( F ) GFP - pho8Δ60 - Pep4 - mRFP - expressing atg5Δ were cultured with ( AmphoB ) or without ( NT ) AmphoB ( 2 . 5 µg / ml , 24 hr ) , localization of GFP - pho8Δ60 Pep4 - mRFP was observed by confocal microscopy . Arrowheads indicate in which GFP - pho8Δ60 was delivered to vacuoles . Scale bars = 2 µm .

O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O B-GENEPROD O O O B-SUBCELLULAR O O O O O O O

(F) GFP-pho8Δ60- and Pep4-mRFP-expressing atg5Δ cells were cultured with (AmphoB) or without (NT) AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-pho8Δ60 and Pep4-mRFP was observed by confocal microscopy. Arrowheads indicate the cells in which GFP-pho8Δ60 was delivered to the vacuoles. Scale bars = 2 µm.


( G , H ) Accumulation of autophagic ( AB ) - like structures by AmphoB in atg5Δpep4Δ . pep4Δ atg5Δpep4Δ were starved for 3 hr or treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Scale bars = 2 µm . containing AB - like structures were counted under phase - contrast microscopy . Representative ( G ) of with AB - like structures ( H ) are shown . * p < 0 . 01 vs value of pep4Δ . ( I - K ) Induction of AB - like structures in atg5Δpep4Δ treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . Representative of quick - frozen replicas ( I ) thin section of frozen - substituted material ( J ) are shown .

O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O B-SUBCELLULAR O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O 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) Accumulation of autophagic body (AB)-like structures by AmphoB in atg5Δpep4Δ cells. pep4Δ and atg5Δpep4Δ cells were starved for 3 hr or treated with AmphoB (2.5 µg/ml, 24 hr). Scale bars = 2 µm. Cells containing AB-like structures were counted under phase-contrast microscopy. Representative images (G) and the percentage of cells with AB-like structures (H) are shown. *p < 0.01 vs the value of pep4Δ cells. (I-K) Induction of AB-like structures in atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr). Representative images of quick-frozen replicas (I) and thin section of the frozen-substituted material (J) are shown.


( I ) AB - like structures accumulated in vacuole . Scale bar = 0 . 5 µm . ( J ) AB - like structures containing lipid particles mitochondria accumulated in cross - section of vacuole . An autophagosome ( AP ) - like double - compartment containing a ribosome Golgigranule is present in cytosol . Scale bar = 0 . 5 µm . A magnified photo is available in Appendix Fig . S4B . ( K ) containing AB - like structures were counted under electron microscopy [ no treatment ( NT ) : n = 20 , AmphoB : n = 192 ] . * p < 0 . 01 vs value of NT .

O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O

(I) AB-like structures accumulated in the vacuole. Scale bar = 0.5 µm. (J) AB-like structures containing lipid particles and mitochondria accumulated in the cross-section of the vacuole. An autophagosome (AP)-like double-membrane compartment containing a ribosome and Golgigranule is present in the cytosol. Scale bar = 0.5 µm. A magnified photo is available in Appendix Fig. S4B. (K) The cells containing AB-like structures were counted under electron microscopy [no treatment (NT): n = 20, AmphoB: n = 192]. * p < 0.01 vs the value of NT.


( A - D ) Generation of Golgi stacks by AmphoB . Stacked Golgicisternae are observed in quick - frozen replicas ( A - C ) substituted thin section ( D ) of atg5Δpep4Δ treated with AmphoB ( 2 . 5 µg / ml ) for indicated time . ( A ) Scale bar = 1 µm . ( B ) magnified image shows indicated by square in ( A ) . Scale bar = 0 . 5 µm . ( C , D ) A representative Golgi stack with four cisternae is shown . Scale bar = 0 . 5 µm .

O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SMALL_MOLECULE O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O

(A-D) Generation of Golgi stacks by AmphoB. Stacked Golgicisternae are observed in quick-frozen replicas (A-C) and substituted thin section (D) of atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml) for the indicated time. (A) Scale bar = 1 µm. (B) The magnified image shows the area indicated by the square in (A). Scale bar = 0.5 µm. (C, D) A representative Golgi stack with four cisternae is shown. Scale bar = 0.5 µm.


( E - G ) Generation of AP - like double - compartment from stacked Golgi . A quick - frozen replica ( E ) a substituted thin section ( F , G ) are shown . Scale bars = 0 . 5 µm . distal stacks are extended , curved , form AP - like structure ( E , F ) . Mitochondria are encircled by Golgi ( F ) . ( G ) AP - like structure was separated away from Golgi stacks .

O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O

(E-G) Generation of AP-like double-membrane compartment from the stacked Golgi. A quick-frozen replica (E) and a substituted thin section (F, G) are shown. Scale bars = 0.5 µm. The distal stacks are extended, curved, and form AP-like structure (E, F). Mitochondria are encircled by the Golgi membrane (F). (G) The AP-like structure was separated away from Golgi stacks.


( H , I ) scene that AP - like structure was fused with vacuole . A magnified image of ( H ) is shown in ( I ) . In atg5Δpep4Δ treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) , outer of AP - like structure ( red arrows ) was fused with vacuolar ( blue arrows ) . Inner membranes of AP - like structure ( yellow arrowheads ) were entered into vacuoles to become AB - like structure . Green arrows indicate fusion site . Scale bars = 0 . 5 µm ( H ) 0 . 2 µm ( I ) .

O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O

(H, I) The scene that AP-like structure was fused with vacuole. A magnified image of (H) is shown in (I). In atg5Δpep4Δ cells treated with AmphoB (2.5 µg/ml, 24 hr), outer membrane of AP-like structure (red arrows) was fused with vacuolar membrane (blue arrows). Inner membranes of AP-like structure (yellow arrowheads) were entered into the vacuoles to become AB-like structure. Green arrows indicate the membrane fusion site. Scale bars = 0.5 µm (H) and 0.2 µm (I).


( J ) number of elongated single cisternae ( green bars ) , Golgi stacks ( red bars ) , AB - like structures ( blue bars ) was counted over time using electron microscopy . Error bars indicate s . e . m . ( 3 hr : n = 21 , 6 hr : n = 20 , 12 hr : n = 193 , 24 hr : n = 192 ) .

O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O B-SUBCELLULAR O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O 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) The number of elongated single cisternae (green bars), Golgi stacks (red bars), and AB-like structures (blue bars) was counted over time using electron microscopy images. Error bars indicate s.e.m. (3 hr: n = 21, 6 hr: n = 20, 12 hr: n = 193, 24 hr: n = 192).


( K ) Translocation of trans - Golgi to vacuolar induced by AmphoB treatment . GFP - Sft2 - expressing atg5Δ ( trans - Golgi marker ) were incubated with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) , localization of GFP - Sft2 was assessed by confocal microscopy . Arrowhead indicates GFP signal on vacuolar . Scale bars = 2 µm .

O O O O O O O O O B-SUBCELLULAR O O B-SMALL_MOLECULE O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O O O B-GENEPROD O O B-SUBCELLULAR O O O O O O O

(K) Translocation of the trans-Golgi protein to the vacuolar membrane induced by AmphoB treatment. GFP-Sft2-expressing atg5Δ cells (trans-Golgi marker protein) were incubated with or without AmphoB (2.5 µg/ml, 24 hr), and localization of GFP-Sft2 was assessed by confocal microscopy. Arrowhead indicates the GFP signal on the vacuolar membrane. Scale bars = 2 µm.


( L - O ) Grh1 Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( L , M ) Representative electron microscopy of grh1Δatg5Δpep4Δ ( L ) gvp36Δatg5Δpep4Δ ( M ) treated with AmphoB ( 2 . 5 µg / ml , 24 hr ) . AP - like AB - like structures were absent . ( L ) A unilamellar Golgi cisterna was observed in cytoplasm ( dotted square ) . Scale bar = 1 µm . ( M ) Formation of a Golgi stack , but not Golgi curvature , was observed ( dotted square ) . Bar = 0 . 5 µm .

O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (L, M) Representative electron microscopy images of grh1Δatg5Δpep4Δ (L) and gvp36Δatg5Δpep4Δ cells (M) treated with AmphoB (2.5 µg/ml, 24 hr). AP-like and AB-like structures were absent. (L) A unilamellar Golgi cisterna was observed in the cytoplasm (dotted square). Scale bar = 1 µm. (M) Formation of a Golgi stack, but not Golgi curvature, was observed (dotted square). Bar = 0.5 µm.


( L - O ) Grh1 Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( N ) Indicated were treated with AmphoB for 24 hr number of containing AB - like structures was counted by phase - contrast microscopy . * p < 0 . 01 vs value of atg5Δpep4Δ .

O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (N) Indicated cells were treated with AmphoB for 24 hr and the number of cells containing AB-like structures was counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( L - O ) Grh1 Gvp36 involvement in AmphoB - induced autophagy - like proteolysis . ( O ) GFP - pho8Δ60 - expressing atg5Δ , grh1Δatg5Δ gvp36Δatg5Δ were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) subjected to western blotting for GFP . An unidentified band at 32kD ( asterisk ) is non - specific band because it was present in Pep4 - lacking grh1Δatg5Δ .

