infected cells at 4 and 6 HPI . Scale bars , 2 µm ( left ) and 200 nm ( zoomed - in , right ) . ( f - g ) Distribution of calculated volumes and sphericities of the Seg3 RNA - containing granules in RV - infected cells at 4 HPI ( N = 704 ) and 6 hpi ( N = 698 ) , shown in ( e ) . Box plots represent the 25th / 75th interquartile range , with whiskers representing the 5th / 95th percentile values . Medians shown as central bands , and means shown as squares . Symbol ' x ' denotes 1 % and 99 % percentile values , and min and max values are shown as ' - ' . At the 0 , 001 level , the two distributions are significantly different between 4 and 6 hpi , assessed by the two - sample Kolmogorov - Smirnov test .

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Figure 7(e) 3D DNA-PAINT analysis of Seg3 RNA foci in RV-infected cells at 4 and 6 HPI. Scale bars, 2 µm (left) and 200 nm (zoomed-in, right). (f-g) Distribution of calculated volumes and sphericities of the Seg3 RNA-containing granules in RV-infected cells at 4 HPI (N=704) and 6 hpi (N=698), shown in (e). Box plots represent the 25th/75th interquartile range, with whiskers representing the 5th/95th percentile values. Medians shown as central bands, and means shown as squares. Symbol 'x' denotes 1% and 99% percentile values, and min and max values are shown as '-'. At the 0,001 level, the two distributions are significantly different between 4 and 6 hpi, assessed by the two-sample Kolmogorov-Smirnov test.


Figure 1C Frequency of C179 + or V5 - tag + 293T cells 24h after transfection of AAV - vector plasmids as measured by flow cytometry . Symbols represent single experiments and bars the mean ± SE ( n = 3 ) . D Immunoblot of 293T cells 72 h after transduction with AAV - vectors at a MOI of 106 . Antigen expression was detected with an anti - V5 - tag antibody . Equal loading was controlled with a GAPDH antibody ( n = 3 ) .

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Figure 1C Frequency of C179+ or V5-tag+ 293T cells 24h after transfection of AAV-vector plasmids as measured by flow cytometry. Symbols represent single experiments and bars the mean ± SE (n = 3).D Immunoblot of 293T cells 72 h after transduction with AAV-vectors at a MOI of 106. Antigen expression was detected with an anti-V5-tag antibody. Equal loading was controlled with a GAPDH antibody (n = 3).


Figure 2 Total IgG ELISA titers expressed as area under the curve ( AUC ) against homologous Cal / 7 / 9 virus of pre - immune and pre - challenge sera of individual animals of the indicated vaccine groups ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) . Background reactivity of pooled AAV - GFP pre - challenge serum is shown as dashed line . ELISAs were performed in technical duplicates . IgA ELISA titers in post - challenge lung homogenates against Cal / 7 / 9 of individual mice of the indicated vaccine groups ( AAV - HA , - cHA , - GFP , - NP , WIV n = 11 ) . of the Cal / 7 / 9 ( blue symbols ) and PR8 low dose ( red symbols ) challenge groups . ELISAs were performed in technical duplicates .

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Figure 2 Total IgG ELISA titers expressed as area under the curve (AUC) against homologous Cal/7/9 virus of pre-immune and pre-challenge sera of individual animals of the indicated vaccine groups (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11). Background reactivity of pooled AAV-GFP pre-challenge serum is shown as dashed line. ELISAs were performed in technical duplicates.  IgA ELISA titers in post-challenge lung homogenates against Cal/7/9 of individual mice of the indicated vaccine groups (AAV-HA, -cHA, -GFP, -NP, WIV n = 11). of the Cal/7/9 (blue symbols) and PR8 low dose (red symbols) challenge groups. ELISAs were performed in technical duplicates.


Figure 3 A Immunoblot analysis of purified H1N1 viruses A / California / 7 / 2009 ( Cal / 7 / 9 ) , A / Brevig Mission / 1 / 1918 ( BM / 1 / 1918 ) , A / Puerto Rico / 8 / 1934 ( PR8 ) or A / Brisbane / 59 / 2007 ( Bris / 59 / 7 ) separated under reducing and denaturing conditions . Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 ) as indicated ( n = 3 ) . C to F Binding of C179 ( C ) , or antibodies present in AAV - HA ( D ) , AAV - cHA ( E ) or WIV ( F ) pooled pre - challenge sera ( n = 18 mice per group ) to Cal / 7 / 9 or PR8 virus after incubation of the virions at pH = 7 . 2 , 5 . 8 , 5 . 4 , 5 . 0 , 4 . 4 or 4 . 4 + DTT to induce conformational changes in HA or remove the HA1 subdomain . Mean ± SD ( n = 3 , in technical triplicates ) . Statistical significance between pH = 7 . 2 and other conditions was determined using Kruskal - Wallis test with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) .

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

Figure 3 A Immunoblot analysis of purified H1N1 viruses A/California/7/2009 (Cal/7/9), A/Brevig Mission/1/1918 (BM/1/1918), A/Puerto Rico/8/1934 (PR8) or A/Brisbane/59/2007 (Bris/59/7) separated under reducing and denaturing conditions. Uncleaved HA0 and cleavage products HA1 and HA2 were detected with pooled pre-challenge sera (AAV-HA, -cHA, -GFP , WIV n = 18, AAV-NP n = 11) as indicated (n = 3). C to F Binding of C179 (C), or antibodies present in AAV-HA (D), AAV-cHA (E) or WIV (F) pooled pre-challenge sera (n = 18 mice per group) to Cal/7/9 or PR8 virus after incubation of the virions at pH = 7.2, 5.8, 5.4, 5.0, 4.4 or 4.4+DTT to induce conformational changes in HA or remove the HA1 subdomain. Mean ± SD (n = 3, in technical triplicates). Statistical significance between pH = 7.2 and other conditions was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05, **p<0.01).


