figf3 ( a ) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Cells were starved of amino acids for 1 h before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( b ) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid ( AA ) stimulation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K into HEK293 cells . Cells were starved of amino acids for 1 h ( - AA ) and either remained in the starvation medium or were stimulated with amino - acid - containing - medium ( + AA ) for 30 min before collection . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . ( c ) RagAT21N and RagCQ120L suppress insulin - induced stimulation of S6K phosphorylation . pcDNA3 ( 200 ng , lanes 1 and 2 ) or each indicated Rag construct was co - transfected with HA - S6K ( 20 ng ) or GST - Akt ( 100 ng ) into HeLa cells . Cells were serum - starved overnight and stimulated with insulin ( 400 nM ) for 30 min . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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 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 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

figf3(a) RagAQ66L and RagCS75N activate TORC1 in the absence of amino acids. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng) into HEK293 cells. Cells were starved of amino acids for 1 h before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(b) RagAT21N and RagCQ120L block S6K phosphorylation in response to amino acid (AA) stimulation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K into HEK293 cells. Cells were starved of amino acids for 1 h (-AA) and either remained in the starvation medium or were stimulated with amino-acid-containing-medium (+AA) for 30 min before collection. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.(c) RagAT21N and RagCQ120L suppress insulin-induced stimulation of S6K phosphorylation. pcDNA3 (200 ng, lanes 1 and 2) or each indicated Rag construct was co-transfected with HA-S6K (20 ng) or GST-Akt (100 ng) into HeLa cells. Cells were serum-starved overnight and stimulated with insulin (400 nM) for 30 min. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf4 ( a ) dRagA positively regulates wing compartment size . Wild - type or mutant dRagA transgenes were expressed in posterior compartments with the en - GAL4 driver . The ratios of representative posterior to anterior compartment areas are shown . Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression . Data are mean ± s . d . , * P = 7 . 32 × 10 − 4 ( n = 7 ) , * * P = 6 . 18 × 10 − 4 ( n = 12 ) , Student ' s two - tailed t - test , ( n is the number of adult wings analysed ) . ( b ) dRagA positively regulates wing cell size . The average area of posterior compartment cells from en - GAL4 UAS - dRagA adult wings is shown . Cells expressing dRagAQ61L are significantly larger and dRagAT61N - expressing cells are smaller than controls . Data are mean ± s . d . , * P = 1 . 15 × 10 − 3 ( n = 7 ) , * * P = 0 . 025 ( n = 12 ) , Student ' s 2 - tailed t - test ( n represents number of adult wings analysed ) . ( c , d ) dRag GTPases positively regulate larval fat body cell size . ( c ) Cell area of clonally - induced dRagA - expressing cells or dRagC homozygous mutant cells relative to neighbouring wild - type control cells is shown . Cell area was determined from phalloidin - stained fixed fat body samples from fed or 48 h starved larvae . Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions . Cells expressing dRagAT61N and dRagC loss - of - function cells were significantly smaller than control cells only under nutrient replete conditions . Data are means ± s . d . , * P = 2 . 04 × 10 − 3 ( n = 5 ) , * * P = 2 . 94 × 10 − 6 ( n = 14 ) , * * * P = 3 . 79 × 10 − 7 ( n = 14 ) , * * * * P = 1 . 36 × 10 − 5 ( n = 30 ) , Student ' s two - tailed t - test . ( d ) Representative examples of fat body cells with altered dRagA activity . Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels , and dRagC homozygous mutant cells are marked by absence of GFP in the right panel . Scale bar represents 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

figf4(a) dRagA positively regulates wing compartment size. Wild-type or mutant dRagA transgenes were expressed in posterior compartments with the en-GAL4 driver. The ratios of representative posterior to anterior compartment areas are shown. Posterior compartment area was significantly increased in response to dRagAQ61L expression and decreased in response to dRagAT61N expression. Data are mean ± s.d., *P = 7.32 × 10−4 (n = 7), **P = 6.18 × 10−4 (n = 12), Student's two-tailed t-test, (n is the number of adult wings analysed).(b) dRagA positively regulates wing cell size. The average area of posterior compartment cells from en-GAL4 UAS-dRagA adult wings is shown. Cells expressing dRagAQ61L are significantly larger and dRagAT61N-expressing cells are smaller than controls. Data are mean ± s.d., *P = 1.15 × 10−3 (n = 7), **P = 0.025 (n = 12), Student's 2-tailed t-test (n represents number of adult wings analysed).(c, d) dRag GTPases positively regulate larval fat body cell size. (c) Cell area of clonally-induced dRagA-expressing cells or dRagC homozygous mutant cells relative to neighbouring wild-type control cells is shown. Cell area was determined from phalloidin-stained fixed fat body samples from fed or 48 h starved larvae. Expression of dRagAWT or dRagAQ61L significantly increased relative cell area under starvation but not fed conditions. Cells expressing dRagAT61N and dRagC loss-of-function cells were significantly smaller than control cells only under nutrient replete conditions. Data are means ± s.d., *P = 2.04 × 10−3 (n = 5), **P = 2.94 × 10−6 (n = 14), ***P = 3.79 × 10−7 (n = 14), ****P = 1.36 × 10−5 (n = 30), Student's two-tailed t-test. (d) Representative examples of fat body cells with altered dRagA activity. Cells expressing dRagA transgenes are marked by the expression of GFP in the left and middle panels, and dRagC homozygous mutant cells are marked by absence of GFP in the right panel. Scale bar represents 50 μm.


figf5 ( a ) Rag acts through TORC1 to regulate S6K phosphorylation . HEK293 cells were transfected with constructs as indicated . Co - expression of of mTORKD construct ( 600 ng ) or rapamycin treatment ( rapa , 20 nM , 30 min ) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation . The protein level of mTORKD was determined by immunoblotting with anti - mTOR antibody . ( b ) RagA / RagC and TSC1 / TSC2 independently regulate S6K phosphorylation . HEK293 cells were transfected with 200 ng of each Rag and / or ′ constructs as indicated . Amino acid starvation for 1 h ( - AA ) is indicated . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated . ( c ) TSC2 and RagA / B independently affect S6K phosphorylation . HA - S6K ( 20 ng ) was transfected into HeLa cells with or without RNAi against human TSC2 , RagA and RagB as indicated . ( d ) RagAT21N and RagCQ120L do not block Rheb - induced S6K phosphorylation . RagAT21N and RagCQ120L ( 200 ng each ) were transfected into HEK293 cells with or without Rheb construct ( 20 ng ) . S6K was included in the co - transfection . Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies , as indicated . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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 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 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

figf5(a) Rag acts through TORC1 to regulate S6K phosphorylation. HEK293 cells were transfected with constructs as indicated. Co-expression of of mTORKD construct (600 ng) or rapamycin treatment (rapa, 20 nM, 30 min) abolished the effect of RagAQ66L and RagCS75N on S6K phosphorylation. The protein level of mTORKD was determined by immunoblotting with anti-mTOR antibody.(b) RagA/RagC and TSC1/TSC2 independently regulate S6K phosphorylation. HEK293 cells were transfected with 200 ng of each Rag and/or ′ constructs as indicated. Amino acid starvation for 1 h (-AA) is indicated. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated.(c) TSC2 and RagA/B independently affect S6K phosphorylation. HA-S6K (20 ng) was transfected into HeLa cells with or without RNAi against human TSC2, RagA and RagB as indicated.(d) RagAT21N and RagCQ120L do not block Rheb-induced S6K phosphorylation. RagAT21N and RagCQ120L (200 ng each) were transfected into HEK293 cells with or without Rheb construct (20 ng). S6K was included in the co-transfection. Phosphorylation and protein levels of the transfected proteins were determined by immunoblotting with appropriate antibodies, as indicated. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf6 ( a , b ) dRagC is not required for Rheb - induced cell growth . ( a ) The area of Rheb - overexpressing cells in control or dRagC mutant ( dRagC − / − ) backgrounds under fed conditions , relative to that of neighbouring control cells which were assigned a value of 1 . Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds . Data are mean ± s . d . , * P = 0 . 034 ( n = 5 ) , * * P = 6 . 1 × 10 − 3 ( n = 5 ) , Student ' s two - tailed t - test ( n represents number of experimental samples ) . ( b ) A representative example of a clone of Rheb - overexpressing cells in a dRagC − / − animal . Rheb transgene - expressing cells are marked by co - expression of GFP . Cell boundaries are labelled by phalloidin staining in red ; nuclei are labelled by DAPI in blue . Scale bar represents 50 μm . ( c , d ) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells . ( c ) Relative area of clonally - induced Rheb26 . 2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body . Clonally induced Rheb26 . 2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild - type and in dRagAQ61L expressing backgrounds . Data are mean ± s . d . , * P = 2 . 91 × 10 − 4 ( n = 7 ) , * * P = 2 . 59 × 10 − 8 ( n = 5 ) , Student ' s two - tailed t - test ( fed conditions where n represents number of experimental samples ) . ( d ) A representative example of Rheb homozygous mutant cells ( marked by lack of GFP , arrows ) in fat body ubiquitously expressing UAS - dRagAQ61L . GFP - positive control cells in this experiment are a mixture of Rheb + / − and Rheb + / + . Scale bar represents 50 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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