O O O O O B-GENEPROD I-GENEPROD O O B-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD I-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O O B-GENEPROD O

(L-O) Grh1 and Gvp36 involvement in AmphoB-induced autophagy-like proteolysis. (O) GFP-pho8Δ60-expressing atg5Δ, grh1Δatg5Δ and gvp36Δatg5Δ cells were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP. An unidentified band at 32kD (asterisk) is non-specific band because it was present in Pep4-lacking grh1Δatg5Δ cells.


( A ) Localization of PI ( 4 ) P . GFP - 2xPHOsh2 - expressing atg5Δ , sac1 - 23 / atg5Δ , pik1 - 83 / atg5Δ were incubated with or without AmphoB ( 2 . 5 µg / ml ) for 6 hr at 37 ° C , their localization was observed by confocal microscopy . Scale bars = 2 µm .

O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O

(A) Localization of cellular PI(4)P. GFP-2xPHOsh2-expressing atg5Δ, sac1-23/atg5Δ, and pik1-83/atg5Δ cells were incubated with or without AmphoB (2.5 µg/ml) for 6 hr at 37 °C, and their localization was observed by confocal microscopy. Scale bars = 2 µm.


( B ) same experiments were performed using GFP - GOLPH3 ( a GolgiPI ( 4 ) P - monitoring ) instead of GFP - 2xPHOsh2 . Scale bars = 2 µm .

O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O

(B) The same experiments were performed using GFP-GOLPH3 (a GolgiPI(4)P-monitoring protein) instead of GFP-2xPHOsh2. Scale bars = 2 µm.


( C , D ) Reduction of AmphoB - induced GOMED by deletion of Golgi PI ( 4 ) phosphatases . ( C ) Indicated expressing GFP - pho8Δ60 were cultured with or without AmphoB ( 2 . 5 µg / ml , 24 hr ) subjected to western blotting for GFP .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O

(C, D) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (C) Indicated cells expressing GFP-pho8Δ60 were cultured with or without AmphoB (2.5 µg/ml, 24 hr) and subjected to western blotting for GFP.


( C , D ) Reduction of AmphoB - induced GOMED by deletion of Golgi PI ( 4 ) phosphatases . ( D ) number of AP - like AB - like structures was counted using EM ( Fig . EV2B , C ) . ( E − H ) Induction of GOMED by deletion of Golgi PI ( 4 ) kinase .

O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O 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) Reduction of AmphoB-induced GOMED by the deletion of Golgi PI(4) phosphatases. (D) The number of AP-like and AB-like structures was counted using EM images (Fig. EV2B, C). (E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase.


( E − H ) Induction of GOMED by deletion of Golgi PI ( 4 ) kinase . ( E ) Indicated were incubated at 37 ° C ( shift ) for 6 hr , containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs value of atg5Δpep4Δ .

O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (E) Indicated cells were incubated at 37 °C (temperature shift) for 6 hr, and cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by deletion of Golgi PI ( 4 ) kinase . ( F ) Representative electron microscopy image of pik1 - 83 / atg5Δpep4Δ at 3 hr after shift . AP - like structure ( arrow ) AB - like structures were generated . Scale bar = 0 . 2 µm . ( G ) number of AP - like AB - like structures was counted using EM . Error bars indicate s . e . m . ( atg5Δpep4Δ at 37 ° C : n = 54 pik1 - 83 / atg5Δpep4Δ at 37 ° C : n = 63 ) . * p < 0 . 01 vs value of atg5Δpep4Δ .

O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O B-GENEPROD O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD O O O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O B-GENEPROD O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (F) Representative electron microscopy image of pik1-83/atg5Δpep4Δ cells at 3 hr after temperature shift. AP-like structure (arrow) and AB-like structures were generated. Scale bar = 0.2 µm. (G) The number of AP-like and AB-like structures was counted using EM images. Error bars indicate s.e.m. (atg5Δpep4Δ cells at 37 °C: n = 54 and pik1-83/atg5Δpep4Δ cells at 37 °C: n = 63). *p < 0.01 vs the value of atg5Δpep4Δ cells.


( E − H ) Induction of GOMED by deletion of Golgi PI ( 4 ) kinase . ( H ) Sec7 - HA - expressing pik1 - 83 / atg5Δ were incubated at 37 ° C ( shift ) for 3 hr , freeze replica immunolabelling was performed as described in Methods . Sec7 - HA - positive signals ( labelled with 15 nm gold particle ) were observed on surface of AP - like structure Golgi . Scale bar = 0 . 1 µm .

O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O

(E−H) Induction of GOMED by the deletion of Golgi PI(4) kinase. (H) Sec7-HA-expressing pik1-83/atg5Δ cells were incubated at 37 °C (temperature shift) for 3 hr, and freeze replica immunolabelling was performed as described in Methods. Sec7-HA-positive signals (labelled with 15 nm gold particle) were observed on the surface membrane of AP-like structure and Golgi membrane. Scale bar = 0.1 µm.


( I − L ) Genetic ( I , J ) pharmacological ( K , L ) alterations in anterograde trafficking from Golgi are required for GOMED . ( I ) Indicated were incubated at 37 ° C ( switch ) for 3 hr , containing AB - like structures were counted by phase - contrast microscopy . * p < 0 . 01 vs value of atg5Δpep4Δ .

O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O B-GENEPROD O

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (I) Indicated cells were incubated at 37 °C (temperature switch) for 3 hr, and the cells containing AB-like structures were counted by phase-contrast microscopy. *p < 0.01 vs the value of atg5Δpep4Δ cells.


( I − L ) Genetic ( I , J ) pharmacological ( K , L ) alterations in anterograde trafficking from Golgi are required for GOMED . ( J ) Indicated were also observed using EM . Arrow indicates AP - like structure . Scale bar = 0 . 5 µm .

O O O O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O O O O O O O O O O O

(I−L) Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (J) Indicated cells were also observed using EM. Arrow indicates AP-like structure. Scale bar = 0.5 µm.


Genetic ( I , J ) pharmacological ( K , L ) alterations in anterograde trafficking from Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) , containing AB - like structures were counted by phase - contrast microscopy ( K ) . * p < 0 . 01 vs value of NT .

O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr), and cells containing AB-like structures were counted by phase-contrast microscopy (K). * p < 0.01 vs the value of NT.


Genetic ( I , J ) pharmacological ( K , L ) alterations in anterograde trafficking from Golgi are required for GOMED . ( K , L ) atg5Δpep4Δ were treated with ( CBM ) or without ( NT ) CBM ( 1 mM , 3 hr ) . were also observed using EM ( L ) . AB - like structures were observed . Scale bar = 0 . 5 µm .

O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O

Genetic (I, J) and pharmacological (K, L) alterations in anterograde trafficking from the Golgi are required for GOMED. (K, L) atg5Δpep4Δ cells were treated with (CBM) or without (NT) CBM (1 mM, 3 hr). Cells were also observed using EM (L). AB-like structures were observed. Scale bar = 0.5 µm.


( M − O ) Disturbance of anterograde trafficking from Golgi to PM in with GOMED . Indicated expressing Hsp150 - HA ( 3 × ) were treated as follows : NT , no treatment ; starve , starvation for 6 hr ; AmphoB , 2 . 5 µg / ml for 24 hr ( M ) ; CBM , 1 mM for 24 hr ( N ) ; shift , from 25 ° C to 37 ° C for 3 hr ( O ) . were then lysed examined for Hsp150 - HA ( 3 × ) expression by western blotting . Accumulation of O - glycosylated Hsp150 - HA ( 3 × ) indicated trafficking failure from Golgi to PM .

O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O 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-GENEPROD O B-GENEPROD O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O