Figure 4 A and B Influenza virus specific activation of the murine FcγRI , FcγRII , FcγRIII and FcγRIV by antibodies in pooled pre - challenge sera ( AAV - HA , - cHA , - GFP , WIV n = 18 , AAV - NP n = 11 mice per group ) . MDCKII cells were infected with Cal / 7 / 9 ( A ) or PR8 ( B ) before mouse serum and FcγR - expressing reporter cells were added . Influenza specific Fc - FcγR interaction induces the production of IL - 2 in the FcγR - expressing reporter cell , which was measured by anti - IL2 ELISA ( n = 3 , technical duplicates ) . Mean ± SE . C HA - specific activation of FcγR by AAV - HA , AAV - cHA or WIV pooled pre - challenge sera ( n = 18 mice per group ) . MDCKII cells were transfected with wildtype HA ( pAAV - HA ) or a stalk - only HA ( pAAV - mHL1 ) before mouse serum and FcγR - I - expressing reporter cells were added ( n = 2 , technical duplicates ) . Mean ± SE .

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Figure 4 A and B Influenza virus specific activation of the murine FcγRI, FcγRII, FcγRIII and FcγRIV by antibodies in pooled pre-challenge sera (AAV-HA, -cHA, -GFP, WIV n = 18, AAV-NP n = 11 mice per group). MDCKII cells were infected with Cal/7/9 (A) or PR8 (B) before mouse serum and FcγR-expressing reporter cells were added. Influenza specific Fc-FcγR interaction induces the production of IL-2 in the FcγR-expressing reporter cell, which was measured by anti-IL2 ELISA (n = 3, technical duplicates). Mean ± SE.  C HA-specific activation of FcγR by AAV-HA, AAV-cHA or WIV pooled pre-challenge sera (n = 18 mice per group). MDCKII cells were transfected with wildtype HA (pAAV-HA) or a stalk-only HA (pAAV-mHL1) before mouse serum and FcγR-I-expressing reporter cells were added (n = 2, technical duplicates). Mean ± SE.


Figure 5 A to F Mice were vaccinated three times with AAV - vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus ( Cal / 7 / 9 ) ( A , B ; n = 5 ) or two different lethal doses of the heterologous PR8 strain ( C , D ; n = 6 and E , F ; n = 7 ) . Survival was monitored over a 14 - day period and is depicted as Kaplan - Meier plot ( A , C , E ) . Weight loss was determined during the 14 - day period , which is shown in relation to the weight at day 0 ( B , D , F ) . Mean ± SE . Statistical significance between negative control group ( AAV - GFP ) or WIV group ( E ) and each vaccine group was determined using Log - Rank ( Mantel - Cox ) test ( * p < 0 . 05 , * * p < 0 . 01 ) .

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Figure 5 A to F Mice were vaccinated three times with AAV-vectors or two times with WIV and then challenged with a lethal dose of homologous influenza virus (Cal/7/9) (A, B; n = 5) or two different lethal doses of the heterologous PR8 strain (C, D; n = 6 and E, F; n = 7). Survival was monitored over a 14-day period and is depicted as Kaplan-Meier plot (A, C, E). Weight loss was determined during the 14-day period, which is shown in relation to the weight at day 0 (B, D, F). Mean ± SE. Statistical significance between negative control group (AAV-GFP) or WIV group (E) and each vaccine group was determined using Log-Rank (Mantel-Cox) test (*p<0.05, **p<0.01).


Figure 6 B Cal / 7 / 9 - specific serum antibody titers in individual sera at indicated time points . Black lines : individual animals , red lines : mean titer . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( * p < 0 . 05 , * * p < 0 . 01 ) . The lower table indicates Cal / 7 / 9 - specific MN50 and HAI mean titers . Assays were performed in technical duplicates . C to E Clinical score ( C ) , body temperature ( D ) , and relative weight ( E ) during challenge period . Statistical significance between pre - immune and immune sera was determined using Friedman test with Dunn ' s multiple comparison testing ( AAV - HA # # p < 0 . 01 ; AAV - cHA § § p < 0 . 01 ; AAV - GFP $ p < 0 . 05 ; $ $ p < 0 . 01 ; QIV * p < 0 . 05 ; ) . Mean ± SD F Virus titers in nasal turbinates ( NT ) , trachea or lung homogenates . Statistical significance between AAV - GFP and vaccine groups was determined using Kruskal - Wallis test with Dunn ' s multiple comparison testing ( * p < 0 . 05 ) . Lines indicate the GMT . Assays were performed in technical duplicates . G and H IHC staining of submucosal glands ( SMG ) ( G ) or bronchi ( H ) with an anti - H1N1 polyclonal antibody ( bar : 100 µm ; red : influenza antigen ( neufuchsin ) ; blue : nuclei ( haematoxylin ) ) . I and J H & E stained histological sections of bronchi ( I ) and lung parenchyma with alveolar epithelial cells ( AEC , J ) . Arrowheads : areas of suppurative bronchitis / bronchiolitis ; arrows : interstitial pneumonia , necrosis of AEC ( bar : ( I ) 100 µm ; ( J ) 50 µm ) .