figf6(a, b) dRagC is not required for Rheb-induced cell growth. (a) The area of Rheb-overexpressing cells in control or dRagC mutant (dRagC−/−) backgrounds under fed conditions, relative to that of neighbouring control cells which were assigned a value of 1. Overexpression of Rheb led to a significant increase in cell area in both control and dRagC mutant backgrounds. Data are mean ± s.d., *P = 0.034 (n = 5), **P = 6.1 × 10−3 (n = 5), Student's two-tailed t-test (n represents number of experimental samples). (b) A representative example of a clone of Rheb-overexpressing cells in a dRagC−/− animal. Rheb transgene-expressing cells are marked by co-expression of GFP. Cell boundaries are labelled by phalloidin staining in red; nuclei are labelled by DAPI in blue. Scale bar represents 50 μm.(c, d) Expression of dRagAQ61L fails to rescue the growth impairment of Rheb mutant cells. (c) Relative area of clonally-induced Rheb26.2 homozygous mutant cells in a control background and in animals expressing dRagAQ61L throughout the fat body. Clonally induced Rheb26.2 homozygous mutant cells were significantly smaller than neighbouring control cells both in wild-type and in dRagAQ61L expressing backgrounds. Data are mean ±s.d., *P = 2.91 × 10−4 (n = 7), **P = 2.59 × 10−8 (n = 5), Student's two-tailed t-test (fed conditions where n represents number of experimental samples). (d) A representative example of Rheb homozygous mutant cells (marked by lack of GFP, arrows) in fat body ubiquitously expressing UAS-dRagAQ61L. GFP-positive control cells in this experiment are a mixture of Rheb+/− and Rheb+/+. Scale bar represents 50 μm.


figf7 ( a - d ) dRagAQ61L suppresses autophagy . ( a ) Drosophila fat body cells clonally expressing dRagAQ61L ( marked in green by GFP expression ) failed to accumulate autolysosomes ( shown in surrounding control cells by punctate Lysotracker Red staining ) in response to starvation for 4 h . Nuclei are marked in blue by DAPI . ( b - d ) Induction of autophagosomes in response to 4 - h starvation is shown by the punctate pattern of GFP - Atg8a expression in control fat body cells ( b ) , but not in cells expressing dRagAQ61L ( c ) . The average number of GFP - Atg8a - marked autophagosomes per cell in control and dRagAQ61L - expressing clones is shown ( d ) . Data are mean ± s . d . , * P = 2 . 91 × 10 − 6 , Students two - tailed t - test ( n = 33 fat body samples imaged per genotype ) . Scale bars represent 25 μm in each panel . ( e ) RagA regulates LC3 conversion in mammalian cells . Myc - LC3 was co - transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated . One day after transfection , cells were cultured in amino - acid - sufficienct medium ( + AA ) or amino - acid - depleted medium ( − AA ) for 4 h before collection . Western blotting for Myc - LC3 and HA - Rag were performed . Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant - negative RagA . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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 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

figf7(a-d) dRagAQ61L suppresses autophagy. (a) Drosophila fat body cells clonally expressing dRagAQ61L (marked in green by GFP expression) failed to accumulate autolysosomes (shown in surrounding control cells by punctate Lysotracker Red staining) in response to starvation for 4 h. Nuclei are marked in blue by DAPI. (b-d) Induction of autophagosomes in response to 4-h starvation is shown by the punctate pattern of GFP-Atg8a expression in control fat body cells (b), but not in cells expressing dRagAQ61L (c). The average number of GFP-Atg8a-marked autophagosomes per cell in control and dRagAQ61L-expressing clones is shown (d). Data are mean ± s.d., *P = 2.91 × 10−6, Students two-tailed t-test (n = 33 fat body samples imaged per genotype). Scale bars represent 25 μm in each panel.(e) RagA regulates LC3 conversion in mammalian cells. Myc-LC3 was co-transfected with RagAQL and RagCSN or RagATN and RagCQL into HEK293 cells as indicated. One day after transfection, cells were cultured in amino-acid-sufficienct medium (+AA) or amino-acid-depleted medium (−AA) for 4 h before collection. Western blotting for Myc-LC3 and HA-Rag were performed. Autophagic conversion of LC3I into the lipidated LC3II form was blocked by active RagA and stimulated by dominant-negative RagA. Full scans of blots are provided in Supplementary Information, Fig. S6.


figf8 ( a , b ) dRagA activation increases sensitivity to starvation . Expression of dRagAQ61L using the fat - body - specific Cg - GAL4 driver significantly decreased survival of adult female flies under starvation ( b ) but not fed ( a ) conditions , relative to controls ( Cg - GAL4 alone ) . Asterisks indicate significant difference , compared with controls . Data are mean ± s . d . , * P 0 . 05 , Students two - tailed t - test ( n = 150 flies / genotype / treatment ) .

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figf8(a, b) dRagA activation increases sensitivity to starvation. Expression of dRagAQ61L using the fat-body-specific Cg-GAL4 driver significantly decreased survival of adult female flies under starvation (b) but not fed (a) conditions, relative to controls (Cg-GAL4 alone). Asterisks indicate significant difference, compared with controls. Data are mean ± s.d., *P 0.05, Students two-tailed t-test (n = 150 flies/genotype/treatment).


Figure 2I Protein complexes involving aggregators . Heatmaps show the absolute difference in solubility change after heat shock between each complex member . Protein complexes with at least 75 % of its members quantified and containing at least 60 % of its members as aggregators are shown . ' DNA repair complex ' = ' DNA repair complex NEIL2 - PNK - Pol ( beta ) - LigIII ( alpha ) - XRCC1 ' .

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Figure 2I Protein complexes involving aggregators. Heatmaps show the absolute difference in solubility change after heat shock between each complex member. Protein complexes with at least 75% of its members quantified and containing at least 60% of its members as aggregators are shown. 'DNA repair complex' = 'DNA repair complex NEIL2-PNK-Pol(beta)-LigIII(alpha)-XRCC1'.


Figure 3 A Line graphs showing solubility after heat shock ( HS ) and during different time points of recovery . Each line corresponds to one protein . Orange lines show the mean solubility . C Different disaggregation profiles for aggregating proteins . Solubility line graphs as in A shown for aggregators with the bottom 10 % , middle 80 % and top 10 % of disaggregation rates . Proteins with bottom 10 % of disaggregation rates and related to DNA repair are highlighted with red . Proteins with top10 % disaggregation rates and directly related to transcription are highlighted with blue . D Protein localization upon heat shock and during recovery as analyzed by immunofluorescence microscopy . HeLa cells were fixed and immunostained with antibodies against indicated target proteins ( green ) at different conditions [ control , after heat shock ( 10 minutes at 44 ° C ) and after five hours of recovery from the heat shock ] . DNA staining ( Hoechst ) is shown in blue . E - H Comparison of disaggregation rates with different protein characteristics enriched in aggregators ( presented in Fig 2B - C and Fig 2E - G ) . Volcano plot presenting correlation coefficients and Benjamini - Hochberg adjusted p - values ( E ; horizontal dashed line shows a p - value of 0 . 05 ) . Scatterplots comparing disaggregation slopes and solubility change after heat shock ( F ) , fraction of intrinsically disordered regions ( G ) and fraction of ( random ) coil - like secondary structure ( H ) . In F - H , scatter plots are shown for correlations with a p - value lower than 0 . 05 . Correlation coefficients ( r ) with p - values are shown for Spearman ' s rank - order correlation .

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

Figure 3 A Line graphs showing solubility after heat shock (HS) and during different time points of recovery. Each line corresponds to one protein. Orange lines show the mean solubility.  C Different disaggregation profiles for aggregating proteins. Solubility line graphs as in A shown for aggregators with the bottom 10%, middle 80% and top 10% of disaggregation rates. Proteins with bottom 10% of disaggregation rates and related to DNA repair are highlighted with red. Proteins with top10% disaggregation rates and directly related to transcription are highlighted with blue. D Protein localization upon heat shock and during recovery as analyzed by immunofluorescence microscopy. HeLa cells were fixed and immunostained with antibodies against indicated target proteins (green) at different conditions [control, after heat shock (10 minutes at 44°C) and after five hours of recovery from the heat shock]. DNA staining (Hoechst) is shown in blue.E-H Comparison of disaggregation rates with different protein characteristics enriched in aggregators (presented in Fig 2B-C and Fig 2E-G). Volcano plot presenting correlation coefficients and Benjamini-Hochberg adjusted p-values (E; horizontal dashed line shows a p-value of 0.05). Scatterplots comparing disaggregation slopes and solubility change after heat shock (F), fraction of intrinsically disordered regions (G) and fraction of (random) coil-like secondary structure (H). In F-H, scatter plots are shown for correlations with a p-value lower than 0.05. Correlation coefficients (r) with p-values are shown for Spearman's rank-order correlation.