(M−O) Disturbance of anterograde trafficking from the Golgi to the PM in the cells with GOMED. Indicated cells expressing Hsp150-HA(3×) were treated as follows: NT, no treatment; starve, starvation for 6 hr; AmphoB, 2.5 µg/ml for 24 hr (M); CBM, 1 mM for 24 hr (N); temperature shift, from 25 °C to 37 °C for 3 hr (O). The cells were then lysed and examined for Hsp150-HA(3×) expression by western blotting. Accumulation of O-glycosylated Hsp150-HA(3×) indicated the trafficking failure from the Golgi to the PM.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( A , B ) EM revealed formation of AP - like AL - like structures . Atg5 KO MEFs were treated with CBM ( 2 mM ) for 3 hr . A representative low - magnification image ( A ) high - magnification ( B ) are shown . Scale bar = 1 µm ( A ) 0 . 2 µm ( B ) . arrowheads arrows indicate AL - like AP - like structures , respectively .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O B-EXP_ASSAY O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O B-SUBCELLULAR O O O O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (A, B) EM analysis revealed the formation of AP-like and AL-like structures. Atg5 KO MEFs were treated with CBM (2 mM) for 3 hr. A representative low-magnification image (A) and high-magnification images (B) are shown. Scale bar = 1 µm (A) and 0.2 µm (B). The arrowheads and arrows indicate AL-like and AP-like structures, respectively.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in generation of AL - like structures . ( C ) CLEM of mCherry - syntaxin 6 - expressing Atg5 KO MEFs . were treated with CBM ( 2 mM ) for 24 hr observed using electron microscopy . mCherry - syntaxin 6puncta merged with AL - like structures . Scale bar = 5 µm . " N " indicates nucleus . A magnified image of dashed square is shown in inset . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (C) CLEM analysis of mCherry-syntaxin 6-expressing Atg5 KO MEFs. Cells were treated with CBM (2 mM) for 24 hr and observed using fluorescence and electron microscopy. mCherry-syntaxin 6puncta merged with the AL-like structures. Scale bar = 5 µm. "N" indicates nucleus. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in generation of AL - like structures . ( D , E ) Requirement of Grasp65 in generation of AL - like structures . Atg5 KO Grasp65 - silenced Atg5 KO MEFs were treated without ( NT ) or with CBM ( 2 mM ) for indicated times followed by immunostaining with an anti - Lamp2 antibody . Representative ( at 24 h ) are shown in ( D ) . Scale bars = 5 µm . A magnified image of dashed square is shown in inset . Scale bars = 1 µm . CBM induced large ring - like Lamp2 . ( E ) Percentages of with large ring - like Lamp2 immunofluorescence ( mean ± s . e . m . , n = 4 ) . * p < 0 . 01 vs value of Atg5 KO MEF .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O B-GENEPROD O O O B-SUBCELLULAR O O O O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O 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-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O O O O O O B-GENEPROD O 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-GENEPROD O O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (D, E) Requirement of Grasp65 in the generation of AL-like structures. Atg5 KO and Grasp65-silenced Atg5 KO MEFs were treated without (NT) or with CBM (2 mM) for the indicated times followed by immunostaining with an anti-Lamp2 antibody. Representative images (at 24 h) are shown in (D). Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 1 µm. CBM induced large ring-like Lamp2 fluorescence. (E) Percentages of cells with large ring-like Lamp2 immunofluorescence (mean ± s.e.m., n = 4). *p < 0.01 vs the value of Atg5 KO MEF.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( C - F ) Involvement of Golgi membranes in generation of AL - like structures . ( F ) Colocalization of Lamp2 GFP - syntaxin 6 in CBM - treated Atg5 KO MEFs . GFP - syntaxin 6 - expressing Atg5 KO MEFs were treated with ( CBM ) or without ( NT ) CBM ( 2 mM ) for 6 hr immunostained with an anti - Lamp2 antibody . ring - like Lamp2 merged with signal for syntaxin 6 . Scale bar = 5 µm . A magnified image of dashed square is shown in inset . Scale bars = 2 µm .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O B-GENEPROD I-GENEPROD O B-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD I-GENEPROD 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−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (C-F) Involvement of Golgi membranes in the generation of AL-like structures. (F) Colocalization of Lamp2 and GFP-syntaxin 6 in CBM-treated Atg5 KO MEFs. GFP-syntaxin 6-expressing Atg5 KO MEFs were treated with (CBM) or without (NT) CBM (2 mM) for 6 hr and immunostained with an anti-Lamp2 antibody. The ring-like Lamp2 fluorescence merged with the signal for syntaxin 6. Scale bar = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( G ) monomeric red ( mRFP ) - green ( GFP ) tandem revealed induction of GOMED . Atg5 KO MEFs stably expressing tandem mRFP - GFP were incubated without ( no treatment ) or with CBM ( 2 mM ) for 24 hr . Red signals indicate acidic compartments . Lysosomes were counterstained with an anti - Lamp2 antibody ( cyan ) . Scale bars = 5 µm . Regions of interest ( ROI ) are indicated by dashed squares . Arrowheads indicate GOMED structure . Scale bars = 2 µm .

O O O O O O B-GENEPROD O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O O O O O O O O O O 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) The monomeric red fluorescent protein (mRFP)-green fluorescent protein (GFP) tandem protein assay revealed the induction of GOMED. Atg5 KO MEFs stably expressing tandem mRFP-GFP were incubated without (no treatment) or with CBM (2 mM) for 24 hr. Red signals indicate acidic compartments. Lysosomes were counterstained with an anti-Lamp2 antibody (cyan). Scale bars = 5 µm. Regions of interest (ROI) are indicated by dashed squares. Arrowheads indicate GOMED structure. Scale bars = 2 µm.


( A − I ) Induction of GOMED in Atg5 KO MEFs by CBM treatment . ( H , I ) Degradation of GFP - fused indicated induction of GOMED . Atg5 KO MEFs stably expressing VSVG - GFP ( H ) or M6PR - GFP ( I ) were incubated with CBM ( 5 mM ) for indicated times . lysates were subjected to immunoblotting with anti - GFP antibody .

O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O B-CELL_LINE O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O O O B-GENEPROD O O

(A−I) Induction of GOMED in Atg5 KO MEFs by CBM treatment. (H, I) Degradation of GFP-fused proteins indicated the induction of GOMED. Atg5 KO MEFs stably expressing VSVG-GFP (H) or M6PR-GFP (I) were incubated with CBM (5 mM) for the indicated times. Cell lysates were subjected to immunoblotting with the anti-GFP antibody.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with indicated small interfering ( si ) RNAs for 24 hr GOMED was assessed by EM ( J - L ) . Representative are shown in ( J , K ) . Arrows indicate AL - like structures . Scale bars = 2 µm . ( L ) number of AP - like AL - like structures was calculated from EM of treated with indicated siRNAs . Error bars indicate s . e . m . ( siControl : n = 23 , siPI4k2α + siPI4k3β : n = 20 , siArfaptin1 : n = 20 ) . * p < 0 . 01 vs value of siControl .

O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O 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−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by EM (J-L). Representative images are shown in (J, K). Arrows indicate AL-like structures. Scale bars = 2 µm. (L) The number of AP-like and AL-like structures was calculated from the EM images of cells treated with the indicated siRNAs. Error bars indicate s.e.m. (siControl: n = 23, siPI4k2α + siPI4k3β: n = 20, siArfaptin1: n = 20). *p < 0.01 vs the value of siControl.


( J − M ) Genetic induction of GOMED in Atg5 KO MEFs . Atg5 KO MEFs were treated with indicated small interfering ( si ) RNAs for 24 hr GOMED was assessed by mRFP - GFP tandem ( M ) . ( M ) same experiments as in ( G ) were performed using treated with indicated siRNAs . Representative are shown . Arrowheads indicate GOMED structure . Scale bars = 5 µm .

O O O O O O O O O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O 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−M) Genetic induction of GOMED in Atg5 KO MEFs. Atg5 KO MEFs were treated with the indicated small interfering (si)RNAs for 24 hr and GOMED was assessed by the mRFP-GFP tandem protein assay (M). (M) The same experiments as in (G) were performed using cells treated with the indicated siRNAs. Representative images are shown. Arrowheads indicate GOMED structure. Scale bars = 5 µm.


( A ) Suppression of insulin secretion in Atg5 knockout ( KO ) MIN6 after glucose deprivation . Atg5 KO MIN6 were incubated with normal culture medium ( NT ) or glucose - depleted medium for 1 hr . amount of secreted insulin is expressed as of that of untreated control ( NT ) ( mean ± s . e . m , n = 3 ) . * p < 0 . 01 vs value of NT .

O O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O O B-CELL_LINE O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O 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) Suppression of insulin secretion in Atg5 knockout (KO) MIN6 cells after glucose deprivation. Atg5 KO MIN6 cells were incubated with normal culture medium (NT) or glucose-depleted medium for 1 hr. The amount of secreted insulin is expressed as the percentage of that of the untreated control (NT) (mean ± s.e.m, n = 3). *p < 0.01 vs the value of NT.


( B , C ) EM revealed induction of GOMED structures . Atg5 KO MIN6 were subjected to glucose deprivation for 1 hr . Representative are shown . arrow black arrows indicate AP - like structure insulinvesicles , respectively . Scale bars = 0 . 2 µm . ( D ) number of AP - like AL - like structures was calculated from EM of left untreated ( NT ) or subjected to glucose deprivation . Error bars indicate s . e . m . ( NT : n = 17 , glucose deprivation : n = 84 ) . * p < 0 . 01 # p < 0 . 05 vs value of NT .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O B-SUBCELLULAR O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE 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, C) EM analysis revealed the induction of GOMED structures. Atg5 KO MIN6 cells were subjected to glucose deprivation for 1 hr. Representative images are shown. The white arrow and black arrows indicate the AP-like structure and insulinvesicles, respectively. Scale bars = 0.2 µm. (D) The number of AP-like and AL-like structures was calculated from the EM images of cells left untreated (NT) or subjected to glucose deprivation. Error bars indicate s.e.m. (NT: n = 17, glucose deprivation: n = 84). *p < 0.01 and #p < 0.05 vs the value of NT.


( E ) Involvement of Golgi in generation of GOMED . Lamp1 - GFP / mCherry - Syntaxin 6 - expressing Atg5 KO MIN6 were subjected to glucose deprivation , analysed by confocal microscopy . CLEM revealed that AL - like structures ( arrows ) merged with Lamp1 - GFP Syntaxin 6 . Scale bars = 5 µm . A magnified image of dashed square is shown in inset . Scale bars = 2 µm .