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

Figure 6 B Cal/7/9-specific serum antibody titers in individual sera at indicated time points. Black lines: individual animals, red lines: mean titer. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (*p<0.05, **p<0.01). The lower table indicates Cal/7/9-specific MN50 and HAI mean titers. Assays were performed in technical duplicates.  C to E Clinical score (C), body temperature (D), and relative weight (E) during challenge period. Statistical significance between pre-immune and immune sera was determined using Friedman test with Dunn's multiple comparison testing (AAV-HA ##p<0.01; AAV-cHA §§p<0.01; AAV-GFP $p<0.05; $$p<0.01; QIV *p<0.05;). Mean ± SD  F Virus titers in nasal turbinates (NT), trachea or lung homogenates. Statistical significance between AAV-GFP and vaccine groups was determined using Kruskal-Wallis test with Dunn's multiple comparison testing (*p<0.05). Lines indicate the GMT. Assays were performed in technical duplicates.  G and H IHC staining of submucosal glands (SMG) (G) or bronchi (H) with an anti-H1N1 polyclonal antibody (bar: 100 µm; red: influenza antigen (neufuchsin); blue: nuclei (haematoxylin)).  I and J H&E stained histological sections of bronchi (I) and lung parenchyma with alveolar epithelial cells (AEC, J). Arrowheads: areas of suppurative bronchitis/bronchiolitis; arrows: interstitial pneumonia, necrosis of AEC (bar: (I) 100 µm; (J) 50 µm).


Figure 1 ( F ) ChIP - seq tracks Brd4 on select genes ( Foxp3 , Nr4a2 , Med1 , Taf10 , Ar , and Fbxw7 ) in Th2 cells , treated with or without JQ1 .

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Figure 1(F) ChIP-seq tracks Brd4 on select genes (Foxp3, Nr4a2, Med1, Taf10, Ar, and Fbxw7) in Th2 cells, treated with or without JQ1.


Figure 2 ( B ) qPCR analysis of Il4 , Il5 , Il13 , Gata3 , Foxp3 and Fbxw7 in mouse Th2 cells infected with sh - Ctrl , sh - Foxp3 , and sh - Fbxw7 lenti - virus . ( C ) Immunoprecipitation of Flag - tagged Fbxw7 with anti - Flag affinity gel , followed by Western blotting of Myc to detect Myc - Gata3 in HEK293T cells transfected with Myc - Gata3 and / or Flag - Fbxw7 plasmids . ( F ) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 ( 500nM ) , and then treated with cycloheximide ( CHX ; 25μg / ml ) for 0h , 0 . 5h , 1h and 2h before harvest to stop protein synthesis .

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Figure 2(B) qPCR analysis of Il4, Il5, Il13, Gata3, Foxp3 and Fbxw7 in mouse Th2 cells infected with sh-Ctrl, sh-Foxp3, and sh-Fbxw7 lenti-virus.(C) Immunoprecipitation of Flag-tagged Fbxw7 with anti-Flag affinity gel, followed by Western blotting of Myc to detect Myc-Gata3 in HEK293T cells transfected with Myc-Gata3 and/or Flag-Fbxw7 plasmids.(F) Western blotting of Gata3 in mouse Th2 cells treated with or without JQ1 (500nM), and then treated with cycloheximide (CHX; 25μg/ml) for 0h, 0.5h, 1h and 2h before harvest to stop protein synthesis.


Figure 3 ( C ) ChIP - qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 ( 250nM ) . ( D ) Immunoprecipitation of Brd4 in mouse Th2 cells , followed by Western blotting of Ezh2 , Suz12 , EED and G9a . ( F ) ChIP - qPCR analysis of Brd4 , Ezh2 , Suz12 and EED binding on the gene loci of Il4 , Il5 , Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 ( 250nM ) .

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Figure 3(C) ChIP-qPCR analysis of histone modifications on the gene loci of Foxp3 and Fbxw7 in mouse Th2 cells treated with or without JQ1 (250nM).(D) Immunoprecipitation of Brd4 in mouse Th2 cells, followed by Western blotting of Ezh2, Suz12, EED and G9a.(F) ChIP-qPCR analysis of Brd4, Ezh2, Suz12 and EED binding on the gene loci of Il4, Il5, Foxp3 and Fbxw7 in mouse Th2 cells treat with or without JQ1 (250nM).


Figure 4 ( B ) HEK293T cells are transfected with Flag - Brd4 and HA - EED , HA - Ezh2 , and Suz12 plasmids . Lysates are immunoprecipitated with Flag - tagged Brd4 , treated in - vitro with JQ1 ( 5μM ) , MS402 ( 5μM ) , and ABBV - 744 ( 5μM ) , followed by Western blotting of HA . ( F ) HEK293T cells are transfected with Flag - Brd4 , HA - EED and HA - EED ( K19R ) plasmids . Lysates are immunoprecipitated with Flag followed by Western blotting of HA , and vice versa .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4(B) HEK293T cells are transfected with Flag-Brd4 and HA-EED, HA-Ezh2, and Suz12 plasmids. Lysates are immunoprecipitated with Flag-tagged Brd4, treated in-vitro with JQ1 (5μM), MS402 (5μM), and ABBV-744 (5μM), followed by Western blotting of HA.(F) HEK293T cells are transfected with Flag-Brd4, HA-EED and HA-EED(K19R) plasmids. Lysates are immunoprecipitated with Flag followed by Western blotting of HA, and vice versa.