Figure 4 A - B Accumulation of newly synthesized proteins after mock shock ( A ) and heat shock ( B ) . Line graphs showing the accumulation of signal intensity ( log2 - transformed ratios to pre - shock control ) in the soluble fraction . Orange lines show the mean ratio . C Amount of newly synthesized proteins after heat shock compared to mock shock . Line graphs show the heat shock to mock shock ratio during recovery . Orange line show the mean ratio . Data information : All proteomics data shown in A - D and F is quantified with at least two unique peptides from at least two biological replicates . Proteomics data shown for newly synthesized proteins ( heavy ) . HS = heat shock . MS = mock shock . F Comparison of heat shock - induced protein synthesis and mRNA synthesis after heat shock ( Vihervaara et al , 2017 ) . Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue ; heat shock proteins are labelled red . HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity ( indicated with a vertical dashed line ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Accumulation of newly synthesized proteins after mock shock (A) and heat shock (B). Line graphs showing the accumulation of signal intensity (log2-transformed ratios to pre-shock control) in the soluble fraction. Orange lines show the mean ratio. C Amount of newly synthesized proteins after heat shock compared to mock shock. Line graphs show the heat shock to mock shock ratio during recovery. Orange line show the mean ratio. Data information: All proteomics data shown in A-D and F is quantified with at least two unique peptides from at least two biological replicates. Proteomics data shown for newly synthesized proteins (heavy). HS = heat shock. MS = mock shock. F Comparison of heat shock-induced protein synthesis and mRNA synthesis after heat shock (Vihervaara et al, 2017). Ten most upregulated proteins in the proteomics analysis are labelled and highlighted in blue; heat shock proteins are labelled red. HSPA1A and HSPA1B could not be distinguished from one another in the proteomics analysis and are plotted as they would have the same intensity (indicated with a vertical dashed line).


Figure 5 B : Volcano plot for the score for thermal stability . Significant ( see ' Materials and Methods ' ) heat shock - induced stabilization and destabilization are indicated with blue and red , respectively . Proteins that aggregate in heat shock ( as defined in Figure 2A ) are shown in black - aggregators with the strongest stabilization are annotated . Members of stress - activated MAPK pathway highlighted in green - alternative names for MAP - kinases are shown in parenthesis . Boxplot on top compares stability scores of aggregators ( as defined in Figure 2A and other proteins ) . The p - value is for non - parametric Wilcoxon test . D Thermal stability changes in protein complex members . Scores for thermal stability shown for proteins from 26S proteasome ( separately for 19S regulatory and 20S core particles ) and CCT chaperonin complex . Boxplots indicate median , first and third quartiles with whiskers extended to 1 . 5 times the interquartile range out from each quartile . Data used in stability score calculation is from two ( mock shock ) and three ( heat shock ) biological replicates .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Volcano plot for the score for thermal stability. Significant (see 'Materials and Methods') heat shock-induced stabilization and destabilization are indicated with blue and red, respectively. Proteins that aggregate in heat shock (as defined in Figure 2A) are shown in black - aggregators with the strongest stabilization are annotated. Members of stress-activated MAPK pathway highlighted in green - alternative names for MAP-kinases are shown in parenthesis. Boxplot on top compares stability scores of aggregators (as defined in Figure 2A and other proteins). The p-value is for non-parametric Wilcoxon test.  D Thermal stability changes in protein complex members. Scores for thermal stability shown for proteins from 26S proteasome (separately for 19S regulatory and 20S core particles) and CCT chaperonin complex. Boxplots indicate median, first and third quartiles with whiskers extended to 1.5 times the interquartile range out from each quartile. Data used in stability score calculation is from two (mock shock) and three (heat shock) biological replicates.


Figure 1 ( A ) miR - 155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB / c mice 6 weeks postinfection ( B ) Murine bone marrow - derived macrophages ( BMDMs ) were infected with BCG at an MOI of 5 for the indicated times , and the expression levels of miR - 155 were measured by real - time PCR . ( C and D ) RAW264 . 7 cells were infected with BCG at an MOI of 5 for the indicated time points ( C ) or at indicated MOIs for 24 h ( D ) . The expression levels of miR - 155 were examined by real - time PCR . ( E and F ) RAW264 . 7 cells were infected with H37Ra at an MOI of 5 for the indicated time ( E ) or at indicated MOI for 24 h ( F ) . The expression levels of miR - 155 were examined by real - time PCR . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 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 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 O O O O O O

Figure 1(A) miR-155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB/c mice 6 weeks postinfection(B) Murine bone marrow-derived macrophages (BMDMs) were infected with BCG at an MOI of 5 for the indicated times, and the expression levels of miR-155 were measured by real-time PCR.(C and D) RAW264.7 cells were infected with BCG at an MOI of 5 for the indicated time points (C) or at indicated MOIs for 24 h (D). The expression levels of miR-155 were examined by real-time PCR.(E and F) RAW264.7 cells were infected with H37Ra at an MOI of 5 for the indicated time (E) or at indicated MOI for 24 h (F). The expression levels of miR-155 were examined by real-time PCR. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; ***, p<0.001.


Figure 2 ( A - C ) RAW264 . 7 cells were transfected with control or miR - 155 mimic for 24 h followed by BCG ( A ) , H37Ra ( B ) or H37Rv ( C ) infection at an MOI of 10 for 1 h , and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . ( D - F ) RAW264 . 7 cells were transfected with control or miR - 155 inhibitor for 24 h followed by BCG ( D ) , H37Ra ( E ) or H37Rv ( F ) infection at an MOI of 10 for 1 h , and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . ( G and H ) RAW264 . 7 cells were transfected with miR - 155 mimic ( G ) or inhibitor ( H ) for 24 h followed by Texas Red - labeled BCG infection at an MOI of 10 for 1 h . Phagocytosis of mycobacteria was determined by flow cytometry . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 .

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 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 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 2(A-C) RAW264.7 cells were transfected with control or miR-155 mimic for 24 h followed by BCG (A), H37Ra (B) or H37Rv (C) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints.(D-F) RAW264.7 cells were transfected with control or miR-155 inhibitor for 24 h followed by BCG (D), H37Ra (E) or H37Rv (F) infection at an MOI of 10 for 1 h, and intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints.(G and H) RAW264.7 cells were transfected with miR-155 mimic (G) or inhibitor (H) for 24 h followed by Texas Red-labeled BCG infection at an MOI of 10 for 1 h. Phagocytosis of mycobacteria was determined by flow cytometry. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01.


Figure 3RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h and then infected with Texas Red - labeled BCG for 1 h . Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG antibody ( A ) The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B ) RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h and then infected with Texas Red - labeled BCG for 1 h . Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG antibody ( A ) , or labeled with a fluorogenic substrate for proteases , DQ - Green ( C ) . The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B ) or DQ - Green labeled ( D ) lysosomes was quantified , respectively . Cells treated with rapamycin were used as a positive control . Arrows indicate the co - localization of BCG with lysosomes ; scale bar = 5 µm . Quantification of data are shown as the mean ± SEM of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A)The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B)RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h and then infected with Texas Red-labeled BCG for 1 h. Lysosomes were immunolabeled with CD63 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG antibody (A), or labeled with a fluorogenic substrate for proteases, DQ-Green (C). The colocalization of BCG with lysosome was detected by confocal microscopy. The percentage of co-localization of BCG with CD63-positive (B) or DQ-Green labeled (D) lysosomes was quantified, respectively. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with lysosomes; scale bar = 5 µm. Quantification of data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01.


Figure 4 ( A - D ) RAW264 . 7 cells were transfected with miR - 155 mimic ( A and C ) or inhibitor ( B and D ) for 24 h and then either left uninfected or infected with BCG . Expression levels of miR - 155 were detected by real - time PCR ( A and B ) . ( A - D ) RAW264 . 7 cells were transfected with miR - 155 mimic ( A and C ) or inhibitor ( B and D ) for 24 h and then either left uninfected or infected with BCG . Expression levels of miR - 155 were detected by real - time PCR ( A and B ) . The LC3 levels were detected by Western - blot , and the ratios of LC3 - II / β - actin were calculated as shown below the representative blot ( C and D ) ( E and F ) RAW264 . 7 cells transfected with miR - 155 mimic ( E ) or inhibitor ( F ) was incubated with DMSO or bafilomycin A1 ( BafA . ) at a concentration of 100 nM for 2 h , and then LC3 levels were detected by Western - blot . The ratios of LC3 - II / β - actin were calculated as shown below the representative blot . * * * , p < 0 . 001 .

