A ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 3 , 7 and 18 months of age . Representative images show brain arteries and capillaries of TgN3R182C150 , sham and NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an anti - perlecan antibody ( green ) . Scale bar = 20µm . B ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 - ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non - vaccinated 3 months old TgN3R182C150 ( n = 3 ) and 7 months old TgN3R182C150 ( n = 6 ) mice and 18 months old TgN3R182C150 ( n = 3 ) . Quantification of NOTCH3 - ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 - ECD stained area and average size per vessel revealed a significant decrease in NOTCH3 - ECD deposition in brain arteries and capillaries between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice . Statistical significance was assessed using a Brown - Forsythe and Welch ANOVA tests followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel area ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0029 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0004 ; 7 m . o . vs . Sham , ns P = 0 . 7326 ; 7 m . o . vs . Vaccinated , * * P = 0 . 003 ; Sham vs . Vaccinated , * * * * P < 0 . 0001 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 3 m . o . vs . 18 m . o . * * P = 0 . 0038 ; 3 m . o . vs . 7 m . o . * * * P = 0 . 0002 ; 7 m . o . vs . Sham , ns P = 0 . 9913 ; 7 m . o . vs . Vaccinated , * P = 0 . 021 ; Sham vs . Vaccinated , * * * P = 0 . 001 ) . NOTCH3 ECD deposits size : 3 m . o . vs . 18 m . o . * P = 0 . 0215 ; 3 m . o . vs . 7 m . o . * * P = 0 . 0029 ; 7 m . o . vs . Sham , ns P = 0 . 3622 ; 7 m . o . vs . Vaccinated , * P = 0 . 0428 ; Sham vs . Vaccinated , * * P = 0 . 0038 , ns = non - significant ) . Dotted lines indicate quartiles and dashed thicker lines are the median .

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

A) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 3, 7 and 18 months of age. Representative images show brain arteries and capillaries of TgN3R182C150, sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an anti-perlecan antibody (green). Scale bar =20µm. B) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries and capillaries between non-vaccinated 3 months old TgN3R182C150 (n=3) and 7 months old TgN3R182C150 (n=6) mice and 18 months old TgN3R182C150 (n=3). Quantification of NOTCH3-ECD deposits (numbers per 1,000 μm2) and NOTCH3-ECD stained area and average size per vessel revealed a significant decrease in NOTCH3-ECD deposition in brain arteries and capillaries between NOTCH3 EGF1-5 -immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice. Statistical significance was assessed using a Brown-Forsythe and Welch ANOVA tests followed by Dunnett's T3 multiple comparisons. (NOTCH3 ECD deposits (% of vessel area): 3 m.o. vs. 18 m.o. **P=0.0029; 3 m.o. vs. 7 m.o. ***P=0.0004; 7 m.o. vs. Sham, ns P=0.7326; 7 m.o. vs. Vaccinated, **P=0.003; Sham vs. Vaccinated, ****P<0.0001). NOTCH3 ECD deposits (number/1000 um2): 3 m.o. vs. 18 m.o. **P=0.0038; 3 m.o. vs. 7 m.o. ***P=0.0002; 7 m.o. vs. Sham, ns P=0.9913; 7 m.o. vs. Vaccinated, *P=0.021; Sham vs. Vaccinated, ***P=0.001). NOTCH3 ECD deposits size: 3 m.o. vs. 18 m.o. *P=0.0215; 3 m.o. vs. 7 m.o. **P=0.0029; 7 m.o. vs. Sham, ns P=0.3622; 7 m.o. vs. Vaccinated, *P=0.0428; Sham vs. Vaccinated, **P=0.0038, ns= non-significant). Dotted lines indicate quartiles and dashed thicker lines are the median.


A ) NOTCH3 ECD was detected in the whole blood serum of the non - treated TgN3R182C150 mice at three months ( n = 9 ) of age and further increased at seven months ( n = 6 ) of age . Serum from Notch3 - / - and C57Bl6 / J WT mice were included as negative controls . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( 3 m . o . vs 7 m . o . * * * P = 0 . 0005 ) . Error bars indicate standard error of the mean ( SEM ) .

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

A) NOTCH3 ECD was detected in the whole blood serum of the non-treated TgN3R182C150 mice at three months (n=9) of age and further increased at seven months (n=6) of age. Serum from Notch3 -/- and C57Bl6/J WT mice were included as negative controls. Statistical analysis was performed using unpaired student t test with Welch's correction. (3 m.o. vs 7 m.o. ***P=0.0005). Error bars indicate standard error of the mean (SEM).


B ) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated ( n = 10 ) TgN3R182C150 mice vs sham ( n = 9 ) . Statistical analysis was performed using unpaired student t test with Welch ' s correction . ( Sham vs Vaccinated * P = 0 . 0196 ) . Error bars indicate standard error of the mean ( SEM ) .

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B) NOTCH3 ECD in the TgN3R182C150 mice was significantly reduced in the vaccinated (n=10) TgN3R182C150 mice vs sham (n=9). Statistical analysis was performed using unpaired student t test with Welch's correction. (Sham vs Vaccinated *P=0.0196). Error bars indicate standard error of the mean (SEM).


A ) Representative images show microglia stained with anti - CD68 antibody ( red ) and Iba1 antibody ( green ) . Scale bar = 20µm . B ) Quantification of CD68 - stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1 - 5 - immunized ( n = 6 ) , sham ( n = 4 ) and non - vaccinated TgN3R182C150 ( n = 5 ) mice . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with CD68 staining : 7 m . o . vs . Sham ns P = 0 . 7393 ; Sham vs . Vaccinated * * P = 0 . 0087 . CD68 staining ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 9131 ; Sham vs . Vaccinated * P = 0 . 0157 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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

A) Representative images show microglia stained with anti-CD68 antibody (red) and Iba1 antibody (green). Scale bar=20µm. B) Quantification of CD68-stained area revealed a significant increase in the % of microglia and microglia area between N3 EGF1-5-immunized (n=6), sham (n=4) and non-vaccinated TgN3R182C150 (n=5) mice. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with CD68 staining: 7 m.o. vs. Sham ns P=0.7393; Sham vs. Vaccinated **P=0.0087. CD68 staining (% of microglia area): 7 m.o. vs. Sham ns P=0.9131; Sham vs. Vaccinated *P=0.0157, ns=non-significant). Error bars indicate standard error of the mean (SEM).


C ) Representative images of TgN3R182C150 , sham - and NOTCH3 EGF1 - 5 - immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) and an antibody against microglia ( Iba1 , green ) . Scale bar = 20µm . D ) Quantification of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) and non - vaccinated TgN3R182C150 ( n = 6 ) mice at 7 months of age . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons test . ( % microglia with NOTCH3 ECD deposits : 7 m . o . vs . Sham ns P = 0 . 7997 ; Sham vs . Vaccinated ns P = 0 . 0526 . NOTCH3 ECD deposits ( % of microglia area ) : 7 m . o . vs . Sham ns P = 0 . 5362 ; Sham vs . Vaccinated ns P = 0 . 8059 . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham ns P = 0 . 8392 ; Sham vs . Vaccinated ns P = 0 . 5777 . NOTCH3 ECD deposits size : 7 m . o . vs . Sham ns P = 0 . 6664 ; Sham vs . Vaccinated ns P = 0 . 8787 , ns = non - significant ) . Error bars indicate standard error of the mean ( SEM ) .

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C) Representative images of TgN3R182C150, sham- and NOTCH3 EGF1-5-immunized mice at 7 months of age stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an antibody against microglia (Iba1, green). Scale bar=20µm. D) Quantification of NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per microglia revealed no alterations between the NOTCH3 EGF1-5-immunized (n=11), sham (n=8) and non-vaccinated TgN3R182C150 (n=6) mice at 7 months of age. Statistical significance was assessed using an ordinary one-way ANOVA followed by Tukey's multiple comparisons test. (% microglia with NOTCH3 ECD deposits: 7 m.o. vs. Sham ns P=0.7997; Sham vs. Vaccinated ns P=0.0526. NOTCH3 ECD deposits (% of microglia area): 7 m.o. vs. Sham ns P=0.5362; Sham vs. Vaccinated ns P=0.8059. NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham ns P=0.8392; Sham vs. Vaccinated ns P=0.5777. NOTCH3 ECD deposits size: 7 m.o. vs. Sham ns P=0.6664; Sham vs. Vaccinated ns P=0.8787, ns=non-significant). Error bars indicate standard error of the mean (SEM).