O O O O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O B-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD 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) Involvement of Golgi membrane in the generation of GOMED. Lamp1-GFP/mCherry-Syntaxin 6-expressing Atg5 KO MIN6 cells were subjected to glucose deprivation, and analysed by TEM and confocal microscopy. CLEM analysis revealed that AL-like structures (arrows) merged with Lamp1-GFP fluorescence and Syntaxin 6 fluorescence. Scale bars = 5 µm. A magnified image of the dashed square is shown in the inset. Scale bars = 2 µm.


( F ) GOMED - mediated degradation of ( pro ) insulin by glucose deprivation of Atg5 KO MIN6 . Atg5 KO MIN6 were incubated in glucose - depleted medium in absence or presence of bafilomycin A1 ( 10 nM ) for indicated times . lysates were then subjected to immunoblot using an anti - ( pro ) insulin antibody .

O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-SMALL_MOLECULE O O B-GENEPROD O B-CELL_LINE O B-GENEPROD O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O

(F) GOMED-mediated degradation of (pro)insulin by glucose deprivation of Atg5 KO MIN6 cells. Atg5 KO MIN6 cells were incubated in glucose-depleted medium in the absence or presence of bafilomycin A1 (10 nM) for the indicated times. The cell lysates were then subjected to immunoblot analysis using an anti-(pro)insulin antibody.


( G ) Immunofluorescence of ( pro ) insulin Lamp2 . Atg5 KO MIN6 were left untreated ( NT ) or subjected to glucose deprivation for 1 hr in presence of E64d ( 25 µM ) or bafilomycin A1 ( 10 nM ) , were stained with anti - ( pro ) insulin anti - Lamp2 ( lysosomal marker ) antibodies observed by microscopy . Scale bar = 1 µm . Arrowheads indicate colocalization of ( pro ) insulin with GOMED structures .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-GENEPROD O B-SUBCELLULAR O O O O O B-EXP_ASSAY O O O O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O O O

(G) Immunofluorescence analysis of (pro)insulin and Lamp2. Atg5 KO MIN6 cells were left untreated (NT) or subjected to glucose deprivation for 1 hr in the presence of E64d (25 µM) or bafilomycin A1 (10 nM), and were stained with anti-(pro)insulin and anti-Lamp2 (lysosomal marker) antibodies and observed by fluorescence microscopy. Scale bar = 1 µm. Arrowheads indicate the colocalization of (pro)insulin with GOMED structures.


( A − D ) EM demonstrated induction of GOMED . Atg7 KO β - were subjected to glucose deprivation for 1 hr . A representative AP - like structure containing insulingranule is shown in ( A ) . An AL - like structure containing insulingranules is shown in ( B ) . Scale bars = 0 . 2 µm .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. A representative AP-like structure containing insulingranule is shown in (A). An AL-like structure containing insulingranules is shown in (B). Scale bars = 0.2 µm.


( A − D ) EM demonstrated induction of GOMED . Atg7 KO β - were subjected to glucose deprivation for 1 hr . ( C , D ) GOMED structures containing Golgi vesicles . An AP - like structure containing multiple Golgi vesicles ( arrows ) ( C ) an AL - like structure fusing with a lysosome ( arrowhead ) ( D ) are shown . Scale bar = 0 . 5 µm . A magnified image of dashed rectangle is shown in inset . Scale bar = 0 . 2 µm . AP - like structure contained double - .

O O O O O B-EXP_ASSAY O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O B-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O

(A−D) EM analysis demonstrated the induction of GOMED. Primary Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. (C, D) GOMED structures containing Golgi vesicles. An AP-like structure containing multiple Golgi vesicles (arrows) (C) and an AL-like structure fusing with a lysosome (arrowhead) (D) are shown. Scale bar = 0.5 µm. A magnified image of the dashed rectangle is shown in the inset. Scale bar = 0.2 µm. AP-like structure contained double-membrane.


( E ) total number of AL - like structure was calculated from EM of WT Atg7 KO β - after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 # p < 0 . 05 vs value of 0 min .

O O O O O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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) The total number of AL-like structure was calculated from the EM images of WT and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( F ) total number of GOMED structure was calculated from EM of Atg7 KO β - after glucose deprivation in absence or presence of bafilomycin A1 ( 10 nM ) . Error bars indicate s . e . m . ( n = 109 − 116 ) . * p < 0 . 01 vs value of 60 min .

O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(F) The total number of GOMED structure was calculated from the EM images of Atg7 KO β-cells after glucose deprivation in the absence or presence of bafilomycin A1 (10 nM). Error bars indicate s.e.m. (n = 109−116). *p < 0.01 vs the value of 60 min.


( G − J ) EM showed induction of mitophagy . WT Atg7 KO β - were subjected to glucose deprivation for 1 hr . Representative showing mitophagy in Atg7 KO β - are shown in ( G ) . Arrowhead ( G ) indicates mitophagy . Scale bars = 0 . 5 µm . ( H , J ) number of structures representative of mitophagy ( H ) per was calculated from EM of β - after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 # p < 0 . 05 vs value of 0 min .

O O O O O B-EXP_ASSAY O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O 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-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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−J) EM analysis showed the induction of mitophagy. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing mitophagy in Atg7 KO β-cells are shown in (G). Arrowhead (G) indicates mitophagy. Scale bars = 0.5 µm. (H, J) The number of structures representative of mitophagy (H) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( G − J ) EM showed induction of crinophagy / SINGD . WT Atg7 KO β - were subjected to glucose deprivation for 1 hr . Representative showing crinophagy / SINGD in WT β - are shown in ( I ) . arrow ( I ) indicates crinophagy / SINGD . Arrows indicate insulingranules ( I ) . Scale bars = 0 . 5 µm . ( H , J ) number of structures representative of crinophagy / SINGD ( J ) per was calculated from EM of β - after glucose depletion . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 # p < 0 . 05 vs value of 0 min .

O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O 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-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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−J) EM analysis showed the induction of crinophagy/SINGD. WT and Atg7 KO β-cells were subjected to glucose deprivation for 1 hr. Representative images showing crinophagy/SINGD in WT β-cells are shown in (I). White arrow (I) indicates crinophagy/SINGD. Arrows indicate insulingranules (I). Scale bars = 0.5 µm. (H, J) The number of structures representative of crinophagy/SINGD (J) per cell was calculated from the EM images of β-cells after glucose depletion. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


( K ) number of ( pro ) insulingranule - containing AL - like structure was calculated from EM of wild - type ( WT ) Atg7 KO β - after glucose deprivation . Error bars indicate s . e . m . ( n = 46 − 163 ) . * p < 0 . 01 # p < 0 . 05 vs value of 0 min .

O O O O O B-GENEPROD I-GENEPROD I-GENEPROD I-GENEPROD O O B-SUBCELLULAR O O O O O O B-EXP_ASSAY O O O O O O O B-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-SMALL_MOLECULE O O O O O O O O O O O O O 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) The number of (pro)insulingranule-containing AL-like structure was calculated from the EM images of wild-type (WT) and Atg7 KO β-cells after glucose deprivation. Error bars indicate s.e.m. (n = 46−163). *p < 0.01 and #p < 0.05 vs the value of 0 min.


Survival curve of N . furzeri in FLI facility . Recording of deaths starts at of 5 wph , which corresponds to sexual maturity , colored dashed lines indicate three groups analysed in this study ( 5 / group ) , namely 5 post hatching ( wph , young , sexual maturity ) , 12 wph ( adult ) 39 wph ( old , past median lifespan ) of a wild - derived that exhibits a median lifespan of 7 - 8 months .

B-EXP_ASSAY O O B-ORGANISM I-ORGANISM I-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Survival curve of N. furzeri in the FLI facility. Recording of deaths starts at age of 5 wph, which corresponds to sexual maturity, and the colored dashed lines indicate the three age groups analysed in this study (5 animals/group), namely 5 weeks post hatching (wph, young, sexual maturity), 12 wph (adult) and 39 wph (old, past median lifespan) of a wild-derived strain that exhibits a median lifespan of 7-8 months.


Global - transcript correlation for each sample , grouped by . RPKM iBAQ values were used to estimate transcript from matched - seq TMT - proteomics data obtained from same . An ANOVA was performed to evaluate significance among groups ( mean correlation at 5 wph : 0 . 48 ; at 12 wph : 0 . 43 ; at 39 wph : 0 . 33 ; p = 3 . 05e - 07 , n = 5 per group ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Global protein-transcript correlation for each sample, grouped by age. RPKM and iBAQ values were used to estimate transcript and protein levels from matched RNA-seq and TMT-based proteomics data obtained from the same animal. An ANOVA test was performed to evaluate significance among the age groups (mean correlation at 5 wph: 0.48; at 12 wph: 0.43; and at 39 wph: 0.33; p = 3.05e-07, n=5 per age group). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Scatter plot of log2 fold changes for genes differentially expressed both at transcript ( adj . p < 0 . 05 ) . color gradients indicate gene density in regions where points overlap . Numbers of genes in each quadrant value of Pearson ' s coefficient of correlation , r , are reported for each graph . Solid lines represent a spline fit ( r = 0 . 505 for genes significantly affected at both transcript , p < 2 . 2 x 10 - 16 , D ) ( r = 0 . 126 , p = 0 . 007 , E ) .