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

Figure 4(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B).(A-D) RAW264.7 cells were transfected with miR-155 mimic (A and C) or inhibitor (B and D) for 24 h and then either left uninfected or infected with BCG. Expression levels of miR-155 were detected by real-time PCR (A and B). The LC3 levels were detected by Western-blot, and the ratios of LC3-II/β-actin were calculated as shown below the representative blot (C and D)(E and F) RAW264.7 cells transfected with miR-155 mimic (E) or inhibitor (F) was incubated with DMSO or bafilomycin A1 (BafA.) at a concentration of 100 nM for 2 h, and then LC3 levels were detected by Western-blot. The ratios of LC3-II/β-actin were calculated as shown below the representative blot. ***, p<0.001.


Figure 5 ( A and B ) RAW264 . 7 cells stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic for 24 h , and GFP - LC3 puncta ( > 1 µm ) were detected by confocal microscopy ( A ) and quantified ( B ) . RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h . Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( Green ) , and LC3 puncta ( > 1 µm ) were detected by confocal microscopy ( C ) and quantified ( D ) . ( E and F ) RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h , and then incubated with a specific fluorescent dye MDC ( 50 µM ) . The MDC - labeled autophagic vacuoles were detected by confocal microscopy ( E ) and cells with MDC - positive autophagic vacuoles were quantified ( F ) . Cells treated with rapamycin were used as a positive control . Arrows indicate the GFP - LC3 puncta or autophagic vacuoles labeled with MDC ; scale bar = 5 µm . Data are shown as the mean ± SEM of three independent experiments ( n = 300 cells ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 O O O O O O O O O O O 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

Figure 5(A and B) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic for 24 h, and GFP-LC3 puncta (>1 µm) were detected by confocal microscopy (A) and quantified (B).RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green), and LC3 puncta (>1 µm) were detected by confocal microscopy (C) and quantified (D).(E and F) RAW264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with a specific fluorescent dye MDC (50 µM). The MDC-labeled autophagic vacuoles were detected by confocal microscopy (E) and cells with MDC-positive autophagic vacuoles were quantified (F). Cells treated with rapamycin were used as a positive control. Arrows indicate the GFP-LC3 puncta or autophagic vacuoles labeled with MDC; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 300 cells). **, p<0.01; ***, p<0.001.


Figure 6 ( A ) RAW264 . 7 cells stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic and then infected with Texas Red - labeled BCG for 1 h . The co - localization of BCG with LC3 was detected by confocal microscopy . ( B ) Quantification of the co - localization of BCG with LC3 - positive autophagosomes is shown . ( C ) RAW264 . 7 cells were transiently transfected with control or miR - 155 mimic , and then infected with Texas Red - labeled BCG for 1 h . Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488 - conjugated goat anti - rabbit IgG ( Green ) . The co - localization of BCG with endogenous LC3 was detected by confocal microscopy . ( D ) Quantification of the co - localization of BCG with LC3 - positive autophagosomes is shown . ( E ) After transient transfection with control or miR - 155 mimic , RAW264 . 7 cells were infected with Texas Red - labeled BCG for 1 h , and then were labeled with a specific fluorescent dye MDC ( 50 µM ) for autophagic vacuoles . The co - localization of BCG with MDC - positive autophagic vacuoles was detected by confocal microscopy . ( F ) Quantification of the co - localization of BCG with MDC - positive autophagosomes is shown . Cells treated with rapamycin were used as a positive control . Arrows indicate the co - localization of BCG with autophagosomes ; scale bar = 5 µm . Data are shown as the mean ± SEM of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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 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 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

Figure 6(A) RAW264.7 cells stably expressing GFP-LC3 were transiently transfected with control or miR-155 mimic and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG with LC3 was detected by confocal microscopy. (B) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.(C) RAW264.7 cells were transiently transfected with control or miR-155 mimic, and then infected with Texas Red-labeled BCG for 1 h. Endogenous LC3 was stained with LC3 antibody followed by Alexa Fluor 488-conjugated goat anti-rabbit IgG (Green). The co-localization of BCG with endogenous LC3 was detected by confocal microscopy. (D) Quantification of the co-localization of BCG with LC3-positive autophagosomes is shown.(E) After transient transfection with control or miR-155 mimic, RAW264.7 cells were infected with Texas Red-labeled BCG for 1 h, and then were labeled with a specific fluorescent dye MDC (50 µM) for autophagic vacuoles. The co-localization of BCG with MDC-positive autophagic vacuoles was detected by confocal microscopy. (F) Quantification of the co-localization of BCG with MDC-positive autophagosomes is shown. Cells treated with rapamycin were used as a positive control. Arrows indicate the co-localization of BCG with autophagosomes; scale bar = 5 µm. Data are shown as the mean ± SEM of three independent experiments (n = 100 phagosomes). **, p<0.01; ***, p<0.001.


Figure 7 ( A and B ) RAW 264 . 7 cells were transiently transfected with control or miR - 155 mimic for 24 h , and then incubated with DMSO or 3 - MA for 2 h . Protein levels of LC3 were detected by Western - blot in uninfected cells ( A ) Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h ( B ) ( C and D ) RAW 264 . 7 cells were transiently co - transfected with control or miR - 155 mimic together with a control siRNA or Atg7 siRNA . The expression levels of Atg7 and LC3 were detected by Western - blot ( C ) . Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h ( D ) . Values of LC3 - II / β - actin ratios are indicated below the representative blot . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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 B-PANEL_START 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 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 and B) RAW 264.7 cells were transiently transfected with control or miR-155 mimic for 24 h, and then incubated with DMSO or 3-MA for 2 h. Protein levels of LC3 were detected by Western-blot in uninfected cells (A)Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B)(C and D) RAW 264.7 cells were transiently co-transfected with control or miR-155 mimic together with a control siRNA or Atg7 siRNA. The expression levels of Atg7 and LC3 were detected by Western-blot (C).Intracellular mycobacterial viability was determined by CFU assay at the indicated time after challenging with BCG for 1 h (D). Values of LC3-II/β-actin ratios are indicated below the representative blot. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


Figure 8 ( A ) Sequences of mouse and human miR - 155 and their predicted interactions with conserved 7 - mer 1A miR - 155 seeds found within the Rheb 3 ′ UTRs of different species are shown . The sequence of the Rheb 3 ′ UTR seed mutant used for the reporter assays and the predicted disruption of the miR - 155 interaction are also shown . B ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic and a wild - type ( WT - Rheb ) or mutated Rheb 3 ′ UTR ( mut - Rheb ) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection . Data are shown as the mean ± SEM of three independent experiments . * * * , p < 0 . 001 ; NS , not significant . ( C - F ) RAW264 . 7 cells were transfected with miR - 155 mimic ( C and E ) or inhibitor ( D and F ) for 24 h and either left uninfected or infected with BCG . Protein expression levels of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below the representative blot ( C and D ) ( C - F ) RAW264 . 7 cells were transfected with miR - 155 mimic ( C and E ) or inhibitor ( D and F ) for 24 h and either left uninfected or infected with BCG . Protein expression levels of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below the representative blot ( C and D ) , and expression levels of Rheb mRNA were detected by RT - PCR ( E and F ) .

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 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 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 8(A) Sequences of mouse and human miR-155 and their predicted interactions with conserved 7-mer 1A miR-155 seeds found within the Rheb 3′UTRs of different species are shown. The sequence of the Rheb 3′UTR seed mutant used for the reporter assays and the predicted disruption of the miR-155 interaction are also shown.B) RAW264.7 cells were co-transfected with control or miR-155 mimic and a wild-type (WT-Rheb) or mutated Rheb 3′UTR (mut-Rheb) luciferase reporter plasmid and assessed for luciferase activity at 24 h after transfection. Data are shown as the mean ± SEM of three independent experiments. ***, p<0.001; NS, not significant.(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D)(C-F) RAW264.7 cells were transfected with miR-155 mimic (C and E) or inhibitor (D and F) for 24 h and either left uninfected or infected with BCG. Protein expression levels of Rheb were detected by Western-blot. Values of Rheb/β-actin ratios are indicated below the representative blot (C and D), and expression levels of Rheb mRNA were detected by RT-PCR (E and F).