C - F Division pattern of seam cells at the postembryonic stage in hermaphrodites . Seam cells V1 - V4 and V6 divide symmetrically at the L2 stage ( highlighted in red ) , thus doubling in number .

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C-F Division pattern of seam cells at the postembryonic stage in hermaphrodites. Seam cells V1-V4 and V6 divide symmetrically at the L2 stage (highlighted in red), thus doubling in number.


( G , H ) dcr ‐ 1 young adults have an increased number of seam cells ( shown in brackets ) and gaps in the alae ( between arrows ) .

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(G,H) dcr‐1 young adults have an increased number of seam cells (shown in brackets) and gaps in the alae (between arrows).


( I , J ) atg ‐ 2 mutations suppress the retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants .

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(I,J) atg‐2 mutations suppress the retarded heterochronic defect in dcr‐1mutants. Complete alae (arrow) are formed in dcr‐1; atg‐2 double mutants.


K - M Confocal images showing expression of hbl ‐ 1 : : gfp : : hbl ‐ 1 reporter in VNC in wild type ( K ) , atg ‐ 2 ( L ) and epg ‐ 6 ( M ) L3 larvae .

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K-M Confocal images showing expression of hbl‐1::gfp::hbl‐1 reporter in VNC in wild type (K), atg‐2 (L) and epg‐6 (M) L3 larvae.


N Distribution of relative fluorescence intensity of HBL ‐ 1 : : GFP in 50 unit areas in VNC in the indicated strains .

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N Distribution of relative fluorescence intensity of HBL‐1::GFP in 50 unit areas in VNC in the indicated strains.


O - Q Confocal images showing the expression of the col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) reporter in wild type ( O ) , atg ‐ 2 ( P ) and atg ‐ 3 ( Q ) late L4 larvae .

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O-Q Confocal images showing the expression of the col‐10::gfp::lin‐41(3′UTR) reporter in wild type (O), atg‐2 (P) and atg‐3 (Q) late L4 larvae.


R Distribution of relative fluorescence intensity of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal cells in the indicated strains .

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R Distribution of relative fluorescence intensity of col‐10::gfp::lin‐41(3′UTR) in 50 unit areas of hypodermal cells in the indicated strains.


S , T Distribution of relative fluorescence intensity of hbl ‐ 1 : : gfp in VNC and col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal cells in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated animals . Black lines in ( N , R - T ) represent the average fluorescence intensity of the reporter in each strain . P values are determined by Student ' s two ‐ tailed unpaired t ‐ test in ( N , R - T ) . ( C , E , G , I ) Scale bar , 20 μm ; ( D , F , H , J - M , O - Q ) scale bar , 10 μm . GFP , green fluorescent protein ; miRISC , miRNA ‐ induced silencing complex ; RNAi , RNA ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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S,T Distribution of relative fluorescence intensity of hbl‐1::gfp in VNC and col‐10::gfp::lin‐41(3′UTR) in hypodermal cells in 50 unit areas in wild type, let‐363(RNAi) or starvation‐treated animals. Black lines in (N,R-T) represent the average fluorescence intensity of the reporter in each strain. P values are determined by Student's two‐tailed unpaired t‐test in (N,R-T). (C,E,G,I) Scale bar, 20 μm; (D,F,H,J-M,O-Q) scale bar, 10  μm. GFP, green fluorescent protein; miRISC, miRNA‐induced silencing complex; RNAi, RNA‐mediated interference; UTR, untranslated region; VNC, ventral nerve cord.


A - C Wild ‐ type and atg ‐ 2 hermaphrodites have a single vulva ( arrow ) . ( B ) let ‐ 60 ( n1046gf ) mutants show a Muv phenotype . Black arrows indicate pseudovulvae .

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A-C Wild‐type and atg‐2 hermaphrodites have a single vulva (arrow). (B) let‐60(n1046gf) mutants show a Muv phenotype. Black arrows indicate pseudovulvae.


D Loss of autophagy activity suppresses the Muv phenotype in let ‐ 60 ( n1046 ) mutants . The number of adult animals examined was : wild type ( n = 450 ) , let ‐ 60 ( n = 406 ) , epg ‐ 6 ; let ‐ 60 ( n = 444 ) , let ‐ 60 ; atg ‐ 2 ( n = 803 ) , epg ‐ 5 ; let ‐ 60 ( n = 672 ) , epg ‐ 5 ; let ‐ 60 ; ain ‐ 1 ( ku322 ) ( n = 635 ) . P values are determined by a two ‐ tailed χ2 test .

