( G ) Representative of intestinal organoids cultured at indicated conditions timepoints . Scale bar : 100 μm . organoids were co - cultured in a mixed medium of supernatant with / without mIslr from CT26 regular organoid culture solution at of 1 : 4 . of organoids diameter at indicated timepoints . n > 150 organoids at each timepoint .

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

(G) Representative images of primary intestinal organoids cultured at indicated conditions and timepoints. Scale bar: 100 μm. The organoids were co-cultured in a mixed medium of the supernatant with/without mIslr from CT26 cells and regular organoid culture solution at the ratio of 1:4. Quantification of organoids diameter at indicated timepoints. n>150 organoids at each timepoint.


( H ) Western blotting for pMst1 / 2 , Mst1 , pMob1 Mob1 , pYap1 Yap1 24 hours after treatment . β - actin was used as a loading control .

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

(H) Western blotting for pMst1/2, Mst1, pMob1 and Mob1, pYap1 and Yap1 24 hours after the treatment. β-actin was used as a loading control.


( I ) qRT - PCR for Ctgf , Cry61 Fstl1 in intestinal organoids 24 hours after treatments . n = 3 .

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

(I) qRT-PCR for Ctgf, Cry61 and Fstl1 in primary intestinal organoids 24 hours after the treatments. n=3.


( J ) Immunofluorescence for Yap1 β - catenin in intestinal organoids 24 hours after indicated treatments . treatments are identical to ( G ) . of Yap1 + versus epithelial was quantified . Scale bar : 50 μm . n = 12 .

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

(J) Immunofluorescence for Yap1 and β-catenin in intestinal organoids 24 hours after the indicated treatments. The treatments are identical to (G). The percentage of nuclear Yap1+ cells versus epithelial cells was quantified. Scale bar: 50 μm. n=12.


B ) Western blot of NOTCH3 EGF1 - 5 WT R133C purified . eluate fractions were visualized by western blot using an α - myc antibody .

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

B) Western blot of the NOTCH3 EGF1-5 WT and R133C purified proteins. The eluate fractions were visualized by western blot using an α-myc antibody.


C ) Western blot of NOTCH3 EGF1 - 5 WT R133C aggregated . incubated fractions of NOTCH3 EGF1 - 5 WT R133C were visualized on a western blot using an α - myc antibody . purified were visualized after 1 - 5 days incubation by western blot using α - myc antibody under non - reducing conditions .

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

C) Western blot of NOTCH3 EGF1-5 WT and R133C aggregated proteins. The incubated fractions of NOTCH3 EGF1-5 WT and R133C were visualized on a western blot using an α-myc antibody. The purified proteins and the aggregates were visualized after 1-5 days incubation by western blot using α-myc antibody under non-reducing conditions.