Figure 9 ( A ) RAW264 . 7 cells were transfected with a plasmid expressing Rheb for 24 h , and then incubated with DMSO or BafA . for 2 h . The expression levels of Rheb and LC3 were detected by Western - blot . Values of LC3 - II / β - actin ratios are indicated below the representative blot . ( B and C ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h , and then infected with Texas Red - labeled BCG for 1 h . The co - localization of BCG and MDC - labeled autophagosome was detected by confocal microscopy . Arrows indicate Quantification of BCG co - localization with autophagosomes described in B is shown ( C ) . D ) RAW264 . 7 cells were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h . Cells were infected with BCG for 1 h , and washed for three times to remove extracellular mycobacteria . Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints . Data are shown as the mean ± SEM of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9(A) RAW264.7 cells were transfected with a plasmid expressing Rheb for 24 h, and then incubated with DMSO or BafA. for 2 h. The expression levels of Rheb and LC3 were detected by Western-blot. Values of LC3-II/β-actin ratios are indicated below the representative blot.(B and C) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h, and then infected with Texas Red-labeled BCG for 1 h. The co-localization of BCG and MDC-labeled autophagosome was detected by confocal microscopy. Arrows indicate Quantification of BCG co-localization with autophagosomes described in B is shown (C).D) RAW264.7 cells were co-transfected with control or miR-155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h. Cells were infected with BCG for 1 h, and washed for three times to remove extracellular mycobacteria. Intracellular mycobacterial viability was determined by CFU assay at the indicated timepoints. Data are shown as the mean ± SEM of three independent experiments. *, p<0.05; **, p<0.01; NS, not significant.


Figure 1 ( b , c ) Immunoblots of recruitment reactions performed as described in ( a ) , with the indicated proteins omitted . In ( c ) , a mid reaction wash in high salt ( 0 . 5 M NaCl ) buffer ( HSW ) was included following DDK phosphorylation as indicated . ( d ) Recruitment reaction performed as in ( a ) , using wild type ( wt ) or a Cdc45 - binding mutant ( 3E1 ) of Sld3 . ( f ) Wild type or mutant FLAG - Sld3 was added to an S - phase protein extract and tested for interaction with Cdc45 . Immunoprecipitated proteins were analysed by immunoblot . ( h ) Reaction performed as in ( g ) . Nascent DNA was separated in a 0 . 7 % alkaline agarose gel in this and all subsequent replication reactions .

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Figure 1(b, c) Immunoblots of recruitment reactions performed as described in (a), with the indicated proteins omitted. In (c), a mid reaction wash in high salt (0.5 M NaCl) buffer (HSW) was included following DDK phosphorylation as indicated.(d) Recruitment reaction performed as in (a), using wild type (wt) or a Cdc45-binding mutant (3E1) of Sld3.(f) Wild type or mutant FLAG-Sld3 was added to an S-phase protein extract and tested for interaction with Cdc45. Immunoprecipitated proteins were analysed by immunoblot.(h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.


Figure 2 ( a ) Reaction performed as in Fig . 1a , except Cdc45 was omitted . ( b ) Reaction performed as in Fig . 1g , with the indicated proteins omitted . ( e ) Reaction performed as in ( a ) , except Sld3 / 7 was incubated with Rad53 before addition to reactions . ATP was omitted from this pre - incubation as indicated . ( f ) Sld3 recruitment reaction performed as in ( a ) . Sld7 was omitted from this experiment .

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Figure 2(a) Reaction performed as in Fig. 1a, except Cdc45 was omitted.(b) Reaction performed as in Fig. 1g, with the indicated proteins omitted.(e) Reaction performed as in (a), except Sld3/7 was incubated with Rad53 before addition to reactions. ATP was omitted from this pre-incubation as indicated.(f) Sld3 recruitment reaction performed as in (a). Sld7 was omitted from this experiment.


Figure 3 ( b ) Recruitment reaction performed as described in ( a ) . ( c ) Sld3 / 7 recruitment reaction conducted as in Fig . 2a . ( d ) Replication reaction performed as in Fig . 1g . ( e ) Representative tetrads from dissection of SLD3 + / sld3 - 6E heterozygotes .

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Figure 3(b) Recruitment reaction performed as described in (a).(c) Sld3/7 recruitment reaction conducted as in Fig. 2a.(d) Replication reaction performed as in Fig. 1g.(e) Representative tetrads from dissection of SLD3+/sld3-6E heterozygotes.


Figure 4 ( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( B ) , either reconstituted Sld3 / 7 complex ( lanes 6 and 7 ) or Sld3 alone ( lanes 8 and 9 ) was coupled to beads . In ( C ) , MCM was treated with Lambda Phosphatase after dissociation from DNA ( lanes 3 and 7 ) . ( b , c , D ) Immunoblot of reactions performed as described in ( a ) . In ( D ) , Sld7 was omitted . ( E ) 30 - residue phosphorylated Mcm6 peptides were bound to streptavidin - coated beads and used as bait to pulldown Sld3 . Peptide - coupled beads were dephosphorylated with Lambda Phosphatase as indicated . ( F ) Reaction conducted as in ( a ) , using wild type ( wt ) or the indicated S / T - A substitution mutant versions of Mcm4 and Mcm6 . Positions of mutations in Mcm6 are summarised in Fig . EV5c .

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Figure 4(b, c, D) Immunoblot of reactions performed as described in (a). In (B), either reconstituted Sld3/7 complex (lanes 6 and 7) or Sld3 alone (lanes 8 and 9) was coupled to beads. In (C), MCM was treated with Lambda Phosphatase after dissociation from DNA (lanes 3 and 7).(b, c, D) Immunoblot of reactions performed as described in (a). In (D), Sld7 was omitted.(E) 30-residue phosphorylated Mcm6 peptides were bound to streptavidin-coated beads and used as bait to pulldown Sld3. Peptide-coupled beads were dephosphorylated with Lambda Phosphatase as indicated.(F) Reaction conducted as in (a), using wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6. Positions of mutations in Mcm6 are summarised in Fig. EV5c.


Figure 5 ( a ) Sld3 / 7 recruitment reaction performed as in Fig . 2a . ( b ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) and ( b ) used MCM containing wild type ( wt ) or the indicated S / T - A substitution mutant versions of Mcm4 and Mcm6 . c ) Quantification of ( b ) , performed using ImageQuant software ( GE Healthcare ) . ( d ) Representative tetrads from diploids heterozygous for mcm4 - 25A and mcm6ΔN + 11A .

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Figure 5(a) Sld3/7 recruitment reaction performed as in Fig. 2a.(b) Replication reaction performed as in Fig. 1g. Experiments in (a) and (b) used MCM containing wild type (wt) or the indicated S/T-A substitution mutant versions of Mcm4 and Mcm6.c) Quantification of (b), performed using ImageQuant software (GE Healthcare).(d) Representative tetrads from diploids heterozygous for mcm4-25A and mcm6ΔN+11A.


Figure 6 ( b ) Immunoblots of reactions performed as in ( a ) part i ( lanes 1 - 3 ) or ii ( lanes 4 - 6 ) . ( c ) Reactions in i and ii were performed as described in ( a ) parts i and ii , respectively .

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Figure 6(b) Immunoblots of reactions performed as in (a) part i (lanes 1-3) or ii (lanes 4-6).(c) Reactions in i and ii were performed as described in (a) parts i and ii, respectively.


Figure 7 ( a , b ) Recruitment reactions performed as in Fig . 2a . Sld7 was omitted in ( b ) . ( c ) Recruitment reaction conducted as in Fig . 3a , with washes performed at 0 . 3 M KCl . ( d ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) - ( d ) used MCM containing wild type ( wt ) or S / T - D substitution mutants of Mcm4 and Mcm6 as indicated . In ( d ) , MCM loading was equalised across samples by using 5 - fold more mutant than wild type MCM protein , as is shown in Fig . EV6c .

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Figure 7(a, b) Recruitment reactions performed as in Fig. 2a. Sld7 was omitted in (b).(c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl.(d) Replication reaction performed as in Fig. 1g. Experiments in (a)-(d) used MCM containing wild type (wt) or S/T-D substitution mutants of Mcm4 and Mcm6 as indicated. In (d), MCM loading was equalised across samples by using 5-fold more mutant than wild type MCM protein, as is shown in Fig. EV6c.