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D Loss of autophagy activity suppresses the Muv phenotype in let‐60(n1046) mutants. The number of adult animals examined was: wild type (n=450), let‐60 (n=406), epg‐6; let‐60 (n=444), let‐60; atg‐2 (n=803), epg‐5; let‐60 (n=672), epg‐5; let‐60; ain‐1(ku322) (n=635). P values are determined by a two‐tailed χ2 test.


E , F lsy ‐ 6 mutants show defects in ASEL fate specification and do not express the ASEL ‐ specific reporter lim ‐ 6pro : : GFP .

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E,F lsy‐6 mutants show defects in ASEL fate specification and do not express the ASEL‐specific reporter lim‐6pro::GFP.


G Expression of lim ‐ 6pro : : GFP in various strains . aCompared with lsy ‐ 6 ( ot150 ) . bCompared with lsy ‐ 6 ( ot150 ) ; ain ‐ 1 ( ku322 ) . P values are determined by a two ‐ tailed χ2 test .

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G Expression of lim‐6pro::GFP in various strains. aCompared with lsy‐6(ot150).bCompared with lsy‐6(ot150); ain‐1(ku322). P values are determined by a two‐tailed χ2 test.


AIN ‐ 1 : : GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild ‐ type animals . ( A ) Nomarski image of the embryo shown in ( B ) . Scale bar , 10 μm for whole embryo . C . elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size . Thus , scale bars for embryos are only shown once in each figure . ( C ) AIN ‐ 1 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos . ( A - C ) are confocal images .

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AIN‐1::GFP is weakly and diffusely expressed in the cytoplasm during embryogenesis in wild‐type animals. (A) Nomarski image of the embryo shown in (B). Scale bar, 10 μm for whole embryo. C. elegans embryos remain the same size during embryogenesis and loss of autophagy activity has no effect on the embryo size. Thus, scale bars for embryos are only shown once in each figure. (C) AIN‐1::GFP forms a large number of aggregates in epg‐6 mutant embryos. (A-C) are confocal images.


( D ) Western blotting shows that endogenous AIN ‐ 1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type .

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(D) Western blotting shows that endogenous AIN‐1 protein levels are dramatically elevated in autophagy mutant embryos compared with wild type.


( E ) No AIN ‐ 1 : : GFP aggregates are formed in sqst ‐ 1 mutant embryos .

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(E) No AIN‐1::GFP aggregates are formed in sqst‐1 mutant embryos.


( F ) AIN ‐ 1 : : GFP accumulates into numerous aggregates in epg ‐ 7 mutant embryos .

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(F) AIN‐1::GFP accumulates into numerous aggregates in epg‐7 mutant embryos.


( G , H ) Number of AIN ‐ 1 : : GFP ( G ) and ALG ‐ 2 : : GFP ( H ) aggregates per focal plane in the indicated strains . Error bars indicate the s . d . of four examined embryos .

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(G,H) Number of AIN‐1::GFP (G) and ALG‐2::GFP (H) aggregates per focal plane in the indicated strains. Error bars indicate the s.d. of four examined embryos.


( I , J ) ALG ‐ 2 : : GFP is diffusely localized in the cytoplasm in wild ‐ type embryos . ( I ) Nomarski image of the embryo shown in ( J ) .

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(I,J) ALG‐2::GFP is diffusely localized in the cytoplasm in wild‐type embryos. (I) Nomarski image of the embryo shown in (J).


( K ) ALG ‐ 2 : : GFP forms a few aggregates in epg ‐ 6 mutant embryos . ( L ) ALG ‐ 2 : : GFP forms a large number of aggregates in epg ‐ 6 mutant embryos under stress conditions . GFP , green fluorescent protein ; s . d . , standard deviation .

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(K) ALG‐2::GFP forms a few aggregates in epg‐6 mutant embryos. (L) ALG‐2::GFP forms a large number of aggregates in epg‐6 mutant embryos under stress conditions. GFP, green fluorescent protein; s.d., standard deviation.


( A - D ) Both AIN ‐ 1 : : GFP and SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed and diffusely localized in wild ‐ type embryos .

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(A-D) Both AIN‐1::GFP and SQST‐1, detected by anti‐SQST‐1 antibody, are very weakly expressed and diffusely localized in wild‐type embryos.