A ) Representative of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 7 months of TgN3R182C150 at 18 months of . Representative show brain arteries of TgN3R182C150 ( 7 18 months ) , sham NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an α - SMA antibody ( green ) . Scale bar = 20µm . B ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per vessel revealed no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of . NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per vessel increases significantly in TgN3R182C150 mice at 18 months ( n = 3 ) of versus NOTCH3 EGF1 - 5 - immunized ( n = 10 ) , sham ( n = 8 ) non - vaccinated ( n = 6 ) TgN3R182C150 mice at 7 months of . Statistical significance was assessed using a Brown - Forsythe Welch ANOVA followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel ) : 7 m . o . vs . Sham , ns P = 0 . 9424 ; 7 m . o . vs . Vaccinated , ns P = 0 . 9809 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0046 ; Sham vs . Vaccinated , ns P = 0 . 9999 ; Sham vs . 18 m . o . * * P = 0 . 0032 ; Vaccinated vs . 18 m . o . * * P = 0 . 0032 ) . NOTCH3 ECD deposits ( number / 1000 um2 ) : 7 m . o . vs . Sham , ns P = 0 . 7877 ; 7 m . o . vs . Vaccinated , ns P = 0 . 8358 ; 7 m . o . vs . 18 m . o . * * P = 0 . 0069 ; Sham vs . Vaccinated , ns P = 0 . 9995 ; Sham vs . 18 m . o . * * P = 0 . 0044 ; Vaccinated vs . 18 m . o . * P = 0 . 0104 ) . NOTCH3 ECD deposits : 7 m . o . vs . Sham , ns P = 0 . 1169 ; 7 m . o . vs . Vaccinated , ns P > 0 . 9999 ; 7 m . o . vs . 18 m . o . * * * P = 0 . 0009 ; Sham vs . Vaccinated , ns P = 0 . 0577 ; Sham vs . 18 m . o . * * P = 0 . 007 ; Vaccinated vs . 18 m . o . * * * * P < 0 . 0001 , ns = non - significant ) . Error bars indicate standard error of mean ( ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7 months of age and TgN3R182C150 at 18 months of age. Representative images show brain arteries of TgN3R182C150 (7 and 18 months), sham and NOTCH3 EGF1-5-immunized mice stained with a monoclonal antibody against NOTCH3 ECD (1E4, red) and an α-SMA 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 no decrease in NOTCH3 ECD deposition in brain arteries between NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. NOTCH3 ECD deposits (numbers per 1,000 μm2) and NOTCH3 ECD stained area and average size per vessel increases significantly in the TgN3R182C150 mice at 18 months (n=3) of age versus NOTCH3 EGF1-5 -immunized (n=10), sham (n=8) and non-vaccinated (n=6) TgN3R182C150 mice at 7 months of age. 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): 7 m.o. vs. Sham, ns P=0.9424; 7 m.o. vs. Vaccinated, ns P=0.9809; 7 m.o. vs. 18 m.o.**P=0.0046; Sham vs. Vaccinated, ns P=0.9999; Sham vs. 18 m.o. **P=0.0032; Vaccinated vs. 18 m.o. **P=0.0032). NOTCH3 ECD deposits (number/1000 um2): 7 m.o. vs. Sham, ns P=0.7877; 7 m.o. vs. Vaccinated, ns P=0.8358; 7 m.o. vs. 18 m.o.**P=0.0069; Sham vs. Vaccinated, ns P=0.9995; Sham vs. 18 m.o. **P=0.0044; Vaccinated vs. 18 m.o. *P=0.0104). NOTCH3 ECD deposits size: 7 m.o. vs. Sham, ns P=0.1169; 7 m.o. vs. Vaccinated, ns P>0.9999; 7 m.o. vs. 18 m.o.***P=0.0009; Sham vs. Vaccinated, ns P=0.0577; Sham vs. 18 m.o. **P=0.007; Vaccinated vs. 18 m.o. ****P<0.0001, ns=non-significant). Error bars indicate standard error of the mean (SEM).


A ) Representative of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 3 , 7 18 months of . Representative show brain arteries capillaries of TgN3R182C150 , sham NOTCH3 EGF1 - 5 - immunized mice stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an anti - perlecan antibody ( green ) . Scale bar = 20µm . B ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 - ECD stained average per vessel revealed a significant increase in NOTCH3 ECD deposition in brain arteries capillaries between non - vaccinated 3 months old TgN3R182C150 ( n = 3 ) 7 months old TgN3R182C150 ( n = 6 ) mice 18 months old TgN3R182C150 ( n = 3 ) . of NOTCH3 - ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 - ECD stained average per vessel revealed a significant decrease in NOTCH3 - ECD deposition in brain arteries capillaries between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) non - vaccinated TgN3R182C150 ( n = 6 ) mice . Statistical significance was assessed using a Brown - Forsythe Welch ANOVA followed by Dunnett ' s T3 multiple comparisons . ( NOTCH3 ECD deposits ( % of vessel ) : 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 : 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 dashed thicker lines are median .