Figure 5 ( B ) qPCR analysis of Il4 , Il5 , Gata3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . ( D ) Western blotting of Gata3 of mouse Th2 cells treated with ABBV - 744 ( 500nM ) and MG132 ( 20μM ) . ( E ) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . ( F ) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV - 744 at 500nM and 1μM . (

O O B-PANEL_START 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-PANEL_START 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) qPCR analysis of Il4, Il5, Gata3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM.(D) Western blotting of Gata3 of mouse Th2 cells treated with ABBV-744 (500nM) and MG132 (20μM).(E) qPCR analysis of Foxp3 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (F) qPCR analysis of Fbxw7 in mouse Th2 cells treated with ABBV-744 at 500nM and 1μM. (


Figure 1Phloroglucinol staining of wild - type seedlings inoculated with PTI - inducing Pst DC3000 hrcC - , virulent Pst DC3000 , and avirulent Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) . Data information : Twelve - day - old seedlings were flood - inoculated with P . syringae at 108 cfu / ml in ( A hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculationB Phloroglucinol staining of wild - type adult leaves inoculated with PTI - inducing Pst DC3000 hrcC - , virulent Pst DC3000 , and avirulent Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) . The upper images are enlarged ones of the lower boxes at 2 dpi . Scale bars , 100 μm . Data information Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml in ( B hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculation ; IS , infected site ; UIS , uninfected site . C Quantification of lignin content in pathogen - treated seedlings Data are shown as means ± SD ( n = 4 ; 20 - 30 seedlings each ) . Data information : Twelve - day - old seedlings were flood - inoculated with P . syringae at 108 cfu / ml Significant differences are indicated by different letters ( Tukey ' s HSD test ; P < 0 . 05 ) Experiments were repeated 3 times with similar results . hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculationD Quantification of lignin content in pathogen - treated wild - type leaves Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Data information : Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml Significant differences are indicated by different letters ( Tukey ' s HSD test ; P < 0 . 05 ) Experiments were repeated 3 times with similar results . hrcC - , Pst DC3000 hrcC - ; DC3000 , Pst DC3000 ; AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculationE Quantification of lignin content in wild - type leaves treated with different titers of Pst DC3000 ( AvrRpm1 ) . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Six - week - old leaves were syringe - infiltrated with P . syringae at the indicated titers in ( E ) . Significant differences are indicated by asterisks ( t test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Experiments were repeated 3 times with similar results . dpi , days post - inoculationF Quantification of lignin content in wild - type and rpm1 - 3 rps2 - 101c leaves treated with avirulent bacteria . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Six - week - old leaves were syringe - infiltrated with P . syringae at 108 cfu / ml Significant differences are indicated by asterisks ( t test ; * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post -

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Phloroglucinol staining of wild-type seedlings inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml in (A hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationB Phloroglucinol staining of wild-type adult leaves inoculated with PTI-inducing Pst DC3000 hrcC-, virulent Pst DC3000, and avirulent Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2). The upper images are enlarged ones of the lower boxes at 2 dpi. Scale bars, 100 μm. Data information Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml in (B hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation; IS, infected site; UIS, uninfected site.C Quantification of lignin content in pathogen-treated seedlings Data are shown as means ± SD (n = 4; 20-30 seedlings each). Data information: Twelve-day-old seedlings were flood-inoculated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationD Quantification of lignin content in pathogen-treated wild-type leaves Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by different letters (Tukey's HSD test; P < 0.05) Experiments were repeated 3 times with similar results. hrcC-, Pst DC3000 hrcC-; DC3000, Pst DC3000; AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculationE Quantification of lignin content in wild-type leaves treated with different titers of Pst DC3000 (AvrRpm1). Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at the indicated titers in (E). Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. dpi, days post-inoculationF Quantification of lignin content in wild-type and rpm1-3 rps2-101c leaves treated with avirulent bacteria. Data are shown as means ± SD (n = 4; 3-9 leaves each). Six-week-old leaves were syringe-infiltrated with P. syringae at 108 cfu/ml Significant differences are indicated by asterisks (t test; *P < 0.05; **P < 0.01; ***P < 0.001). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation


Figure 2B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection . Data are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 108 cfu / ml for experiments . Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation . C Measurements of Pst DC3000 ( AvrRpm1 ) growth . Data are shown as means ± SD ( n = 3 ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 105 cfu / ml for growth assays Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation . D Cell death phenotypes of leaves inoculated with Pst DC3000 ( AvrRpm1 ) . E Quantification of leaves ( n ≥ 30 ) with spreading cell death as in ( D ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 105 cfu / ml for growth assays M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; dpi , days post - inoculation . F Quantification of total and free SA in Col - 0 and palQ leaves after the indicated treatments and pathogen infection . Data are shown as means ± SD ( n = 5 ; 6 leaves each ) . Data information : Six - week - old leaves were inoculated with Pst DC3000 ( AvrRpm1 ) at 108 cfu / ml for experiments . Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . M , mock ; PA , piperonylic acid dpi , days post - inoculation .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B Quantification of lignin content in palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 4; 3-9 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.C Measurements of Pst DC3000 (AvrRpm1) growth. Data are shown as means ± SD (n = 3). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.D Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). E Quantification of leaves (n ≥ 30) with spreading cell death as in (D). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 105 cfu/ml for growth assays M, mock; PA, piperonylic acid; CA, coniferyl alcohol; dpi, days post-inoculation.F Quantification of total and free SA in Col-0 and palQ leaves after the indicated treatments and pathogen infection. Data are shown as means ± SD (n = 5; 6 leaves each). Data information: Six-week-old leaves were inoculated with Pst DC3000 (AvrRpm1) at 108 cfu/ml for experiments. Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. M, mock; PA, piperonylic acid dpi, days post-inoculation.