( E - H ) AIN ‐ 1 : : GFP aggregates colocalize with SQST ‐ 1 aggregates in atg ‐ 3 mutant embryos . Scale bars , 10 μm for insets that show a magnified view .

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(E-H) AIN‐1::GFP aggregates colocalize with SQST‐1 aggregates in atg‐3 mutant embryos. Scale bars, 10 μm for insets that show a magnified view.


( I - L ) AIN ‐ 1 : : GFP aggregates are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant embryos .

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(I-L) AIN‐1::GFP aggregates are separable from PGL granules, detected by anti‐SEPA‐1, in atg‐3 mutant embryos.


( M - P ) Colocalization of AIN ‐ 1 : : GFP aggregates with LGG ‐ 1 puncta in epg ‐ 6 mutant embryos .

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(M-P) Colocalization of AIN‐1::GFP aggregates with LGG‐1 puncta in epg‐6 mutant embryos.


( Q ) % of AIN ‐ 1 : : GFP aggregates colocalized with SQST ‐ 1 aggregates , SEPA ‐ 1 aggregates and LGG ‐ 1 puncta in autophagy mutants . Error bars indicate the s . d . of four examined embryos .

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(Q) % of AIN‐1::GFP aggregates colocalized with SQST‐1 aggregates, SEPA‐1 aggregates and LGG‐1 puncta in autophagy mutants. Error bars indicate the s.d. of four examined embryos.


( R ) A co ‐ IP assay shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo .

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(R) A co‐IP assay shows that AIN‐1 associates with LGG‐1 in vivo.


( S ) Direct interaction between AIN ‐ 1 and LGG ‐ 1 in an in vitro pull ‐ down assay . 5 % of the His fusion proteins used for pull ‐ down are shown as input . Corresponding Ponceau ‐ stained blots show the amount of proteins used for pull ‐ down .

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(S) Direct interaction between AIN‐1 and LGG‐1 in an in vitro pull‐down assay. 5% of the His fusion proteins used for pull‐down are shown as input. Corresponding Ponceau‐stained blots show the amount of proteins used for pull‐down.


( T ) A fragment of AIN ‐ 1 directly binds to LGG ‐ 1 in an in vitro pull ‐ down assay .

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(T) A fragment of AIN‐1 directly binds to LGG‐1 in an in vitro pull‐down assay.


( a ) Knockdown of dRagA and dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 cells untreated ( lane 2 ) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies .

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(a) Knockdown of dRagA and dRagC decreased dS6K phosphorylation (Thr 398). Drosophila S2 cells untreated (lane 2) or treated with the indicated RNAi were starved of amino acids for 1 h followed by amino acid stimulation for 30 min Phosphorylation and protein levels of dS6K were determined by immunoblotting with the indicated antibodies.


( b ) dRagA and dRagC are not required for dAkt phosphorylation . Drosophila S2 cells untreated ( lanes 1 - 3 ) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min . Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated . NC , negative control RNAi .

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(b) dRagA and dRagC are not required for dAkt phosphorylation. Drosophila S2 cells untreated (lanes 1-3) or treated with the indicated RNAi were starved of amino acids for 1 h and stimulated with amino acids for 30 min. Phosphorylation and protein levels of dAkt were determined by immunoblotting with appropriate antibodies as indicated. NC, negative control RNAi.


( c ) dRagA and dRagC function in parallel with TSC2 and PTEN . dRagA and / or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) and this increase was compromised by dRagA or / and dRagC RNAi . Full scans of blots are provided in Supplementary Information , Fig . S6 .

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(c) dRagA and dRagC function in parallel with TSC2 and PTEN. dRagA and/or dRagC RNAi was added to S2 cells in combination with dTSC2 or dPTEN RNAi, as indicated. dTSC2 or dPTEN RNAi treatment increased pdS6K (Thr 398) and this increase was compromised by dRagA or/and dRagC RNAi. Full scans of blots are provided in Supplementary Information, Fig. S6.


( a ) Constitutively active RagA and RagB stimulate S6K phosphorylation . 200 ng of each mammalian RagA , RagB , RagC or RagD construct was co - transfected with HA - S6K ( 20 ng ) into HEK293 cells . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) and constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies , as indicated .