O O O O O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Scatter plot of log2 fold changes for genes differentially expressed both at transcript and protein levels (adj. p < 0.05). The color gradients indicate gene density in the regions where individual points overlap. Numbers of genes in each quadrant and the value of Pearson's coefficient of correlation, r, are reported for each graph. Solid lines represent a spline fit (r = 0.505 for genes significantly affected at both transcript and protein levels, p < 2.2 x 10-16, D) (r = 0.126, p = 0.007, E).


Mechanisms affecting abundance during aging . Bar plots are on affected in either one of comparisons ( adj . p < 0 . 05 ) . were divided in following five groups : ( i ) transcripts with significant consistent changes ( brown ) , ( ii ) with significant changes , with consistent changes of transcripts ( brown ) , ( iii ) with no transcripts detected ( gray ) , ( iv ) with transcripts whose translation is potentially regulated by miRNAs ( gray ) , as assessed by workflow displayed in ( Figure EV2D ) , ( v ) remaining that we classified as regulated by other post - transcriptional mechanisms ( violet ) . pgs = groups .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Mechanisms affecting protein abundance during aging. Bar plots are based on all the proteins affected in either one of the age comparisons (adj. p < 0.05). Proteins were divided in the following five groups: (i) proteins and transcripts with significant and consistent changes (dark brown), (ii) proteins with significant changes, and with consistent changes of the transcripts (light brown), (iii) proteins with no transcripts detected (dark gray), (iv) proteins with transcripts whose translation is potentially regulated by miRNAs (light gray), as assessed by the workflow displayed in (Figure EV2D), (v) all the remaining proteins that we classified as regulated by other post-transcriptional mechanisms (violet). pgs = protein groups.


Abundance changes of ribosomal their transcripts during aging . ribosomal of large small subunits are displayed separately changes are shown for both comparisons as box plots . Transcripts are displayed as blue as blue boxes . Changes of are displayed as dots ; orange dots identify significant cases ( adj . p < 0 . 05 , n = 4 per group for proteome n = 5 per group for transcriptome ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abundance changes of ribosomal proteins and their transcripts during aging. Cytoplasmic ribosomal proteins of large and small subunits are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots; orange dots identify significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Brains from young ( 5 wph ) old ( 39 wph ) were homogenized clarified lysates separated by - exclusion chromatography ( SEC ) . For each group , two pools of brains were processed separately . For each experiment ( 4 in total ) , 39 fractions were collected along chromatogram , digested into peptides analysed by Data Independent Acquisition ( DIA ) quantitative mass spectrometry .

O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O O O O O O O O B-TISSUE O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY O

Brains from young (5 wph) and old (39 wph) were homogenized and clarified lysates separated by size-exclusion chromatography (SEC). For each age group, two pools of brains were processed separately. For each experiment (4 in total), 39 fractions were collected along the chromatogram, digested into peptides and analysed by Data Independent Acquisition (DIA) quantitative mass spectrometry.


Co - elution profiles for selected complexes . For each , median abundance of quantified subunits was used to generate profile across fractions ( Dataset EV5 ) . profiles are scaled to max value ( set to 1 ) to make profiles comparable across experiments . estimated molecular of displayed complexes is indicated in brackets .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Co-elution profiles for selected protein complexes. For each complex, the median abundance of all the quantified subunits was used to generate the complex profile across fractions (Dataset EV5). All the complex profiles are scaled to the max value (set to 1) to make profiles comparable across experiments. The estimated molecular weight of the displayed complexes is indicated in brackets.


Statistics of complexes undergoing stoichiometry changes with aging . Only complexes that had at least 5 members quantified were considered for each comparison . Complexes were considered affected if at least two members showed significant stoichiometry change ( adj . p < 0 . 05 absolute log2 fold change > 0 . 5 ) . complete list of stoichiometry changes is available in Dataset EV6 .

O O B-SUBCELLULAR O B-EXP_ASSAY I-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O

Statistics of protein complexes undergoing stoichiometry changes with aging. Only protein complexes that had at least 5 members quantified were considered for each comparison. Complexes were considered affected if at least two members showed significant stoichiometry change (adj. p < 0.05 and absolute log2 fold change > 0.5). The complete list of stoichiometry changes is available in Dataset EV6.


Violin plots depicting interquartile ranges ( IQRs ) of members of complexes during aging . IQR for each considered in G was calculated using transcript ( total dataset , blue ) or ( blue ) log2 fold changes between two groups ( n = 4 per group for proteome n = 5 per group for transcriptome ) . * p < 0 . 05 , Wilcoxon Rank Sum . central of violin plots indicates median value .

B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Violin plots depicting interquartile ranges (IQRs) of individual members of protein complexes during aging. The IQR for each protein complex considered in G was calculated using transcript (total RNA dataset, light blue) or protein (dark blue) log2 fold changes between two age groups (n=4 per age group for proteome and n=5 per age group for transcriptome). * p < 0.05, Wilcoxon Rank Sum test. The central line of the violin plots indicates the median value.


Heat map showing relative fold changes for members of selected complexes affected by aging . Names of significantly affected members in 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 absolute log2 fold change > 0 . 5 ) are highlighted in bold with a star . Related to Figure EV3 Dataset EV5 EV6 .

O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map showing relative protein fold changes for members of selected complexes affected by aging. Names of significantly affected members in the 39 wph vs. 12 wph comparison (adj. p < 0.05 and absolute log2 fold change > 0.5) are highlighted in bold with a star. Related to Figure EV3 and Dataset EV5 and EV6.


Representative Coomassie - stained SDS - PAGE gel showing isolation of SDS insoluble from mouse brain lysates . TH = total homogenate , SUP = supernatant , W1 = SDS - soluble fraction , Pellet = formic - acid soluble fraction ( see Figure EV4A ) . of yield of SDS - insoluble from young old brain lysates was on densitometry of Coomassie - stained gel bands obtained from different , n = 5 per group ( Figure EV4B ) ; * * p < 0 . 01 , unpaired t - . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-ORGANISM B-TISSUE O O B-SUBCELLULAR O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O B-SMALL_MOLECULE O B-SUBCELLULAR I-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O B-SMALL_MOLECULE O O O O O B-TISSUE O O O B-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Representative Coomassie-stained SDS-PAGE gel showing the isolation of SDS insoluble aggregates from mouse brain lysates. TH = total homogenate, SUP = supernatant, W1 = SDS-soluble fraction, Pellet = formic-acid soluble fraction (see Figure EV4A). Quantification of the yield of SDS-insoluble aggregates from young and old brain lysates was based on densitometry analysis of Coomassie-stained gel bands obtained from different animals, n=5 per age group (Figure EV4B); ** p < 0.01, unpaired t-test. In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Volcano plot on by label - free mass spectrometry depicting enrichment of specific in . x - axis indicates log2 between abundance in ( Pellet ) starting total homogenate ( TH ) . horizontal dashed indicates a p value cut - off of 0 . 05 vertical lines a log2 fold change cut off of ± 0 . 5 . Selected are highlighted as colored dots as indicated in figure legend . was on samples obtained from 3 independent isolations .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Volcano plot based on protein quantification by label-free mass spectrometry depicting the enrichment of specific proteins in protein aggregates. The x-axis indicates the log2 ratio between protein abundance in aggregates (Pellet) and starting total homogenate (TH). The horizontal dashed line indicates a p value cut-off of 0.05 and vertical lines a log2 fold change cut off of ± 0.5. Selected proteins are highlighted as colored dots as indicated in the figure legend. Protein quantification was based on samples obtained from 3 independent isolations.


Double - labelling of telencephalic sections of N . furzeri with anti - RPS6 ( green ) as ribosomal marker Proteostat as a marker for aggregated ( red ) . counterstaining was performed with DAPI ( blue ) . Scale bar = 10 μm .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM I-ORGANISM I-ORGANISM O O O B-GENEPROD O O O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-SMALL_MOLECULE O O O O O O O O O O

Double-labelling of telencephalic sections of N. furzeri with anti-RPS6 (green) as ribosomal marker and Proteostat as a marker for aggregated proteins (red). Nuclear counterstaining was performed with DAPI (blue). Scale bar = 10 μm.


Abundance changes of proteasome their transcripts during aging . Members of 19S 20S are displayed separately changes are shown for both comparisons as box plots . Transcripts are displayed as blue as blue boxes . Changes of are displayed as dots , orange dots represent significant cases ( adj . p < 0 . 05 , n = 4 per group for proteome n = 5 per group for transcriptome ) . In boxplots , horizontal represents median , bottom top of box 25th 75th percentile , respectively , whiskers extend 1 . 5 folds interquartile range .

O O O B-SUBCELLULAR O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Abundance changes of proteasome proteins and their transcripts during aging. Members of the 19S and 20S complex are displayed separately and changes are shown for both the age comparisons as box plots. Transcripts are displayed as light blue and proteins as dark blue boxes. Changes of individual proteins are displayed as dots, orange dots represent significant cases (adj. p < 0.05, n=4 per age group for proteome and n=5 per age group for transcriptome). In boxplots, the horizontal line represents the median, the bottom and top of the box the 25th and 75th percentile, respectively, and the whiskers extend 1.5 folds the interquartile range.