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

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

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

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of TgN3R182C150 , sham - NOTCH3 EGF1 - 5 - immunized mice at 7 months of stained with a monoclonal antibody against NOTCH3 ECD ( 1E4 , red ) an antibody against microglia ( Iba1 , green ) . Scale bar = 20µm . D ) of NOTCH3 ECD deposits ( numbers per 1 , 000 μm2 ) NOTCH3 ECD stained average per microglia revealed no alterations between NOTCH3 EGF1 - 5 - immunized ( n = 11 ) , sham ( n = 8 ) non - vaccinated TgN3R182C150 ( n = 6 ) mice at 7 months of . Statistical significance was assessed using an ordinary one - way ANOVA followed by Tukey ' s multiple comparisons . ( % 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 ) : 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 : 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 mean ( ) .

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

C) Representative 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 at postembryonic stage in hermaphrodites . Seam V1 - V4 V6 divide symmetrically at L2 stage ( highlighted in red ) , thus doubling in number .

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

C-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 ( shown in brackets ) gaps in alae ( between arrows ) .

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

(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 retarded heterochronic defect in dcr ‐ 1mutants . Complete alae ( arrow ) are formed in dcr ‐ 1 ; atg ‐ 2 double mutants .

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

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

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

K-M 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 of HBL ‐ 1 : : GFP in 50 unit areas in VNC in indicated strains .

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

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


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

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

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 of col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in 50 unit areas of hypodermal in indicated strains .

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

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 of hbl ‐ 1 : : gfp in VNC col ‐ 10 : : gfp : : lin ‐ 41 ( 3 ′ UTR ) in hypodermal in 50 unit areas in wild type , let ‐ 363 ( RNAi ) or starvation ‐ treated . Black lines in ( N , R - T ) represent average of reporter in each . P values are determined by Student ' s two ‐ unpaired t ‐ 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 ; miRISC , miRNA ‐ induced silencing ; RNAi , ‐ mediated interference ; UTR , untranslated region ; VNC , ventral nerve cord .

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

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 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 Muv phenotype in let ‐ 60 ( n1046 ) mutants . number of adult 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 ‐ χ2 .

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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 do not express 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 ‐ χ2 .

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

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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 are dramatically elevated in autophagy mutant 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 are formed in sqst ‐ 1 mutant .

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


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

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


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

(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 cytoplasm in wild ‐ type . ( I ) Nomarski image of 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 in epg ‐ 6 mutant . ( L ) ALG ‐ 2 : : GFP forms a large number of in epg ‐ 6 mutant under stress conditions . GFP , green ; 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 SQST ‐ 1 , detected by anti ‐ SQST ‐ 1 antibody , are very weakly expressed diffusely localized in wild ‐ type .

(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 colocalize with SQST ‐ 1 in atg ‐ 3 mutant . Scale bars , 10 μm for insets that show a magnified view .

(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 are separable from PGL granules , detected by anti ‐ SEPA ‐ 1 , in atg ‐ 3 mutant .

(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 with LGG ‐ 1 in epg ‐ 6 mutant .

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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 colocalized with SQST ‐ 1 , SEPA ‐ 1 LGG ‐ 1 in autophagy mutants . Error bars indicate s . d . of four examined .

(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 shows that AIN ‐ 1 associates with LGG ‐ 1 in vivo .