Figure 3B Representative TIRF - M of DCP1 - GFP ( DCP1pro : DCP1 - GFP transgene ) in the lateral PM of epidermal cells showing the transient attachment of DCP1 - positive puncta to the PM . Yellow arrowheads denote a PB that shows motility at the PM focal plane ; blue arrowheads show a PB that transiently localizes to the PM . Scale bars : 2 μm . The corresponding kymographs are shown to the right . Right : distribution of immobile and mobile DCP1 molecules relative to the motility log ( D ) value of - 0 . 75 ( threshold ; see Material and methods for details ) , in NS or HS conditions ( D , diffusion coefficient ) . Inset : individual trajectories of mobile DCP1 - GFP in NS and HS conditions ( 500 frames , n = 120 ) , showing a combination of directional and Brownian motion for both NS / HS . The green arrowheads denote the beginning of the NS and HS tracks for DCP1 - GFP . C Representative confocal micrographs from lines co - expressing RPS5apro : HF - mScarlet - DCP1 and PIN2pro : PIN2 - GFP ( epidermal cells , region 3 , Scale bars : 5 μm ) . Bottom : polarity index of DCP1 in root meristematic cells ( compared to propidium iodide ( PI ) and tubulin staining of root cells ; N , biological replicates = 3 roots , n = 13 cells ) . Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells . The arrowhead in PIN2 indicates the cell plate or PM in DCP1 . Right : representative confocal micrographs showing that PM localization is independent of the promoter used ( DCP1pro , 35Spro ; region 2 , epidermal cells , or RPS5apro on the lower right ) . The details from the inset show increased localization at the cell edge ( discussed later ) . mSc , mScarlet .

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Figure 3B Representative TIRF-M of DCP1-GFP (DCP1pro:DCP1-GFP transgene) in the lateral PM of epidermal cells showing the transient attachment of DCP1-positive puncta to the PM. Yellow arrowheads denote a PB that shows motility at the PM focal plane; blue arrowheads show a PB that transiently localizes to the PM. Scale bars: 2 μm. The corresponding kymographs are shown to the right. Right: distribution of immobile and mobile DCP1 molecules relative to the motility log(D) value of -0.75 (threshold; see Material and methods for details), in NS or HS conditions (D, diffusion coefficient). Inset: individual trajectories of mobile DCP1-GFP in NS and HS conditions (500 frames, n = 120), showing a combination of directional and Brownian motion for both NS/HS. The green arrowheads denote the beginning of the NS and HS tracks for DCP1-GFP.C Representative confocal micrographs from lines co-expressing RPS5apro:HF-mScarlet-DCP1 and PIN2pro:PIN2-GFP (epidermal cells, region 3, Scale bars: 5 μm). Bottom: polarity index of DCP1 in root meristematic cells (compared to propidium iodide (PI) and tubulin staining of root cells; N, biological replicates = 3 roots, n = 13 cells). Polarity index calculated as the ratio of average of apical and basal VS lateral side of fluorescence signal intensity of the root epidemies cells. The arrowhead in PIN2 indicates the cell plate or PM in DCP1. Right: representative confocal micrographs showing that PM localization is independent of the promoter used (DCP1pro, 35Spro; region 2, epidermal cells, or RPS5apro on the lower right). The details from the inset show increased localization at the cell edge (discussed later). mSc, mScarlet.


Figure 4B Representative confocal micrograph of DCP1 - GFP ( DCP1pro : DCP1 - GFP , transgene ) in root meristematic cells under NS / HS conditions ( region 2 ) . Scale bars , 5 μm . Note the depletion of DCP1 from the PM upon HS , but the increased edge / vertex signal ( yellow arrowheads denote the vertex signal ) . Right : DCP1 signal intensity at the PM or edge / vertex ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 23 PMs or edges / vertices ) . C Representative confocal images showing fusion ( coarsening ) , fission , and growth of PBs ( DCP1 - positive ) at the PM ( region 3 ) . Right : states of PBs ( dynamic : fusion and fission and non - dynamic : stable ; N , biological replicates = 2 , n ( pooled data of 3 biological replicates ) = 6 - 8 PBs ) . As a cautionary note , the " stable " PBs may not show dynamicity in the imaging time used ( ~ 3 - 5 min ) but later , may do .

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Figure 4B Representative confocal micrograph of DCP1-GFP (DCP1pro:DCP1-GFP, transgene) in root meristematic cells under NS/HS conditions (region 2). Scale bars, 5 μm. Note the depletion of DCP1 from the PM upon HS, but the increased edge/vertex signal (yellow arrowheads denote the vertex signal). Right: DCP1 signal intensity at the PM or edge/vertex (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-23 PMs or edges/vertices).C Representative confocal images showing fusion (coarsening), fission, and growth of PBs (DCP1-positive) at the PM (region 3). Right: states of PBs (dynamic: fusion and fission and non-dynamic: stable; N, biological replicates = 2, n (pooled data of 3 biological replicates) = 6-8 PBs). As a cautionary note, the "stable" PBs may not show dynamicity in the imaging time used (~3-5 min) but later, may do.


Figure 5Confocal micrographs showing single optical sections PLA - assays producing signal that resembles spots . The antibodies used were anti - FLAG / anti - GFP detecting the HF - mScarlet - DCP1 / DCP2 - YFP , respectively ( RPS5apro : HF - mScarlet - DCP1 and 35Spro : DCP2 - YFP ) . Inset 1 : magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals ( colocalization and Pearson ' s correlation coefficient PCC value for the spots shown ) . The dotted white line in the PLA channel corresponds to the PM plane . Inset 2 : positive PLA signal for DCP1 and DCP2 in the nucleus . " n1 - n3 " correspond to nuclei regions ( green circles ) . On the right , note the PLA spot nearby the nucleus ( " detail of PLA signal " ) . The chart shows the quantification of PLA spots per cell at puncta ( cytoplasm ) or on the PM ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 16 - 33 cells ) . As a cautionary technical note , the cytoplasmic , nuclear or PM " spots " do not connote physiologically relevant puncta , condensates or PM clusters . Scale bar : 20 μm / 10 μm for the insets .

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Figure 5Confocal micrographs showing single optical sections PLA-assays producing signal that resembles spots. The antibodies used were anti-FLAG/anti-GFP detecting the HF-mScarlet-DCP1/ DCP2-YFP, respectively (RPS5apro:HF-mScarlet-DCP1 and 35Spro:DCP2-YFP). Inset 1: magnification showing the colocalization of PLA spots with DCP1 or DCP2 signals (colocalization and Pearson's correlation coefficient PCC value for the spots shown). The dotted white line in the PLA channel corresponds to the PM plane. Inset 2: positive PLA signal for DCP1 and DCP2 in the nucleus. "n1-n3" correspond to nuclei regions (green circles). On the right, note the PLA spot nearby the nucleus ("detail of PLA signal"). The chart shows the quantification of PLA spots per cell at puncta (cytoplasm) or on the PM (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 16-33 cells). As a cautionary technical note, the cytoplasmic, nuclear or PM "spots" do not connote physiologically relevant puncta, condensates or PM clusters. Scale bar: 20 μm / 10 μm for the insets.


Figure 6B Representative confocal micrographs with acceptor photobleaching - FRET efficiency between SCAR2 - mCherry ( up ) or BRK1 - mRuby ( down ) and DCP1 - GFP ( epidermal cells , regions 1 and 3 ) . Right : normalized FRET efficiency between SCAR2 or BRK1 and DCP1 , respectively , among the different developmental root regions ( N , biological replicates = 2 , n = 16 cells ) . D Representative confocal micrographs showing DCP1 localization detected by α - DCP1 in the wild type ( WT ) or the scar1 scar2 scar3 scar4 ( scar1234 ) quadruple mutant or in live - cell imaging of mScarlet - DCP1 ( RPS5apro : HF - mScarlet - DCP1 ) in WT or brk1 mutant ( bottom right ; epidermal cells , region 3 for α - DCP1 and 2 for live - cell imaging ) . The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge / vertex . Small panels ( insets ) at right show details corresponding to the regions delineated by dashed lines , where arrowhead denotes the edge signal of DCP1 in WT ; scale bars , 1 μm . Bottom : percentage of cells with proper edge / vertex localization and quantification of PB numbers ( DCP1 - foci ; N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 35 cells ) .

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Figure 6B Representative confocal micrographs with acceptor photobleaching-FRET efficiency between SCAR2-mCherry (up) or BRK1-mRuby (down) and DCP1-GFP (epidermal cells, regions 1 and 3). Right: normalized FRET efficiency between SCAR2 or BRK1 and DCP1, respectively, among the different developmental root regions (N, biological replicates = 2, n = 16 cells).D Representative confocal micrographs showing DCP1 localization detected by α-DCP1 in the wild type (WT) or the scar1 scar2 scar3 scar4 (scar1234) quadruple mutant or in live-cell imaging of mScarlet-DCP1 (RPS5apro:HF-mScarlet-DCP1) in WT or brk1 mutant (bottom right; epidermal cells, region 3 for α-DCP1 and 2 for live-cell imaging). The arrowheads denote the lack of robust DCP1 localization in scar1234 at the edge/vertex. Small panels (insets) at right show details corresponding to the regions delineated by dashed lines, where arrowhead denotes the edge signal of DCP1 in WT; scale bars, 1 μm. Bottom: percentage of cells with proper edge/vertex localization and quantification of PB numbers (DCP1-foci; N, biological replicates = 3, n (pooled data of 3 biological replicates) = 18-35 cells).