Heat map showing transcript fold changes for members of 26 proteasome ( 19S 12S complexes ) . Genes are annotated according to significance of their changes at level of transcript ( adj . p < 0 . 05 : black , adj . p > 0 . 05 : ) or ( adj . p < 0 . 05 : green , adj . p > 0 . 05 : ) .

O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Heat map showing transcript and protein fold changes for members of the 26 proteasome (19S and 12S complexes). Genes are annotated according to significance of their changes at the level of transcript (adj. p < 0.05: black, adj. p > 0.05: white) or protein (adj. p < 0.05: green, adj. p > 0.05: white).


In - gel proteasome following native gel electrophoresis ( top ) immunoblotting of proteasome complexes ( 30S , 26S 20S ) ( bottom ) in young ( 5wph ) , adult ( 12 wph ) old ( 39 wph ) killifish brains . For additional samples low exposure pictures see Figure EV5B EV5C .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O B-EXP_ASSAY O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O B-ORGANISM B-TISSUE O O O O O O O O O O O O

In-gel proteasome assay following native gel electrophoresis (top) and immunoblotting of proteasome complexes (30S, 26S and 20S) (bottom) in young (5wph), adult (12 wph) and old (39 wph) killifish brains. For additional samples and low exposure pictures see Figure EV5B and EV5C.


Bar plots depicting of chymotrypsin - like ( CT - L ) activity from native gels calculated for doubly capped ( 30S ) or singly capped ( 26S ) proteasomes . n > = 5 per sample group ; error bars indicate standard error of mean . * p < 0 . 05 , * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison . For each sample group , mean value of activity in young samples ( 5 wph ) was set to 100 % .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O

Bar plots depicting the quantification of chymotrypsin-like (CT-L) activity from native gels calculated for doubly capped (30S) or singly capped (26S) proteasomes. n >=5 per sample group; error bars indicate standard error of the mean. * p < 0.05, ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of activity in young samples (5 wph) was set to 100%.


( % ) of chymotrypsin - like ( CT - L ) , trypsin - like ( T - L ) peptidylglutamyl peptide hydrolysing or caspase - like ( PGPH ) proteasome activities in brain extracts of killifish of different ages . n > = 6 per sample group ; error bars indicate standard error of mean . * * p < 0 . 005 , * * * p = 0 . 0001 , * * * * p < 0 . 001 , one - way ANOVA , Holm - Sidak ' s multiple comparison . For each sample group , mean value of each activity in young samples ( 5 wph ) was set to 100 % .

O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-EXP_ASSAY O B-SUBCELLULAR B-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-EXP_ASSAY O O O O O O O O O O O O O

Percentage (%) of chymotrypsin-like (CT-L), trypsin-like (T-L) and peptidylglutamyl peptide hydrolysing or caspase-like (PGPH) proteasome activities in brain extracts of killifish of different ages. n >=6 per sample group; error bars indicate standard error of the mean. ** p < 0.005, *** p=0.0001, **** p < 0.001, one-way ANOVA, Holm-Sidak's multiple comparison test. For each sample group, the mean value of each activity in young samples (5 wph) was set to 100%.


- related changes of involved in ubiquitin cycle . displayed showed significant level changes in 39 wph vs . 12 wph comparison ( adj . p < 0 . 05 ) .

O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O

Age-related changes of proteins involved in the ubiquitin cycle. All the displayed proteins showed significant protein level changes in the 39 wph vs. 12 wph comparison (adj. p < 0.05).


Chymotrypsin - like ( CT - L ) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors . activity was measured at day 4 after beginning of treatment . n = 8 per sample group ; error bars indicate standard deviation . * * p < 0 . 01 , * * * * p < 0 . 0001 , one - way ANOVA followed by Holm - Sidak ' s multiple comparisons .

B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O B-TISSUE O O B-ORGANISM O O O B-SUBCELLULAR O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Chymotrypsin-like (CT-L) proteasome activity in brain extracts of killifish treated with different proteasome inhibitors. The activity was measured at day 4 after the beginning of treatment. n=8 per sample group; error bars indicate standard deviation. ** p < 0.01, **** p < 0.0001, one-way ANOVA followed by Holm-Sidak's multiple comparisons test.


Proteasome inhibition induces proteasome activators ( PSME4 ) , assembly factors ( POMP ) ubiquitin ligases ( UBE3C ) , mediators of autophagosome formation ( ATG2B ) , heat shock chaperones in killifish brain . abundances were quantified by DIA mass spectrometry they are shown relative to mean value of vehicle control samples ( DMSO ) set to 1 , n = 10 per sample group . Adj . p < 0 . 05 for displayed . Error bars indicate mean + / - SD .

B-SMALL_MOLECULE O O B-SMALL_MOLECULE O O B-GENEPROD O O O O O B-GENEPROD O O O O B-GENEPROD O O O O B-SUBCELLULAR O O B-GENEPROD O O O O O O B-ORGANISM B-TISSUE O O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Proteasome inhibition induces proteasome activators (PSME4), assembly factors (POMP) ubiquitin ligases (UBE3C), mediators of autophagosome formation (ATG2B), heat shock proteins and chaperones in killifish brain. Protein abundances were quantified by DIA mass spectrometry and they are shown relative to the mean value of vehicle control samples (DMSO) set to 1, n=10 per sample group. Adj. p < 0.05 for all the displayed proteins. Error bars indicate mean +/-SD.


Bortezomib treatment affects stoichiometry of a subset of complexes . number of affected ( adj . p < 0 . 25 ) for each is indicated . Only complexes that had at least two members affected are shown .

B-SMALL_MOLECULE O O B-EXP_ASSAY O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O B-SUBCELLULAR O O O O O O O O O O

Bortezomib treatment affects the stoichiometry of a subset of protein complexes. The number of affected proteins (adj. p < 0.25) for each protein complex is indicated. Only protein complexes that had at least two members affected are shown.


Members of mitochondrial ribosomes affected by Bortezomib treatment . Relative abundances ( normalized to mean of to which they belong ) are shown . mean value of vehicle control samples ( DMSO ) was set to 1 . n = 10 per sample group . Adj . p < 0 . 25 for displayed . Error bars indicate mean + / - SD .

O O B-SUBCELLULAR I-SUBCELLULAR O O B-SMALL_MOLECULE 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-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Members of the mitochondrial and cytoplasmic ribosomes affected by Bortezomib treatment. Relative protein abundances (normalized to the mean of the protein complex to which they belong) are shown. The mean value of vehicle control samples (DMSO) was set to 1. n=10 per sample group. Adj. p < 0.25 for all the displayed proteins. Error bars indicate mean +/-SD.


Distribution of change in expression for proteasome transcripts across entire cohort of 159 fish . Lifespan was compared among fish that showed extreme changes in proteasome between 10 20 wph ( 32 fish showing most pronounced decreases , shown in black vs . 32 fish showing most pronounce increases ) . * * * p < 0 . 001 Log - rank .

O O O O B-EXP_ASSAY O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O B-ORGANISM O O O O O B-SUBCELLULAR O O O O O O B-ORGANISM O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O

Distribution of change in expression for proteasome transcripts across the entire cohort of 159 fish. Lifespan was compared among fish that showed extreme changes in proteasome levels between 10 and 20 wph (32 fish showing the most pronounced decreases, shown in black vs. 32 fish showing the most pronounce increases). *** p<0.001 Log-rank test.


HeLa co - transfected with EMTB - CFP - FRB YFP - FKBP were treated with 100 nM rapamycin ( Rapa ) . Addition of rapamycin rapidly translocated YFP - FKBP onto EMTB - CFP - FRB - labeled microtubules increased FRET signal . scale for FRET / CFP is shown . Scale bar , 10 μm . normalized of FRET / CFP in before after rapamycin ( blue ) 0 . 1 % DMSO ( control ; red ) treatment . n = 6 10 in rapamycin DMSO groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O B-SMALL_MOLECULE O B-EXP_ASSAY B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-EXP_ASSAY O O O O B-EXP_ASSAY O B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O

HeLa cells co-transfected with EMTB-CFP-FRB and YFP-FKBP were treated with 100 nM rapamycin (Rapa). Addition of rapamycin rapidly translocated YFP-FKBP onto EMTB-CFP-FRB-labeled microtubules and increased the FRET signal. The scale for the FRET/CFP intensity ratio is shown. Scale bar, 10 μm. The normalized intensity of FRET/CFP in cells before and after rapamycin (blue) and 0.1% DMSO (control; red) treatment. n = 6 and 10 cells in the rapamycin and DMSO groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with indicated constructs were treated with nocodazole ( Noc , 3 . 3 µM ; left ) or rapamycin ( Rapa , 100 nM ; right ) for 0 , 30 , 60 min . Transfected were then fixed labeled with anti - acetylated tubulin ( red ) . Dotted lines indicate boundary of transfected . Scale bars , 10 μm . of acetylated tubulin in transfected with EMTB - CFP - FRB ( orange ) or cotransfected with EMTB - CFP - FRB dNSpastin3Q - YFP - FKBP ( blue ) after addition of nocodazole ( Noc , 3 . 3 µM ) or rapamycin ( Rapa , 100 nM ) for indicated times . n = 123 81 in nocodazole dNSpastin3Q groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M . Student ' s t - were performed , with p - values indicated .