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


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

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


( a ) Knockdown of dRagA dRagC decreased dS6K phosphorylation ( Thr 398 ) . Drosophila S2 untreated ( lane 2 ) or treated with indicated RNAi were starved of for 1 h followed by acid stimulation for 30 min Phosphorylation of dS6K were determined by immunoblotting with 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 dRagC are not required for dAkt phosphorylation . Drosophila S2 untreated ( lanes 1 - 3 ) or treated with indicated RNAi were starved of for 1 h stimulated with for 30 min . Phosphorylation 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 dRagC function in parallel with TSC2 PTEN . dRagA / or dRagC RNAi was added to S2 in combination with dTSC2 or dPTEN RNAi , as indicated . dTSC2 or dPTEN RNAi treatment increased pdS6K ( Thr 398 ) this increase was compromised by dRagA or / 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 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 . Their corresponding dominant - negative mutants ( RagAT21N , RagBT54N , RagCS75N , RagDS76N ) constitutively active mutants ( RagAQ66L , RagBQ99L , RagCQ120L , RagDQ121L ) were also tested . Phosphorylation were determined by immunoblotting with 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 ) . different Rag mutants used in transfection are indicated at 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 were determined by immunoblotting with 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 RagCS75N activate TORC1 in absence of . Each Rag construct ( 200 ng ) indicated was co - transfected with HA - S6K ( 20 ng ) into HEK293 . were starved of for 1 h before collection . Phosphorylation were determined by immunoblotting with 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 RagCQ120L block S6K phosphorylation in response to acid ( AA ) stimulation . pcDNA3 ( 200 ng , lanes 1 2 ) or each indicated Rag construct was co - transfected with HA - S6K into HEK293 . were starved of for 1 h ( - AA ) either remained in starvation medium or were stimulated with - acid - containing - medium ( + AA ) for 30 min before collection . Phosphorylation were determined by immunoblotting with 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 RagCQ120L suppress insulin - induced stimulation of S6K phosphorylation . pcDNA3 ( 200 ng , lanes 1 2 ) or each indicated Rag construct was co - transfected with HA - S6K ( 20 ng ) or GST - Akt ( 100 ng ) into HeLa . were serum - starved overnight stimulated with insulin ( 400 nM ) for 30 min . Phosphorylation were determined by immunoblotting with 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 . Wild - type or mutant dRagA transgenes were expressed in posterior compartments with en - GAL4 driver . ratios of representative posterior to anterior compartment areas are shown . Posterior compartment was significantly increased in response to dRagAQ61L expression 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 - t - , ( n is 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 . average of posterior compartment from en - GAL4 UAS - dRagA adult wings is shown . expressing dRagAQ61L are significantly larger dRagAT61N - expressing are smaller than controls . Data are mean ± s . d . , * P = 1 . 15 × 10 − 3 ( n = 7 ) , * * P = 0 . 025 ( n = 12 ) , Student ' s 2 - t - ( 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 . ( c ) of clonally - induced dRagA - expressing or dRagC homozygous mutant relative to neighbouring wild - type control is shown . was determined from phalloidin - stained fixed fat samples from fed or 48 h starved larvae . Expression of dRagAWT or dRagAQ61L significantly increased relative under starvation but not fed conditions . expressing dRagAT61N dRagC loss - of - function were significantly smaller than control 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 - t - . ( d ) Representative examples of fat with altered dRagA activity . expressing dRagA transgenes are marked by expression of GFP in left middle panels , dRagC homozygous mutant are marked by absence of GFP in 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(c, d) 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 were transfected with constructs as indicated . Co - expression of of mTORKD construct ( 600 ng ) or rapamycin treatment ( rapa , 20 nM , 30 min ) abolished effect of RagAQ66L RagCS75N on S6K phosphorylation . 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 TSC1 / TSC2 independently regulate S6K phosphorylation . HEK293 were transfected with 200 ng of each Rag / or ′ constructs as indicated . acid starvation for 1 h ( - AA ) is indicated . Phosphorylation of transfected 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 RagA / B independently affect S6K phosphorylation . HA - S6K ( 20 ng ) was transfected into HeLa with or without RNAi against human TSC2 , RagA 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 RagCQ120L do not block Rheb - induced S6K phosphorylation . RagAT21N RagCQ120L ( 200 ng each ) were transfected into HEK293 with or without Rheb construct ( 20 ng ) . S6K was included in co - transfection . Phosphorylation of transfected 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 growth . ( a ) of Rheb - overexpressing in control or dRagC mutant ( dRagC − / − ) backgrounds under fed conditions , relative to that of neighbouring control which were assigned a value of 1 . Overexpression of Rheb led to a significant increase in in both control dRagC mutant backgrounds . Data are mean ± s . d . , * P = 0 . 034 ( n = 5 ) , * * P = 6 . 1 × 10 − 3 ( n = 5 ) , Student ' s two - t - ( n represents number of experimental samples ) . ( b ) A representative example of a clone of Rheb - overexpressing in a dRagC − / − . Rheb transgene - expressing are marked by co - expression of GFP . boundaries are labelled by phalloidin 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 growth impairment of Rheb mutant . ( c ) Relative of clonally - induced Rheb26 . 2 homozygous mutant in a control background in expressing dRagAQ61L throughout fat . Clonally induced Rheb26 . 2 homozygous mutant were significantly smaller than neighbouring control both in wild - type 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 - t - ( fed conditions where n represents number of experimental samples ) . ( d ) A representative example of Rheb homozygous mutant ( marked by lack of GFP , arrows ) in fat ubiquitously expressing UAS - dRagAQ61L . GFP - positive control in this experiment are a mixture of Rheb + / − Rheb + / + . 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.