Figure 7A Representative confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct - mCherry in lines co - expressing DCP1pro : DCP1 - GFP ( or variants ) and UBQ10pro : LifeAct - mCherry ( cell edges are indicated by yellow arrowheads ) . Right : relative signal intensity of actin at cell edges / vertices ( normalized to the PM ) in epidermal cells ( N , biological replicates = 3 , n = 15 - 30 , regions 2 - 3 ) .

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Figure 7A Representative confocal micrographs showing colocalization between DCP1 or two DCP1 phosphovariants with LifeAct-mCherry in lines co-expressing DCP1pro:DCP1-GFP (or variants) and UBQ10pro:LifeAct-mCherry (cell edges are indicated by yellow arrowheads). Right: relative signal intensity of actin at cell edges/vertices (normalized to the PM) in epidermal cells (N, biological replicates = 3, n = 15-30, regions 2-3).


Figure 8B Representative confocal micrographs showing actin localization in WT , dcp1 - 1 , dcp1 - 3 , scar1234 , dcp2 - 1 , dcp5 and pat1 upon phalloidin staining and graph ( right ) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes ( N , biological replicates = 3 , n = 7 - 9 roots , bars show means + s . d . ) .

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Figure 8B Representative confocal micrographs showing actin localization in WT, dcp1-1, dcp1-3, scar1234, dcp2-1, dcp5 and pat1 upon phalloidin staining and graph (right) indicating the percentage of cells in region 3 with an accumulation of actin at edges in various genotypes (N, biological replicates = 3, n = 7-9 roots, bars show means + s.d.).


Figure 9A SE - FRET efficiency between DCP1 - GFP or its phosphovariants with SCAR2 - mCherry ( among the three different root regions ; mainly epidermal cells ) . Scale bar : 50 μm . The arrowhead denotes high FRET efficiency at edges / vertices of region 3 . Right : signal quantification of SE - FRET efficiency between the indicated combinations at the epidermis of 3 regions ( N , biological replicates = 6 , n ( pooled data of 3 biological replicates ) = 10 ) . C Representative confocal micrographs showing SCAR2 - mCherry localization in WT and dcp1 - 3 mutant , respectively ( root region 2 , epidermal cells ) and quantification of edge / vertex with SCAR2 a confined signal ( N , biological replicates = 3 , n = 5 - 8 cells ) . D Representative confocal micrographs showing the colocalization between DCP1 - GFP or phosphovariants and SCAR2 - mCherry in root meristematic cells ( root region 2 , epidermal cells ) . Scale bars : 10 μm . The insets show details of colocalization ; the white arrowhead denotes the expansion of the SCAR2 / DCP1 domain , while the yellow arrowheads the restricted edge / vertex SCAR2 / DCP1 domains . Scale bars : 3 μm . The graph indicates the relative signal intensity for the indicated combinations ( as Pearson ' s correlation coefficient ; N , biological replicates = 3 , n = 5 at edges / vertices : spread edges were not considered in calculations ) .

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 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 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

Figure 9A SE-FRET efficiency between DCP1-GFP or its phosphovariants with SCAR2-mCherry (among the three different root regions; mainly epidermal cells). Scale bar: 50 μm. The arrowhead denotes high FRET efficiency at edges/vertices of region 3. Right: signal quantification of SE-FRET efficiency between the indicated combinations at the epidermis of 3 regions (N, biological replicates = 6, n (pooled data of 3 biological replicates) = 10).C Representative confocal micrographs showing SCAR2-mCherry localization in WT and dcp1-3 mutant, respectively (root region 2, epidermal cells) and quantification of edge/vertex with SCAR2 a confined signal (N, biological replicates = 3, n = 5-8 cells).D Representative confocal micrographs showing the colocalization between DCP1-GFP or phosphovariants and SCAR2-mCherry in root meristematic cells (root region 2, epidermal cells). Scale bars: 10 μm. The insets show details of colocalization; the white arrowhead denotes the expansion of the SCAR2/DCP1 domain, while the yellow arrowheads the restricted edge/vertex SCAR2/DCP1 domains. Scale bars: 3 μm. The graph indicates the relative signal intensity for the indicated combinations (as Pearson's correlation coefficient; N, biological replicates = 3, n = 5 at edges/vertices: spread edges were not considered in calculations).


Figure 10A Representative images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR - WAVE components ( 5 - day - old seedlings ) . The arrowheads show the growth defects of homozygous dcp1 - 1 or dcp2 - 1 mutants ( denoted - / - ; heterozygous denoted dcp1 - 1 / DCP1 ; details are also shown ) . Lower : graph showing relative root length ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 4 roots , bars show means + s . d ) . B DCP1 regulates cell expansion anisotropy . Representative confocal micrographs showing FM4 - 64 staining of the WT , dcp1 - 1 and dcp2 - 1 mutants ( 2 μM , 10 min ) . Scale bars , 20 μm . Right : percentage of isotropic cells per root meristem ( % , epidermal cells ) in each genotype ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 3 - 5 roots , bars show means ± s . d ) . Examples of isotropic or anisotropic cells are shown , along with the developmental axis offset at the x - and y - axes .

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 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

Figure 10A Representative images showing the phenotypes of dcp1 mutants and mutants in other PB core components or SCAR-WAVE components (5-day-old seedlings). The arrowheads show the growth defects of homozygous dcp1-1 or dcp2-1 mutants (denoted -/-; heterozygous denoted dcp1-1/DCP1; details are also shown). Lower: graph showing relative root length (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-4 roots, bars show means + s.d).B DCP1 regulates cell expansion anisotropy. Representative confocal micrographs showing FM4-64 staining of the WT, dcp1-1 and dcp2-1 mutants (2 μM, 10 min). Scale bars, 20 μm. Right: percentage of isotropic cells per root meristem (%, epidermal cells) in each genotype (N, biological replicates = 3, n (pooled data of 3 biological replicates) = 3-5 roots, bars show means ± s.d). Examples of isotropic or anisotropic cells are shown, along with the developmental axis offset at the x- and y-axes.


Figure 1 ( A , B and C ) Polyphenols TA , PGG and EGCG activate rhpR - lux in wild type but not rhpS deletion Psph strains . Data information : , * indicate significant differences between mock group and strains under the different test conditions . ( Student ' s t test . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) . ( D , E and F ) Polyphenols repress T3SS induction in P . syringae in MM . Transcripts of the indicated genes in the presence of 100 - μM TA ( D ) , 100 - µM PGG ( E ) or 2 - mM EGCG ( F ) were determined by RT - qPCR 6 hours after culturing in MM . Data information : , * indicate significant differences between mock group and strains under the different test conditions . ( Student ' s t test . n . s . , not significant , * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

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 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

Figure 1(A, B and C) Polyphenols TA , PGG and EGCG activate rhpR-lux in wild type but not rhpS deletion Psph strains. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).(D, E and F) Polyphenols repress T3SS induction in P. syringae in MM. Transcripts of the indicated genes in the presence of 100-μM TA (D), 100-µM PGG (E) or 2-mM EGCG (F) were determined by RT-qPCR 6 hours after culturing in MM. Data information: , * indicate significant differences between mock group and strains under the different test conditions. (Student's t test. n.s., not significant, *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


Figure 2 ( D , E and F ) Polyphenols inhibit the phosphatase activity of RhpS . The GST - RhpR was phosphorylated by adding 5 - μM PSPPH _ 3550c protein as well as 10 - μCi [ γ - 32P ] - ATP for 10 minutes . The RhpS - FL protein was treated in the presence of different concentrations of TA ( D ) , PGG ( E ) , or EGCG ( F ) for 10 min . Then , the full - length RhpS protein was added into the mixture phosphorylated GST - RhpR for 1 hour before stopping the reaction . The phosphorylated proteins were separated by 12 % SDS - PAGE . After electrophoresis , the SDS - PAGE gels were dried with gel - dryer and then exposed to a medical X - ray film overnight . This experiment was repeated twice , yielding similar results . Data information : the reaction was stopped by adding 5 × loading buffer before SDS - PAGE . Abbreviation is as follows : CBB , coomassie bright blue stain ; CFU , colony - forming unit . ( I , J and K ) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants . Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants . Disease symptoms were recorded 5 days after inoculation . Leaf disks ( 1 cm2 ) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs . The experiment was repeated three times . Data information : , * indicate significant differences between mock group and strains under the different test conditions as indicated . ( Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) . Error bars represent standard deviations ( n = 3 biological replicates ) .