Figure 3A Colonization patterns of GFP - Pst DC3000 and Pst DC3000 ( AvrRpt2 ) in wild - type plants . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100 μmB Time - lapse images of GFP - Pst DC3000 and Pst DC3000 ( AvrRpt2 ) captured at 0 ( magenta ) , 15 , ( yellow ) , and 30 ( green ) sec Time in seconds in the movies is shown at the top of images . Blue color was used for chlorophyll autofluorescence . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . Scale bars , 10 μmC Colonization patterns of GFP - Pst DC3000 ( AvrRpt2 ) in wild - type and palQ plants after PA and CA pretreatments . Data information : Leaves were inoculated with GFP - labeled bacteria at 108 cfu / ml for 2 days . M , mock ; PA , piperonylic acid ; CA , coniferyl alcohol ; IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Colonization patterns of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) in wild-type plants. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μmB Time-lapse images of GFP-Pst DC3000 and Pst DC3000 (AvrRpt2) captured at 0 (magenta), 15, (yellow), and 30 (green) sec Time in seconds in the movies is shown at the top of images. Blue color was used for chlorophyll autofluorescence. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. Scale bars, 10 μmC Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in wild-type and palQ plants after PA and CA pretreatments. Data information: Leaves were inoculated with GFP-labeled bacteria at 108 cfu/ml for 2 days. M, mock; PA, piperonylic acid; CA, coniferyl alcohol; IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm


Figure 4A CANBD - and CADMAC - incorporated lignin polymerization . CANBD ( green ) and CADMAC ( blue ) fluorescence was observed in the lignified infection site at 2 dpi , as revealed by phloroglucinol staining . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site White dash lines indicate the boundary between IS and UIS . Scale bars , 500 μmB Cross - sections of CANBD - and Pst DC3000 ( AvrRpm1 ) - treated leaves . Data information : Bacterial inoculum was at 108 cfu / ml . hpi , hours post - inoculation Scale bars , 40 μmC Cross - section of the entire leaf with one half leaf infected . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site ; hpi , hours post - inoculation Scale bars , 120 μmD CADMAC - incorporated lignin deposition in the GFP - Pst DC3000 ( AvrRpt2 ) - infected site at 2 dpi . Data information : Bacterial inoculum was at 108 cfu / ml . IS , infected site ; UIS , uninfected site DC3000 , Pst DC3000 ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; Chl , chlorophyll . White dash lines indicate the boundary between IS and UIS . Scale bars , 100 μmE Cellular observation of CADMAC - incorporated lignin formation against infecting GFP - Pst DC3000 ( AvrRpt2 ) . Data information : Bacterial inoculum was at 108 cfu / ml . hpi , hours post - inoculation Scale bars , 20 μmF 3D image of CANBD - polymerized structure . Images were taken at 24 hpi Data information : Bacterial inoculum was at 108 cfu / ml . Scale bars , 20

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A CANBD- and CADMAC-incorporated lignin polymerization. CANBD (green) and CADMAC (blue) fluorescence was observed in the lignified infection site at 2 dpi, as revealed by phloroglucinol staining. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site White dash lines indicate the boundary between IS and UIS. Scale bars, 500 μmB Cross-sections of CANBD- and Pst DC3000 (AvrRpm1)-treated leaves. Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 40 μmC Cross-section of the entire leaf with one half leaf infected. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site; hpi, hours post-inoculation Scale bars, 120 μmD CADMAC-incorporated lignin deposition in the GFP-Pst DC3000 (AvrRpt2)-infected site at 2 dpi. Data information: Bacterial inoculum was at 108 cfu/ml. IS, infected site; UIS, uninfected site DC3000, Pst DC3000; AvrRpt2, Pst DC3000 (AvrRpt2); Chl, chlorophyll. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μmE Cellular observation of CADMAC-incorporated lignin formation against infecting GFP-Pst DC3000 (AvrRpt2). Data information: Bacterial inoculum was at 108 cfu/ml. hpi, hours post-inoculation Scale bars, 20 μmF 3D image of CANBD-polymerized structure. Images were taken at 24 hpi Data information: Bacterial inoculum was at 108 cfu/ml. Scale bars, 20 μm