( a - d ) dRagAQ61L suppresses autophagy . ( a ) Drosophila fat clonally expressing dRagAQ61L ( marked in green by GFP expression ) failed to accumulate autolysosomes ( shown in surrounding control by punctate Lysotracker Red ) 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 punctate pattern of GFP - Atg8a expression in control fat ( b ) , but not in expressing dRagAQ61L ( c ) . average number of GFP - Atg8a - marked autophagosomes per in control dRagAQ61L - expressing clones is shown ( d ) . Data are mean ± s . d . , * P = 2 . 91 × 10 − 6 , Students two - t - ( n = 33 fat 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 . Myc - LC3 was co - transfected with RagAQL RagCSN or RagATN RagCQL into HEK293 as indicated . One day after transfection , were cultured in - acid - sufficienct medium ( + AA ) or - acid - depleted medium ( − AA ) for 4 h before collection . Western blotting for Myc - LC3 HA - Rag were performed . Autophagic conversion of LC3I into lipidated LC3II form was blocked by active RagA 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 fat - - specific Cg - GAL4 driver significantly decreased survival of adult 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 - t - ( 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 complexes involving aggregators . Heatmaps show absolute difference in solubility change after heat shock between each member . complexes with at least 75 % of its members quantified containing at least 60 % of its members as aggregators are shown . ' repair ' = ' repair 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 graphs showing solubility after heat shock ( HS ) during different time points of recovery . Each corresponds to one . Orange lines show 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 . Solubility graphs as in A shown for aggregators with bottom 10 % , middle 80 % top 10 % of disaggregation rates . with bottom 10 % of disaggregation rates related to repair are highlighted with red . with top10 % disaggregation rates 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 localization upon heat shock during recovery as analyzed by immunofluorescence microscopy . HeLa were fixed immunostained with antibodies against indicated target ( green ) at different conditions [ control , after heat shock ( 10 minutes at 44 ° C ) after five hours of recovery from heat shock ] . ( 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 characteristics enriched in aggregators ( presented in Fig 2B - C Fig 2E - G ) . Volcano plot presenting correlation coefficients Benjamini - Hochberg adjusted p - values ( E ; horizontal dashed shows a p - value of 0 . 05 ) . Scatterplots comparing disaggregation slopes solubility change after heat shock ( F ) , fraction of intrinsically disordered regions ( G ) 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 after mock shock ( A ) heat shock ( B ) . graphs showing accumulation of signal ( log2 - transformed ratios to pre - shock control ) in soluble fraction . Orange lines show mean . C Amount of newly synthesized after heat shock compared to mock shock . graphs show heat shock to mock shock during recovery . Orange show mean . Data information : proteomics data shown in A - D F is quantified with at least two unique peptides from at least two biological replicates . Proteomics data shown for newly synthesized ( 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 after mock shock ( A ) heat shock ( B ) . graphs showing accumulation of signal ( log2 - transformed ratios to pre - shock control ) in soluble fraction . Orange lines show mean .