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(a) Constitutively active RagA and RagB stimulate S6K phosphorylation. 200 ng of each mammalian RagA, RagB, RagC or RagD construct was co-transfected with HA-S6K (20 ng) into HEK293 cells. Their corresponding dominant-negative mutants (RagAT21N, RagBT54N, RagCS75N, RagDS76N) and constitutively active mutants (RagAQ66L, RagBQ99L, RagCQ120L, RagDQ121L) were also tested. Phosphorylation and protein levels were determined by immunoblotting with the appropriate antibodies, as indicated.


( b ) RagA has a dominant role over RagC in regulating S6K phosphorylation . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) . The different Rag mutants used in the transfection are indicated at the top of each lane .

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(b) RagA has a dominant role over RagC in regulating S6K phosphorylation. Each Rag construct (200 ng) indicated was co-transfected with HA-S6K (20 ng). The different Rag mutants used in the transfection are indicated at the top of each lane.


( c ) Rag regulates TORC1 but not TORC2 activity . Each indicated Rag construct ( 200 ng ) was co - transfected with either HA - S6K ( 20 ng ) , Myc - 4EBP1 ( 20 ng ) or of GST - Akt ( 100 ng ) . 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 .

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(c) Rag regulates TORC1 but not TORC2 activity. Each indicated Rag construct (200 ng) was co-transfected with either HA-S6K (20 ng), Myc-4EBP1 (20 ng) or of GST-Akt (100 ng). 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.


( 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 .

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(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 .

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(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 .

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(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.


( 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 ) .

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(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 ) .

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

(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.


( 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 .

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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.


( 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 .

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(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 .

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(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.


( 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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(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).


I 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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I 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'.


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 .

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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 .

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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 .

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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 .

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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.


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 .

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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.


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 .

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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.


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 ) .

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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).


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 .

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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 .

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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.


( A ) miR - 155 expression levels were examined in the lungs of normal uninfected or H37Rv infected BALB / c mice 6 weeks

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(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 .

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(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 .

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(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 .

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(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.


( 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 .

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(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 .

(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 .

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(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.


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 ) The colocalization of BCG with lysosome was detected by confocal microscopy . The percentage of co - localization of BCG with CD63 - positive ( B )

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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)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 .

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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.


( 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 ) .

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

(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 )

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

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

(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.


( 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 ) .

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(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 ) .

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

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

(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.


( 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 .

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(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 .

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

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

(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.


( 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 )

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(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 )

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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 ) .

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(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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

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.


( 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 .

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(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 .

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

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 )

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

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

(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).


( 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 .

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

(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 ) .

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

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

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.


( 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 .

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(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 .

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(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 .

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(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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(h) Reaction performed as in (g). Nascent DNA was separated in a 0.7% alkaline agarose gel in this and all subsequent replication reactions.


( a ) Reaction performed as in Fig . 1a , except Cdc45 was omitted .

(a) Reaction performed as in Fig. 1a, except Cdc45 was omitted.


( b ) Reaction performed as in Fig . 1g , with the indicated proteins 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 .

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(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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(f) Sld3 recruitment reaction performed as in (a). Sld7 was omitted from this experiment.


( b ) Recruitment reaction performed as described in ( a ) .

(b) Recruitment reaction performed as described in (a).


( c ) Sld3 / 7 recruitment reaction conducted as in Fig . 2a .

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(c) Sld3/7 recruitment reaction conducted as in Fig. 2a.


( d ) Replication reaction performed as in Fig . 1g .

(d) Replication reaction performed as in Fig. 1g.


( e ) Representative tetrads from dissection of SLD3 + / sld3 - 6E heterozygotes .

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(e) Representative tetrads from dissection of SLD3+/sld3-6E heterozygotes.


( 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 ) .

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(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 .

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(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 .

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(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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(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.


( a ) Sld3 / 7 recruitment reaction performed as in Fig . 2a .

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(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 .

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(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 ) .

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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(d) Representative tetrads from diploids heterozygous for mcm4-25A and mcm6ΔN+11A.


( b ) Immunoblots of reactions performed as in ( a ) part i ( lanes 1 - 3 ) or ii ( lanes 4 - 6 ) .

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

(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 .

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

(c) Reactions in i and ii were performed as described in (a) parts i and ii, respectively.


( a , b ) Recruitment reactions performed as in Fig . 2a . Sld7 was omitted in ( b ) .

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

(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 .

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

(c) Recruitment reaction conducted as in Fig. 3a, with washes performed at 0.3 M KCl.