Figure 5A Time course expression profiles of CASPLs in response to Pst DC3000 ( AvrRpm1 ) treatment . Analysis was based on the Genevestigator database ( http : / / www . genevestigator . ethz . ch / ) . The darker color corresponds to stronger expression . ND , not determined . Quantification of lignin content in wild - type and caspl mutant leaves after Pst DC3000 ( AvrRpm1 ) inoculation . ata are shown as means ± SD ( n = 4 ; 3 - 9 leaves each ) . ata information : Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; dpi , days post - inoculation . C Cell death phenotypes of leaves inoculated with Pst DC3000 ( AvrRpm1 ) . Data information : AvrRpm1 , Pst DC3000 ( AvrRpm1 ) dpi , days post - inoculation . D Quantification of leaves ( n ≥ 30 ) with spreading cell death after Pst DC3000 ( AvrRpm1 ) inoculation . dpi , days post - inoculation . E Measurements of Pst DC3000 ( AvrRpm1 ) and Pst DC3000 ( AvrRpt2 ) growth . Data are shown as means ± SD ( n = 3 ) . Data information : Different letters indicate significant differences ( Tukey ' s HSD test ; P < 0 . 05 ) . Experiments were repeated 3 times with similar results . AvrRpm1 , Pst DC3000 ( AvrRpm1 ) ; AvrRpt2 , Pst DC3000 ( AvrRpt2 ) ; dpi , days post - inoculation . F Colonization patterns of GFP - Pst DC3000 ( AvrRpt2 ) in caspl mutant plants . IS , infected site ; UIS , uninfected site . White dash lines indicate the boundary between IS and UIS . Scale bars , 100 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A Time course expression profiles of CASPLs in response to Pst DC3000 (AvrRpm1) treatment. Analysis was based on the Genevestigator database (http://www.genevestigator.ethz.ch/). The darker color corresponds to stronger expression. ND, not determined.Quantification of lignin content in wild-type and caspl mutant leaves after Pst DC3000 (AvrRpm1) inoculation. ata are shown as means ± SD (n = 4; 3-9 leaves each). ata information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); dpi, days post-inoculation.C Cell death phenotypes of leaves inoculated with Pst DC3000 (AvrRpm1). Data information: AvrRpm1, Pst DC3000 (AvrRpm1) dpi, days post-inoculation.D Quantification of leaves (n ≥ 30) with spreading cell death after Pst DC3000 (AvrRpm1) inoculation. dpi, days post-inoculation.E Measurements of Pst DC3000 (AvrRpm1) and Pst DC3000 (AvrRpt2) growth. Data are shown as means ± SD (n = 3). Data information: Different letters indicate significant differences (Tukey's HSD test; P < 0.05). Experiments were repeated 3 times with similar results. AvrRpm1, Pst DC3000 (AvrRpm1); AvrRpt2, Pst DC3000 (AvrRpt2); dpi, days post-inoculation.F Colonization patterns of GFP-Pst DC3000 (AvrRpt2) in caspl mutant plants. IS, infected site; UIS, uninfected site. White dash lines indicate the boundary between IS and UIS. Scale bars, 100 μm.


Figure 6A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence . Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) . mCh , mCherry ; Chl , chlorophyll ; hpi , hours post - inoculation . Scale bars , 20 μm ( A ) B Magnified views of CASPL4D1 and lignin localization . Images in boxes were enlarged in the 2nd to 4th columns . Arrows indicate lignin deposition between cells . Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) . mCh , mCherry ; Chl , chlorophyll ; hpi , hours post - inoculation . Scale bars 10 μm ( B ) . C Relative fluorescence intensity profiles of CASPL1D1 ( mCherry ) and lignin ( CADMAC ) across transects indicated by dotted lines Data information : pCASPL4D1 : : CASPL4D1 - mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 ( AvrRpm1 ) . D A model showing morphological changes of CASPL - and lignin - deposited cells during the HR response .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O

Figure 6A Visualization of CASPL4D1 proteins and lignin by mCherry and CADMAC fluorescence. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars, 20 μm (A)B Magnified views of CASPL4D1 and lignin localization. Images in boxes were enlarged in the 2nd to 4th columns. Arrows indicate lignin deposition between cells. Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1). mCh, mCherry; Chl, chlorophyll; hpi, hours post-inoculation. Scale bars 10 μm (B).C Relative fluorescence intensity profiles of CASPL1D1 (mCherry) and lignin (CADMAC) across transects indicated by dotted lines Data information: pCASPL4D1::CASPL4D1-mCherry plants were pretreated with CADMAC and then inoculated with Pst DC3000 (AvrRpm1).D A model showing morphological changes of CASPL- and lignin-deposited cells during the HR response.


Figure 1 . ( A and B ) ARPE - 19 cells were transfected with siRNA duplexes to raptor , RagA + B , p18 , or nontarget . 60 h after transfection , cells were infected with adenovirus expressing either TFEB - FLAG - WT ( A ) or TFEB - FLAG - S211A ( B ) . 12 h later , cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against FLAG ( used to detect TFEB ) and mTOR . Bars , 10 µm . ( C ) Quantification of A . ( D ) Quantification of B . Values are means ± SD of three independent experiments . * * * , P < 0 . 001 .

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Figure 1.(A and B) ARPE-19 cells were transfected with siRNA duplexes to raptor, RagA+B, p18, or nontarget. 60 h after transfection, cells were infected with adenovirus expressing either TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG (used to detect TFEB) and mTOR. Bars, 10 µm. (C) Quantification of A. (D) Quantification of B. Values are means ± SD of three independent experiments. ***, P < 0.001.


Figure 2 . ( A ) ARPE - 19 cells were nucleofected with the indicated Rag - expressing plasmids . 6 h after nucleofection , cells were infected with adenovirus expressing TFEB - FLAG - WT . 12 h later , cells were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG ( used to detect TFEB - WT ) . ( B ) ARPE - 19 cells were nucleofected with the indicated Rag - and TFEB - GFP - expressing plasmids . 18 h later , cells were lysed and subjected to immunoprecipitation with the anti - HA antibody ( used to immunoprecipitate Rag proteins ) . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP ( used to detect Rag proteins and TFEB - WT , respectively ) . ( C ) ARPE - 19 coexpressing TFEB and the indicated Rag - expressing plasmids were lysed and subjected to immunoprecipitation with the anti - FLAG antibody . The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG ( used to detect Rag proteins and TFEB - WT , respectively ) . ( D ) Immunoblotting analysis of coimmunoprecipitated TFEB - FLAG - S211A with Rag heterodimers . IP , immunoprecipitation .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O