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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 synthesis mRNA synthesis after heat shock ( Vihervaara et al , 2017 ) . Ten most upregulated in proteomics are labelled highlighted in blue ; heat shock are labelled red . HSPA1A HSPA1B could not be distinguished from one another in proteomics are plotted as they would have same ( indicated with a vertical dashed ) .

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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 score for thermal stability . Significant ( see ' Materials Methods ' ) heat shock - induced stabilization destabilization are indicated with blue red , respectively . that aggregate in heat shock ( as defined in Figure 2A ) are shown in black - aggregators with 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 other ) . p - value is for non - parametric Wilcoxon .

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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 members . Scores for thermal stability shown for from 26S proteasome ( separately for 19S regulatory 20S particles ) CCT chaperonin . Boxplots indicate median , first third quartiles with whiskers extended to 1 . 5 times interquartile range out from each quartile . Data used in stability score calculation is from two ( mock shock ) 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 were examined in lungs of normal uninfected or H37Rv infected BALB / c mice 6

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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 indicated times , expression 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 D ) RAW264 . 7 were infected with BCG at an MOI of 5 for indicated time points ( C ) or at indicated MOIs for 24 h ( D ) . expression 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 F ) RAW264 . 7 were infected with H37Ra at an MOI of 5 for indicated time ( E ) or at indicated MOI for 24 h ( F ) . expression of miR - 155 were examined by real - time PCR . Data are shown as mean ± 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 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 , intracellular mycobacterial viability was determined by CFU at 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 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 , intracellular mycobacterial viability was determined by CFU at 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 H ) RAW264 . 7 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 mean ± 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 were transiently transfected with control or miR - 155 mimic for 24 h 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 ) colocalization of BCG with lysosome was detected by confocal microscopy . 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 were transiently transfected with control or miR - 155 mimic for 24 h 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 ) . colocalization of BCG with lysosome was detected by confocal microscopy . of co - localization of BCG with CD63 - positive ( B ) or DQ - Green labeled ( D ) lysosomes was quantified , respectively . treated with rapamycin were used as a positive control . Arrows indicate co - localization of BCG with lysosomes ; scale bar = 5 µm . of data are shown as mean ± 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 were transfected with miR - 155 mimic ( A C ) or inhibitor ( B D ) for 24 h then either left uninfected or infected with BCG . Expression of miR - 155 were detected by real - time PCR ( A B ) .

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(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 were transfected with miR - 155 mimic ( A C ) or inhibitor ( B D ) for 24 h then either left uninfected or infected with BCG . Expression of miR - 155 were detected by real - time PCR ( A B ) . LC3 were detected by Western - blot , ratios of LC3 - II / β - actin were calculated as shown below representative blot ( C D )

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(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 F ) RAW264 . 7 transfected with miR - 155 mimic ( E ) or inhibitor ( F ) was incubated with DMSO or bafilomycin A1 ( BafA . ) at a of 100 nM for 2 h , then LC3 were detected by Western - blot . ratios of LC3 - II / β - actin were calculated as shown below representative blot . * * * , p < 0 . 001 .

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(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 B ) RAW264 . 7 stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic for 24 h , GFP - LC3 ( > 1 µm ) were detected by confocal microscopy ( A ) quantified ( B ) .

(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 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 ) , LC3 ( > 1 µm ) were detected by confocal microscopy ( C ) quantified ( D ) .