B-CELL_LINE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

HeLa cells co-transfected with the indicated constructs were treated with nocodazole (Noc, 3.3 µM; left) or rapamycin (Rapa, 100 nM; right) for 0, 30, and 60 min. Transfected cells were then fixed and labeled with anti-acetylated tubulin (red). Dotted lines indicate the boundary of transfected cells. Scale bars, 10 μm. The intensity of acetylated tubulin in cells transfected with EMTB-CFP-FRB (orange) or cotransfected with EMTB-CFP-FRB and dNSpastin3Q-YFP-FKBP (blue) after addition of nocodazole (Noc, 3.3 µM) or rapamycin (Rapa, 100 nM) for the indicated times. n = 123 and 81 cells in the nocodazole and dNSpastin3Q groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M. Student's t-tests were performed, with p-values indicated.


NIH3T3 fibroblasts co - transfected with 5HT6 - mCherry ( 5HT6 - mCh ; a ciliary marker ; red ) , CFP - FRB - MAP4m ( blue ) , dNSpastin3Q - YFP - FKBP ( green ) were serum - starved for 24 hr to induce ciliogenesis . Ciliated were then treated with rapamycin ( Rapa , 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to axonemal microtubules . Dotted lines indicate boundaries . Scale bar , 5 μm . normalized of axonemes ( blue ) , cilia ( red ) , dNSpastin3Q - YFP - FKBP in cilia ( green ) upon rapamycin treatment . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE B-CELL_TYPE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O B-TISSUE O O O O O O O O O B-CELL_TYPE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

NIH3T3 fibroblasts co-transfected with 5HT6-mCherry (5HT6-mCh; a ciliary membrane marker; red), CFP-FRB-MAP4m (blue), and dNSpastin3Q-YFP-FKBP (green) were serum-starved for 24 hr to induce ciliogenesis. Ciliated cells were then treated with rapamycin (Rapa, 100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to axonemal microtubules. Dotted lines indicate the cell boundaries. Scale bar, 5 μm. The normalized length of axonemes (blue), primary cilia (red), and dNSpastin3Q-YFP-FKBP in cilia (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with H2B - mCherry ( H2B - mCh ; a chromosome marker ; red ) , CFP - FRB - MAP4m ( a marker of mitotic spindles ; blue ) , dNSpastin3Q - YFP - FKBP ( green ) were synchronized in metaphase treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to mitotic spindles . Dotted lines indicate boundaries . Scale bar , 10 μm . normalized of mitotic spindle ( blue ) of dNSpastin3Q - YFP - FKBP in mitotic spindle ( green ) upon rapamycin treatment . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

HeLa cells co-transfected with H2B-mCherry (H2B-mCh; a chromosome marker; red), CFP-FRB-MAP4m (a marker of mitotic spindles; blue), and dNSpastin3Q-YFP-FKBP (green) were synchronized in metaphase and treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to mitotic spindles. Dotted lines indicate the cell boundaries. Scale bar, 10 μm. The normalized area of the mitotic spindle (blue) and intensity of dNSpastin3Q-YFP-FKBP in the mitotic spindle (green) upon rapamycin treatment. n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


HeLa co - transfected with H2B - mCherry ( red ) , CFP - FRB - MAP4m ( intercellular bridges ; blue ) , dNSpastin3Q - YFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) to induce dNSpastin3Q - YFP - FKBP recruitment to intercellular bridges . Arrows indicate intercellular bridges . Dotted lines indicate boundaries . Scale bar , 10 μm . normalized of intercellular bridges ( blue ) of dNSpastin3Q - YFP - FKBP at intercellular bridges ( green ) upon rapamycin treatment . n = 5 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR I-SUBCELLULAR O O O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SUBCELLULAR O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

HeLa cells co-transfected with H2B-mCherry (red), CFP-FRB-MAP4m (intercellular bridges; blue), and dNSpastin3Q-YFP-FKBP (green) were treated with rapamycin (100 nM) to induce dNSpastin3Q-YFP-FKBP recruitment to intercellular bridges. Arrows indicate the intercellular bridges. Dotted lines indicate the cell boundaries. Scale bar, 10 μm.   The normalized area of intercellular bridges (blue) and intensity of dNSpastin3Q-YFP-FKBP at intercellular bridges (green) upon rapamycin treatment. n = 5 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 transfected with TagRFP - FRB - A1AY1 ( red ) were labeled by anti - α - tubulin antibody ( green ; upper panel ) , anti - tyrosinated tubulin antibody ( green ; middle panel ) , or anti - detyrosinated tubulin antibody ( green ; lower panel ) , respectively . Scale bar , 10 µm . profiles of TagRFP - FRB - A1AY1 ( red ) tubulin with indicated PTMs ( green ) along dotted lines drawn in ( A ) . Pearson ' s correlation coefficients for TagRFP - FRB - A1AY1 indicated tubulin ( tub ) subtypes were calculated , data are shown as mean ± S . E . M . n = 16 , 13 , 15 from left to right , respectively ; three independent experiments .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

COS7 cells transfected with TagRFP-FRB-A1AY1 (red) were labeled by anti-α-tubulin antibody (green; upper panel), anti-tyrosinated tubulin antibody (green; middle panel), or anti-detyrosinated tubulin antibody (green; lower panel), respectively. Scale bar, 10 µm. The intensity profiles of TagRFP-FRB-A1AY1 (red) and tubulin with the indicated PTMs (green) along dotted lines drawn in (A). The Pearson's correlation coefficients for TagRFP-FRB-A1AY1 and indicated tubulin (tub) subtypes were calculated, and data are shown as the mean ± S.E.M. n = 16, 13, and 15 cells from left to right, respectively; three independent experiments.


COS7 co - transfected with TagRFP - FRB - A1AY1 ( red ) TagCFP - FKBP ( green ) were treated with rapamycin ( 100 nM ) . addition of rapamycin rapidly recruits TagCFP - FKBP from cytosol onto A1AY1 - labeled microtubules ( arrows ) . Scale bar , 10 µm . normalized of TagCFP - FKBP at A1AY1 - labeled microtubules upon rapamycin treatments . n = 7 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O B-GENEPROD O B-SUBCELLULAR O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-SUBCELLULAR O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and TagCFP-FKBP (green) were treated with rapamycin (100 nM). The addition of rapamycin rapidly recruits TagCFP-FKBP from cytosol onto A1AY1-labeled microtubules (arrows). Scale bar, 10 µm. The normalized intensity of TagCFP-FKBP at A1AY1-labeled microtubules upon rapamycin treatments. n=7 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 co - transfected with TagRFP - FRB - A1AY1 ( red ) dNSpastin3Q - TagCFP - FKBP ( cyan ) were treated with 0 . 1 % DMSO or rapamycin ( 100 nM ) for 1 hr followed by immunostaining with anti - tyrosinated tubulin antibody ( green ) . Dotted lines highlight transfected . Scale bar , 20 μm . normalized of tyrosinated microtubules in TagRFP - FRB - A1AY1 dNSpastin3Q - TagCFP - FKBP co - transfected upon 0 . 1 % DMSO or rapamycin treatment for 1 hr . n = 31 26 in DMSO rapamycin treated groups , respectively , from three independent experiments . Data ( blue ) represent as mean ± S . E . M . Student ' s t - were performed with p values indicated .

B-CELL_LINE O O O O O O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O B-EXP_ASSAY O O O B-EXP_ASSAY B-GENEPROD O O O O O O O O O O O O O O O O O O B-EXP_ASSAY B-SUBCELLULAR O O O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

COS7 cells co-transfected with TagRFP-FRB-A1AY1 (red) and dNSpastin3Q-TagCFP-FKBP (cyan) were treated with 0.1% DMSO or rapamycin (100 nM) for 1 hr and followed by immunostaining with anti-tyrosinated tubulin antibody (green). Dotted lines highlight the transfected cells. Scale bar, 20 μm. The normalized intensity of tyrosinated microtubules in TagRFP-FRB-A1AY1 and dNSpastin3Q-TagCFP-FKBP co-transfected cells upon 0.1% DMSO or rapamycin treatment for 1 hr. n=31 and 26 cells in DMSO and rapamycin treated groups, respectively, from three independent experiments. Data (blue) represent as mean ± S.E.M. Student's t-tests were performed with p values indicated.


COS7 co - transfected with EMTB - YFP - CIBN mCh - Cry2 ( red ) were incubated with SPY650 - tubulin ( SPY650 - tub ) to visualize microtubules . were illuminated by blue within a specified region ( indicated by dotted circle ) for indicated time period . Scale bar , 10 μm . normalized of mCh - Cry2 at microtubules in illuminated regions ( red ) non - illuminated regions ( green ) . n = 6 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-GENEPROD O B-SMALL_MOLECULE O O O O O B-SUBCELLULAR O O O O 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-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize microtubules. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. The normalized intensity of mCh-Cry2 at microtubules in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. Data are shown as the mean ± S.E.M.