Figure 2.(A) ARPE-19 cells were nucleofected with the indicated Rag-expressing plasmids. 6 h after nucleofection, cells were infected with adenovirus expressing TFEB-FLAG-WT. 12 h later, cells were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against Rag proteins and FLAG (used to detect TFEB-WT).(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-GFP-expressing plasmids. 18 h later, cells were lysed and subjected to immunoprecipitation with the anti-HA antibody (used to immunoprecipitate Rag proteins). The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and GFP (used to detect Rag proteins and TFEB-WT, respectively).(C) ARPE-19 coexpressing TFEB and the indicated Rag-expressing plasmids were lysed and subjected to immunoprecipitation with the anti-FLAG antibody. The immunoprecipitates were analyzed by immunoblotting with antibodies against GST and FLAG (used to detect Rag proteins and TFEB-WT, respectively).(D) Immunoblotting analysis of coimmunoprecipitated TFEB-FLAG-S211A with Rag heterodimers. IP, immunoprecipitation.


Figure 3 . ( A ) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin - 1 for 1 h . Cells were then lysed and subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) and actin . ( B ) HeLa cells were treated as indicated in A , and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation . Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB ( used to detect endogenous TFEB ) , Lamp1 , or Histone H3 . ( C ) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin - 1 as indicated in A . ( D ) HeLa cells were starved in serum - and amino acid - free medium ( starvation ) for 3 h and analyzed by immunoblotting with antibodies against TFEB . ( E ) HeLa cells were incubated in normal medium ( control ) and serum - and amino acid - free medium ( Starvation ) for 4 h or starved for 4 h followed by restimulation with amino acids ( starvation + amino acids ) for 30 min and analyzed by immunofluorescence with antibodies against TFEB ( endogenous TFEB is shown in green ) and Lamp1 ( red ) . The region within the dotted box is magnified in the insets . ( F ) HeLa cells were nucleofected with the indicated Rag - expressing plasmids . 18 h later , cells were lysed , and RagB / D heterodimers were pulled down using glutathione - Sepharose beads . Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST ( used to detect endogenous TFEB and Rag proteins , respectively ) . Bars , 10 µm .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O 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-PANEL_START O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3.(A) The indicated cell lines were incubated in medium containing DMSO or 250 nM Torin-1 for 1 h. Cells were then lysed and subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB) and actin.(B) HeLa cells were treated as indicated in A, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from different fractions were subjected to immunoblotting with antibodies against TFEB (used to detect endogenous TFEB), Lamp1, or Histone H3.(C) Immunofluorescence confocal microscopy showing nuclear and lysosomal localization of endogenous TFEB upon treatment of HeLa cells with Torin-1 as indicated in A.(D) HeLa cells were starved in serum- and amino acid-free medium (starvation) for 3 h and analyzed by immunoblotting with antibodies against TFEB.(E) HeLa cells were incubated in normal medium (control) and serum- and amino acid-free medium (Starvation) for 4 h or starved for 4 h followed by restimulation with amino acids (starvation + amino acids) for 30 min and analyzed by immunofluorescence with antibodies against TFEB (endogenous TFEB is shown in green) and Lamp1 (red). The region within the dotted box is magnified in the insets.(F) HeLa cells were nucleofected with the indicated Rag-expressing plasmids. 18 h later, cells were lysed, and RagB/D heterodimers were pulled down using glutathione-Sepharose beads. Proteins bound to the beads were analyzed by immunoblotting with antibodies against TFEB and GST (used to detect endogenous TFEB and Rag proteins, respectively). Bars, 10 µm.


Figure 4 . ( A ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - WT . 16 h later , cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 3 h . Cells were then fixed , permeabilized with 0 . 2 % saponin , and double stained with antibodies against TFEB ( used to detect recombinant TFEB ) and Lamp1 . ( B ) ARPE - 19 cells were infected with adenovirus expressing TFEB - FLAG - S211A . 12 h later , cells were starved in serum - and amino acid - free medium for 4 h ( starvation ) or kept in complete medium ( control ) . Cells were then fixed , permeabilized with 0 . 2 % Triton X - 100 , and stained with antibodies against FLAG ( used to detect TFEB - S211A ) . ( C ) HeLa cells were incubated with 250 nM Torin - 1 for 1 h or starved in serum - and amino acid - free medium for 4 h with the addition of Torin - 1 during the last hour of starvation . Cells were then washed , fixed , permeabilized with 0 . 2 % Triton X - 100 , and double stained with antibodies against TFEB ( used to detect endogenous TFEB ) and Lamp1 . Regions within the dotted boxes are magnified in the insets .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4.(A) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-WT. 16 h later, cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 3 h. Cells were then fixed, permeabilized with 0.2% saponin, and double stained with antibodies against TFEB (used to detect recombinant TFEB) and Lamp1.(B) ARPE-19 cells were infected with adenovirus expressing TFEB-FLAG-S211A. 12 h later, cells were starved in serum- and amino acid-free medium for 4 h (starvation) or kept in complete medium (control). Cells were then fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB-S211A).(C) HeLa cells were incubated with 250 nM Torin-1 for 1 h or starved in serum- and amino acid-free medium for 4 h with the addition of Torin-1 during the last hour of starvation. Cells were then washed, fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against TFEB (used to detect endogenous TFEB) and Lamp1. Regions within the dotted boxes are magnified in the insets.