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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 F ) RAW264 . 7 were transiently transfected with control or miR - 155 mimic for 24 h , then incubated with a specific dye MDC ( 50 µM ) . MDC - labeled autophagic vacuoles were detected by confocal microscopy ( E ) with MDC - positive autophagic vacuoles were quantified ( F ) . treated with rapamycin were used as a positive control . Arrows indicate GFP - LC3 or autophagic vacuoles labeled with MDC ; scale bar = 5 µm . Data are shown as mean ± of three independent experiments ( n = 300 ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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(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 stably expressing GFP - LC3 were transiently transfected with control or miR - 155 mimic then infected with Texas Red - labeled BCG for 1 h . co - localization of BCG with LC3 was detected by confocal microscopy . ( B ) of 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 were transiently transfected with control or miR - 155 mimic , 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 ) . co - localization of BCG with endogenous LC3 was detected by confocal microscopy . ( D ) of co - localization of BCG with LC3 - positive autophagosomes is shown .

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(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 were infected with Texas Red - labeled BCG for 1 h , then were labeled with a specific dye MDC ( 50 µM ) for autophagic vacuoles . co - localization of BCG with MDC - positive autophagic vacuoles was detected by confocal microscopy . ( F ) of co - localization of BCG with MDC - positive autophagosomes is shown . treated with rapamycin were used as a positive control . Arrows indicate co - localization of BCG with autophagosomes ; scale bar = 5 µm . Data are shown as mean ± of three independent experiments ( n = 100 phagosomes ) . * * , p < 0 . 01 ; * * * , p < 0 . 001 .

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

(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 B ) RAW 264 . 7 were transiently transfected with control or miR - 155 mimic for 24 h , then incubated with DMSO or 3 - MA for 2 h . of LC3 were detected by Western - blot in uninfected ( A )

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

(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 indicated timepoints by CFU after BCG challenge for 1 h ( B )

Intracellular mycobacterial viability was determined at the indicated timepoints by CFU assay after BCG challenge for 1 h (B)


( C D ) RAW 264 . 7 were transiently co - transfected with control or miR - 155 mimic together with a control siRNA or Atg7 siRNA . expression of Atg7 LC3 were detected by Western - blot ( C ) .

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

(C 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 at indicated time after challenging with BCG for 1 h ( D ) . Values of LC3 - II / β - actin ratios are indicated below representative blot . Data are shown as mean ± of three independent experiments . * , p < 0 . 05 ; * * , p < 0 . 01 ; NS , not significant .

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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 human miR - 155 their predicted interactions with conserved 7 - mer 1A miR - 155 seeds found within Rheb 3 ′ UTRs of different species are shown . of Rheb 3 ′ UTR seed mutant used for reporter assays predicted disruption of miR - 155 interaction are also shown .

(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 were co - transfected with control or miR - 155 mimic a wild - type ( WT - Rheb ) or mutated Rheb 3 ′ UTR ( mut - Rheb ) luciferase reporter plasmid assessed for luciferase activity at 24 h after transfection . Data are shown as mean ± of three independent experiments . * * * , p < 0 . 001 ; NS , not significant .

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 were transfected with miR - 155 mimic ( C E ) or inhibitor ( D F ) for 24 h either left uninfected or infected with BCG . expression of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below representative blot ( C 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)


( C - F ) RAW264 . 7 were transfected with miR - 155 mimic ( C E ) or inhibitor ( D F ) for 24 h either left uninfected or infected with BCG . expression of Rheb were detected by Western - blot . Values of Rheb / β - actin ratios are indicated below representative blot ( C D ) , expression of Rheb mRNA were detected by RT - PCR ( E F ) .

(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 were transfected with a plasmid expressing Rheb for 24 h , then incubated with DMSO or BafA . for 2 h . expression of Rheb LC3 were detected by Western - blot . Values of LC3 - II / β - actin ratios are indicated below representative blot .