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 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

Figure 2(D, E and F) Polyphenols inhibit the phosphatase activity of RhpS. The GST-RhpR was phosphorylated by adding 5-μM PSPPH_3550c protein as well as 10-μCi [γ-32P]-ATP for 10 minutes. The RhpS-FL protein was treated in the presence of different concentrations of TA (D), PGG (E), or EGCG (F) for 10 min. Then, the full-length RhpS protein was added into the mixture phosphorylated GST-RhpR for 1 hour before stopping the reaction. The phosphorylated proteins were separated by 12% SDS-PAGE. After electrophoresis, the SDS-PAGE gels were dried with gel-dryer and then exposed to a medical X-ray film overnight. This experiment was repeated twice, yielding similar results. Data information: the reaction was stopped by adding 5 × loading buffer before SDS-PAGE. Abbreviation is as follows: CBB, coomassie bright blue stain; CFU, colony-forming unit.(I, J and K) Polyphenols inhibits the virulence levels and in planta growth of Psph in bean plants. Bacterial virulence levels were estimated by inoculating Psph strains with or without polyphenols onto host bean plants. Disease symptoms were recorded 5 days after inoculation. Leaf disks (1 cm2) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001). Error bars represent standard deviations (n=3 biological replicates).


Figure 3 ( B , C and D ) Polyphenols bind to the RhpS - FL protein . The concentration of RhpS - FL protein was consistently measured as 0 . 6 - μM . The binding curves of TA ( B ) , PGG ( C ) and EGCG ( D ) are shown . The individual data points were shown ( n = 2 biological replicates ) . ( E ) Alanine - scanning mutagenesis of the RhpS sensor . RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector . LUX activity was estimated in the absence ( black frame ) and presence TA ( 50 - μM ) , PGG ( 100 - μM ) or EGCG ( 2 - mM ) . Error bars represent standard deviations ( n = 3 biological replicates ) . Data information : , * indicate significant differences between mock group and strains under the different test conditions as indicated . ( Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 ) .

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 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

Figure 3(B, C and D) Polyphenols bind to the RhpS-FL protein. The concentration of RhpS-FL protein was consistently measured as 0.6-μM. The binding curves of TA (B), PGG (C) and EGCG (D) are shown. The individual data points were shown (n=2 biological replicates).(E) Alanine-scanning mutagenesis of the RhpS sensor. RhpS containing a corresponding mutation was provided in the rhpS deletion strain on a pHM1 vector. LUX activity was estimated in the absence (black frame) and presence TA (50-μM), PGG (100-μM) or EGCG (2-mM). Error bars represent standard deviations (n=3 biological replicates). Data information: , * indicate significant differences between mock group and strains under the different test conditions as indicated. (Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001).


Figure 4 ( C and D ) The rhpS / PSPPH _ 3550 / PSPPH _ 3736 / PSPPH _ 5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain . The wild type , rhpS deletion and quadruple deletion strains were cultured in MM ( C ) for 6 hours or KB ( D ) to OD600 = 0 . 6 . Transcripts of the indicated genes were determined by RT - qPCR . Data information : , * indicate significant differences between rhpS mutant and quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 . ( F ) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain . Bacterial virulence levels were estimated by inoculating wild type , rhpS deletion and quadruple deletion strains onto host bean plants . Disease symptoms were recorded 5 days after inoculation . The experiment was repeated three times . ( G ) Leaf disks ( 1 cm2 ) from ( G ) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs . The experiment was repeated three times . Data information : , * indicate significant differences between rhpS mutant and quadruple mutant . Error bars represent standard deviations ( n = 3 biological replicates ) . Student ' s t test . * , P ≤ 0 . 05 , * * , P ≤ 0 . 01 , and * * * , P ≤ 0 . 001 .

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

Figure 4(C and D) The rhpS/PSPPH_3550/PSPPH_3736/PSPPH_5115 quadruple deletion strain restored the T3SS induction of rhpS deletion strain. The wild type, rhpS deletion and quadruple deletion strains were cultured in MM (C) for 6 hours or KB (D) to OD600=0.6. Transcripts of the indicated genes were determined by RT-qPCR. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.(F) The quadruple mutant strain restored the pathogenicity of rhpS deletion strain. Bacterial virulence levels were estimated by inoculating wild type, rhpS deletion and quadruple deletion strains onto host bean plants. Disease symptoms were recorded 5 days after inoculation. The experiment was repeated three times.(G) Leaf disks (1 cm2) from (G) were removed 5 days after inoculation and diluted in sterile water to count bacterial CFUs. The experiment was repeated three times. Data information: , * indicate significant differences between rhpS mutant and quadruple mutant. Error bars represent standard deviations (n=3 biological replicates). Student's t test. *, P ≤ 0.05, **, P ≤ 0.01, and ***, P ≤ 0.001.


Figure 1A Cytokine release by PBMCs challenged with heat inactivated S . aureus ( hiSa ) , and / or RNase A ( RA ) treated solution , or Sa19 ( TLR13 activating ORN ) upon TLR2 blockade ( T2 . 5 ; n . p . , not performed ; * * , p ≤ 0 . 01 ; n = 3 ) . C Cytokine release of PBMCs transfected with ORNs including TLR7 / 8 ligand RNA40 ( n = 3 ) . D , E Activity of PBMCs upon bacterial RNA challenge , pretreatment with chloroquine in ( D ) only ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) . F Activity of PBMCs upon mitochondrial ( mt ) RNA challenge ( Hs , human ; triangle , increasing doses ; LFA , Lipofectamine 2000 ; n = 3 ) . G Activity of PBMCs challenged with ORNs and total S . aureus ( Sa ) RNA ( Myd88d / d , mutant MyD88 expression not impairing LPS driven IL - 8 production ; n = 2 ) . H Responsiveness of undifferentiated ( undiff ) and 3 - or 8 days PMA differentiated ( ddi ) THP1 cells to Sa19 challenge ( n = 3 ) . I Activity of parental and Unc93b1 - / - - 3ddiTHP - 1 cells challenged with ORNs ( n = 3 ) .

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 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 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 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 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

Figure 1A Cytokine release by PBMCs challenged with heat inactivated S. aureus (hiSa), and/or RNase A (RA) treated solution, or Sa19 (TLR13 activating ORN) upon TLR2 blockade (T2.5; n.p., not performed; **, p ≤ 0.01; n = 3).C Cytokine release of PBMCs transfected with ORNs including TLR7/8 ligand RNA40 (n = 3).D, E Activity of PBMCs upon bacterial RNA challenge, pretreatment with chloroquine in (D) only (S, Svedberg; r, ribosomal; tot, total; n = 3).F Activity of PBMCs upon mitochondrial (mt) RNA challenge (Hs, human; triangle, increasing doses; LFA, Lipofectamine 2000; n = 3).G Activity of PBMCs challenged with ORNs and total S. aureus (Sa) RNA (Myd88d/d, mutant MyD88 expression not impairing LPS driven IL-8 production; n = 2).H Responsiveness of undifferentiated (undiff) and 3- or 8 days PMA differentiated (ddi) THP1 cells to Sa19 challenge (n = 3).I Activity of parental and Unc93b1-/--3ddiTHP-1 cells challenged with ORNs (n = 3).


Figure 2A Cytokine release of 3 days differentiated ( ddi ) THP1 cells toward stimulation with TLR ligands and bacterial RNAs ( S , Svedberg ; r , ribosomal ; tot , total ; n = 3 ) . B , C Responsiveness of Unc93b1 - / - - 3ddiTHP1 cells treated with T2 . 5 and challenged with heat inactivated ( hi ) bacteria ( triangle , decreasing doses ; n = 3 ) . D Ratios of constitutive mRNA amounts in 3 - versus 8ddi ( grey bar ) and of each of both versus undifferentiated THP1 cells ( black or white bar , respectively ) according to a transcriptome profiling result ( n = 1 ) . E , F NF - κB driven relative luciferase activity ( Rel . luc . act . ) of hTLR8 overexpressing HEK293 and murine RAW264 . 7 cells , respectively , upon challenges ( vector , empty plasmid ; n = 3 ) . G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness ( n = 3 ) .

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

Figure 2A Cytokine release of 3 days differentiated (ddi) THP1 cells toward stimulation with TLR ligands and bacterial RNAs (S, Svedberg; r, ribosomal; tot, total; n = 3).B, C Responsiveness of Unc93b1-/--3ddiTHP1 cells treated with T2.5 and challenged with heat inactivated (hi) bacteria (triangle, decreasing doses; n=3).D Ratios of constitutive mRNA amounts in 3- versus 8ddi (grey bar) and of each of both versus undifferentiated THP1 cells (black or white bar, respectively) according to a transcriptome profiling result (n = 1).E, F NF-κB driven relative luciferase activity (Rel. luc. act.) of hTLR8 overexpressing HEK293 and murine RAW264.7 cells, respectively, upon challenges (vector, empty plasmid; n = 3).G Transfection of TLR8 siRNA impairs 3ddiTHP1 cell responsiveness (n = 3).