Figure 5 . ( A and B ) ARPE - 19 cells were transfected with either active or inactive RagB / D heterodimers and infected with adenovirus expressing TFEB - FLAG - WT ( A ) or TFEB - FLAG - S211A ( B ) . After 12 h , cells were double stained with antibodies against FLAG and GST ( used to detect TFEB or Rag proteins , respectively ) . ( C ) Quantification of TFEB - WT nuclear localization from A . ( D ) Quantification of TFEB - S211A lysosomal localization from B . ( E and F ) ARPE - 19 cells were cotransfected with plasmids expressing TFEB - GFP together with inactive ( E ) or active Rag heterodimers ( F ) . Cells were then incubated with 250 nM Torin - 1 for 1 h ( E ) or starved in serum - and amino acid - free medium for 3 h ( F ) . Anti - GST antibodies were used to detect Rag proteins . Arrows point to cells that do not express Rag heterodimers .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5.(A and B) ARPE-19 cells were transfected with either active or inactive RagB/D heterodimers and infected with adenovirus expressing TFEB-FLAG-WT (A) or TFEB-FLAG-S211A (B). After 12 h, cells were double stained with antibodies against FLAG and GST (used to detect TFEB or Rag proteins, respectively). (C) Quantification of TFEB-WT nuclear localization from A. (D) Quantification of TFEB-S211A lysosomal localization from B.(E and F) ARPE-19 cells were cotransfected with plasmids expressing TFEB-GFP together with inactive (E) or active Rag heterodimers (F). Cells were then incubated with 250 nM Torin-1 for 1 h (E) or starved in serum- and amino acid-free medium for 3 h (F). Anti-GST antibodies were used to detect Rag proteins. Arrows point to cells that do not express Rag heterodimers.


Figure 6 . ( A ) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE - 19 cells treated with either DMSO or Torin - 1 . ( B ) ARPE - 19 cells were nucleofected with the indicated Rag - and TFEB - expressing plasmids . After 12 h , cell lysates were immunoprecipitated with the anti - FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST ( used to detect TFEB and Rag proteins , respectively ) . ( C ) FLAG - tagged TFEB - WT or TFEB deletion mutants were immunoprecipitated with the anti - FLAG antibody and analyzed by immunoblotting with antibodies against FLAG , 14 - 3 - 3 binding motif , or 14 - 3 - 3 . ( D ) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB - WT and TFEB - S3A / R4A mutant upon incubation with DMSO ( vehicle ) or 250 nM Torin - 1 for 1 h . Cells were fixed , permeabilized with 0 . 2 % Triton X - 100 , and stained with antibodies against FLAG ( used to detect TFEB ) . ( E ) ARPE - 19 cells expressing either TFEB - S211A or TFEB - S211A - Δ30 were double stained with antibodies against TFEB and Lamp1 . Regions within the dotted boxes are magnified in the insets .

O O O B-PANEL_START O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6.(A) Summary of the nuclear and lysosomal distribution of several TFEB amino acid and deletion mutants in ARPE-19 cells treated with either DMSO or Torin-1.(B) ARPE-19 cells were nucleofected with the indicated Rag- and TFEB-expressing plasmids. After 12 h, cell lysates were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG and GST (used to detect TFEB and Rag proteins, respectively).(C) FLAG-tagged TFEB-WT or TFEB deletion mutants were immunoprecipitated with the anti-FLAG antibody and analyzed by immunoblotting with antibodies against FLAG, 14-3-3 binding motif, or 14-3-3.(D) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-WT and TFEB-S3A/R4A mutant upon incubation with DMSO (vehicle) or 250 nM Torin-1 for 1 h. Cells were fixed, permeabilized with 0.2% Triton X-100, and stained with antibodies against FLAG (used to detect TFEB).(E) ARPE-19 cells expressing either TFEB-S211A or TFEB-S211A-Δ30 were double stained with antibodies against TFEB and Lamp1. Regions within the dotted boxes are magnified in the insets.


Figure 7 . ( A ) ARPE - 19 cells coexpressing active RagB / D heterodimer and the indicated TFEB plasmids were fixed , permeabilized with 0 . 2 % saponin , and stained with antibodies against GST ( used to detect Rag proteins ) . Regions within the dotted boxes are magnified in the insets . Bars , 10 µm . ( B ) ARPE - 19 cells were cotransfected with active RagB / D heterodimers and the indicated TFEB constructs . After 18 h , cell lysates were immunoprecipitated with the anti - HA antibody ( used to immunoprecipitate Rag proteins ) and immunoblotted with antibodies against GFP and GST ( used to detect TFEB - GFP and Rag proteins , respectively ) . The white line indicates that intervening lanes have been spliced out . ( C ) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB - ( 1 - 30 ) - GFP in ARPE - 19 cells coexpressing active RagB / D heterodimers ( antibodies against GST were used to detect Rags ) . Bar , 4 µm . ( D ) Relative RT - PCR analysis of the mRNA expression of autophagy ( ATG9B and UVRAG ) and lysosomal ( MCOLN1 ) genes in ARPE - 19 cells infected with the indicated adenovirus for 48 h . The values are expressed as a ratio to RNA from cells infected with control adenovirus ( Ad . - Null ) . Values are means ± SD of two independent experiments . * * * , P < 0 . 001 ; * * , P < 0 . 01 . ( E ) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies . IP , immunoprecipitation .

O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O

Figure 7.(A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm.(B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out.(C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1-30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm.(D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01.(E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.