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(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 C ) RAW264 . 7 were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h , then infected with Texas Red - labeled BCG for 1 h . co - localization of BCG MDC - labeled autophagosome was detected by confocal microscopy . Arrows indicate 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 were co - transfected with control or miR - 155 mimic together with a control plasmid or a plasmid expressing Rheb for 24 h . were infected with BCG for 1 h , washed for three times to remove extracellular mycobacteria . Intracellular mycobacterial viability was determined by CFU at indicated timepoints . Data are shown as mean ± 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 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 indicated 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 extract tested for interaction with Cdc45 . Immunoprecipitated were analysed by immunoblot .

(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 was separated in a 0 . 7 % alkaline agarose gel in this 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 indicated 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 .

(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 .

(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 .

(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 ( lanes 6 7 ) or Sld3 alone ( lanes 8 9 ) was coupled to beads . In ( C ) , MCM was treated with Lambda Phosphatase after dissociation from ( lanes 3 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 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 indicated S / T - A substitution mutant versions of Mcm4 Mcm6 . Positions of mutations in Mcm6 are summarised in Fig . EV5c .

(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 .

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


( b ) Replication reaction performed as in Fig . 1g . Experiments in ( a ) ( b ) used MCM containing wild type ( wt ) or indicated S / T - A substitution mutant versions of Mcm4 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 ) 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 mcm6ΔN + 11A .

(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 ) .

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


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

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(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 ) .

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(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 .

(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 Mcm6 as indicated . In ( d ) , MCM loading was equalised across samples by using 5 - fold more mutant than wild type MCM , as is shown in Fig . EV6c .

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(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.


B Representative TIRF - M of DCP1 - GFP ( DCP1pro : DCP1 - GFP transgene ) in lateral PM of epidermal showing transient attachment of DCP1 - positive to PM . Yellow arrowheads denote a PB that shows motility at PM focal plane ; blue arrowheads show a PB that transiently localizes to PM . Scale bars : 2 μm . corresponding kymographs are shown to right . Right : distribution of immobile mobile DCP1 molecules relative to motility log ( D ) value of - 0 . 75 ( threshold ; see Material methods for details ) , in NS or HS conditions ( D , diffusion coefficient ) . Inset : trajectories of mobile DCP1 - GFP in NS HS conditions ( 500 frames , n = 120 ) , showing a combination of directional Brownian motion for both NS / HS . green arrowheads denote beginning of NS HS tracks for DCP1 - GFP .

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B 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 from lines co - expressing RPS5apro : HF - mScarlet - DCP1 PIN2pro : PIN2 - GFP ( epidermal , region 3 , Scale bars : 5 μm ) . Bottom : polarity index of DCP1 in root meristematic ( compared to propidium iodide ( PI ) tubulin of root ; N , biological replicates = 3 roots , n = 13 ) . Polarity index calculated as of average of apical basal VS lateral side of signal of root epidemies . arrowhead in PIN2 indicates plate or PM in DCP1 . Right : representative confocal showing that PM localization is independent of promoter used ( DCP1pro , 35Spro ; region 2 , epidermal , or RPS5apro on lower right ) . details from inset show increased localization at edge ( discussed later ) . mSc , mScarlet .

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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.


B Representative confocal micrograph of DCP1 - GFP ( DCP1pro : DCP1 - GFP , transgene ) in root meristematic under NS / HS conditions ( region 2 ) . Scale bars , 5 μm . Note depletion of DCP1 from PM upon HS , but increased edge / vertex signal ( yellow arrowheads denote vertex signal ) . Right : DCP1 signal at PM or edge / vertex ( N , biological replicates = 3 , n ( pooled data of 3 biological replicates ) = 18 - 23 PMs or edges / vertices ) .

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B 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 showing fusion ( coarsening ) , fission , growth of PBs ( DCP1 - positive ) at PM ( region 3 ) . Right : states of PBs ( dynamic : fusion fission non - dynamic : stable ; N , biological replicates = 2 , n ( pooled data of 3 biological replicates ) = 6 - 8 PBs ) . As a cautionary note , " stable " PBs may not show dynamicity in imaging time used ( ~ 3 - 5 min ) but later , may do .

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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.