COS7 co - transfected with EMTB - YFP - CIBN dNSpastin3Q - mCh - Cry2 were incubated with SPY650 - tubulin to visualize microtubules . were illuminated by blue as in ( A ) . Scale bar , 10 μm . normalized of dNSpastin3Q - mCh - Cry2 mCh - Cry2 at microtubules normalized of SPY650 - tubulin in illuminated regions non - illuminated regions . n = 6 6 in dNSpastin3Q - mCh - Cry2 mCh - Cry2 groups , respectively , from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O B-GENEPROD O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O B-SUBCELLULAR O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O

COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastin3Q-mCh-Cry2 were incubated with SPY650-tubulin to visualize microtubules. The cells were illuminated by blue light as in (A). Scale bar, 10 μm. The normalized intensity of dNSpastin3Q-mCh-Cry2 and mCh-Cry2 at microtubules and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastin3Q-mCh-Cry2 and mCh-Cry2 groups, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.


E - F . COS7 co - transfected with EMTB - CFP - FRB , dNSpastin3Q - mCh - FKBP , Tom20 - Neon were treated with rapamycin ( 100 nM ) for acute microtubule disruption . of mitochondria at different of microtubule disruption is shown . Scale bar , 10 µm . of long axis to short axis of each mitochondrion microtubule filament was determined . n = 4 different , three independent experiments . Data are shown as mean ± S . E . M . Data information : Student ' s t - were performed with p values indicated .

O O O O B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O O O O O O O O O O O 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-F. COS7 cells co-transfected with EMTB-CFP-FRB, dNSpastin3Q-mCh-FKBP, and Tom20-Neon were treated with rapamycin (100 nM) for acute microtubule disruption. The morphology of mitochondria at different levels of microtubule disruption is shown. Scale bar, 10 µm. The ratio of the long axis to the short axis of each mitochondrion and microtubule filament area was determined. n= 4 different cells, three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


G - H . normalized of MitoTracker Red in non - transfected ( Mock ) or co - transfected with EMTB - CFRP - FRB dNSpastin3Q - YFP - FKBP or dNSpastin3QED - YFP - FKBP after rapamycin ( Rapa , 100 nM ) treatment for indicated time . Scale bar , 10 µm . n = 41 , 6 , 18 from three independent experiments . Data are shown as mean ± S . E . M . Data information : Student ' s t - were performed with p values indicated .

O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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. The normalized intensity of MitoTracker Red in non-transfected cells (Mock) or cells co-transfected with EMTB-CFRP-FRB and dNSpastin3Q-YFP-FKBP or dNSpastin3QED-YFP-FKBP after rapamycin (Rapa, 100 nM) treatment for the indicated time. Scale bar, 10 µm. n = 41, 6, and 18 cells from three independent experiments. Data are shown as the mean ± S.E.M. Data information: Student's t-tests were performed with p values indicated.


COS7 co - transfected with EMTB - CFP - FRB ( blue ) , dNSpastin3Q - mCh - FKBP ( red ) , Lifeact - Neon ( green ) were treated with rapamycin ( Rapa , 100 nM ) to induce microtubule disassembly . Arrows arrowheads indicate lamellipodia stress , respectively . Insets show of lamellipodia from regions corresponding to dotted squares with overexposed CFP mCherry signals . Scale bar , 10 µm . normalized level of microtubule , lamellipodia , filopodia density of COS - 7 treated as in ( A ) . n = 4 from three independent experiments . Data are shown as mean ± S . E . M .

B-CELL_LINE O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SUBCELLULAR O O O O O B-SUBCELLULAR I-SUBCELLULAR O O O O O O B-SUBCELLULAR O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O B-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O O O O O O O O O O O O O O O O O O O O O O

COS7 cells co-transfected with EMTB-CFP-FRB (blue), dNSpastin3Q-mCh-FKBP (red), and Lifeact-Neon (heatmap and green) were treated with rapamycin (Rapa, 100 nM) to induce microtubule disassembly. Arrows and arrowheads indicate lamellipodia and stress fibers, respectively. Insets show images of lamellipodia morphology from regions corresponding to the dotted squares with overexposed CFP and mCherry signals. Scale bar, 10 µm. The normalized level of microtubule area, lamellipodia length, and filopodia density of COS-7 cells treated as in (A). n = 4 cells from three independent experiments. Data are shown as the mean ± S.E.M.


B . Carfilzomib , Bortezomib MLN9708 activate luciferase activity in M2 macrophages derived from Il - 1β - luciferase transgenic mice . BMDMs from IL - 1β - luciferase transgenic mice were treated by IL - 4 ( 20 ng / mL ) for 24 hours to differentiate into mature M2 macrophages , then used for screening of FDA compounds ( 5 μM ) . X axis stands for code of drug .

O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-EXP_ASSAY I-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-ORGANISM O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O B-ORGANISM O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-CELL_TYPE I-CELL_TYPE O O O O O O O O O O O O O O O O O O O O O

B. Carfilzomib, Bortezomib and MLN9708 activate luciferase activity in M2 macrophages derived from Il-1β-luciferase transgenic mice. BMDMs from IL-1β-luciferase transgenic mice were treated by IL-4 (20 ng/mL) for 24 hours to differentiate into mature M2 macrophages, and then used for screening of FDA compounds (5 μM). The X axis stands for the code of the drug.


F , G . Carfilzomib , Bortezomib MLN9708 promote mRNA expression of Il - 1β in M2 macrophages . BMDMs ( F ) or Raw264 . 7 ( G ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then treated by DMSO , Carfilzomib ( 1 μM ) , Bortezomib ( 1 μM ) , or MLN9708 ( 2 μM ) . was extracted from BMDMs or Raw264 . 7 6 hours after stimulation expression of Il - 1β were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * * p < 0 . 001 ( student ' s t - ) . H , I . Carfilzomib , Bortezomib MLN9708 promote secretion of inflammatory cytokine IL - 1β in M2 macrophages . BMDMs ( H ) or Raw264 . 7 ( I ) were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO , Carfilzomib ( 500 nM ) , Bortezomib ( 500 nM ) or MLN9708 ( 500 nM ) . Secretion of IL - 1β were measured through ELISA 24 hours later . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * p < 0 . 01 , * * * p < 0 . 001 ( student ' s t - ) .

O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O O O O O O O B-CELL_TYPE O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O B-EXP_ASSAY O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O 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-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-CELL_TYPE I-CELL_TYPE O B-CELL_TYPE O O O O B-CELL_LINE I-CELL_LINE I-CELL_LINE O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F, G. Carfilzomib, Bortezomib and MLN9708 promote the mRNA expression of Il-1β in M2 macrophages. BMDMs (F) or Raw264.7 cells (G) were pretreated by IL-4 (20 ng/mL) for 24 hours, then treated by DMSO, Carfilzomib (1 μM), Bortezomib (1 μM), or MLN9708 (2 μM). RNA was extracted from BMDMs or Raw264.7 cells 6 hours after stimulation and the expression of Il-1β were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test). H, I. Carfilzomib, Bortezomib and MLN9708 promote the secretion of inflammatory cytokine IL-1β in M2 macrophages. BMDMs (H) or Raw264.7 cells (I) were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO, Carfilzomib (500 nM), Bortezomib (500 nM) or MLN9708 (500 nM). Secretion of IL-1β were measured through ELISA 24 hours later. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. **p < 0.01, ***p < 0.001 (student's t-test).


A . most effective one among candidate compounds , Carfilzomib , significantly promotes expression of M1 macrophage markers , as well as reduces expression of M2 macrophage markers . BMDMs were pretreated by IL - 4 ( 20 ng / mL ) for 24 hours , then stimulated by DMSO or Carfilzomib ( 1 μM ) . was extracted from 6 hours after stimulation expression of M1 ( Il - 6 / Inos ) or M2 ( Cd206 / Arg1 ) macrophage markers were quantified through RT - qPCR . Data from three experiments are presented as mean ± SD . T was used for statistical of differences between groups . * * * p < 0 . 001 ( student ' s t - ) .

O O O O O O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O O O O O B-EXP_ASSAY O B-CELL_TYPE I-CELL_TYPE O O B-CELL_TYPE O O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O B-EXP_ASSAY O B-CELL_TYPE O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O B-CELL_TYPE O B-GENEPROD O B-GENEPROD O B-CELL_TYPE O O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

A. The most effective one among the candidate compounds, Carfilzomib, significantly promotes the expression of M1 macrophage markers, as well as reduces the expression of M2 macrophage markers. BMDMs were pretreated by IL-4 (20 ng/mL) for 24 hours, then stimulated by DMSO or Carfilzomib (1 μM). RNA was extracted from cells 6 hours after stimulation and the expression of M1 (Il-6/Inos) or M2 (Cd206/Arg1) macrophage markers were quantified through RT-qPCR. Data from three experiments are presented as the mean ± SD. T test was used for statistical analysis of differences between groups. ***p < 0.001 (student's t-test).


D . Statistics for proportion of CD86 , CD80 , MHC - II or CD206 positive in BMDMs . Data are means ± SD of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( student ' s t - ) .

O O O O O O B-GENEPROD O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR I-SUBCELLULAR O B-GENEPROD O O B-CELL_TYPE O O O O O O O O O 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. Statistics for proportion of CD86, CD80, MHC-II or CD206 positive cells in BMDMs. Data are means ± SD of three independent experiments. *p < 0.05, **p < 0.01 (student's t-test).