Subcutaneous adipose tissue ( sWAT ) ( G ) epididymal adipose tissue ( eWAT ) ( H ) mass development at 10wks , 20wks , 45wks 95wks of . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 10wks ( WT n = 6 , TG n = 7 , TGxKO n = 5 ) , 20wks ( WT n = 10 , TG n = 10 , TGxKO n = 10 ) , 45wks ( WT n = 5 , TG n = 6 , TGxKO n = 7 ) 95wks ( WT n = 8 , TG n = 5 , TGxKO n = 5 ) .

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Subcutaneous white adipose tissue (sWAT) (G) and epididymal white adipose tissue (eWAT) (H) mass development at 10wks, 20wks, 45wks and 95wks of age. Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 10wks (WT n=6, TG n=7, TGxKO n=5), 20wks (WT n=10, TG n=10, TGxKO n=10), 45wks (WT n=5, TG n=6, TGxKO n=7) and 95wks (WT n=8, TG n=5, TGxKO n=5).


Representative H & E histological of sWAT at 20wks of ( scale bars represent 50 μm ) Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Representative H&E histological staining of sWAT at 20wks of age (scale bars represent 50 μm) Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


relative mRNA expression profile in sWAT of mice ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) ( B ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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relative mRNA expression profile in sWAT of male mice (WT n=7, TG n=8, TGxKO n=8) (B). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Representative immunoblots ( C ) ( D ) of UCP1 expression in sWAT of mice at 20wks of ( WT n = 4 , TG n = 6 , TGxKO n = 4 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Representative immunoblots (C) and quantification (D) of UCP1 protein expression in sWAT of male mice at 20wks of age (WT n=4, TG n=6, TGxKO n=4). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Relative mRNA expression profile in eWAT ( WT n = 7 , TG n = 8 , TGxKO n = 8 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Relative mRNA expression profile in eWAT (WT n=7, TG n=8, TGxKO n=8). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Plasma FGF21 from mice at 20wks of ( n = 10 per genotype ) . Plasma Leptin from mice at 20wks of ( n = 5 per genotype ) . Post - absorptive plasma insulin at 20wks of ( WT n = 8 , TG n = 9 , TG n = 9 ) . : Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Plasma FGF21 levels from male mice at 20wks of age (n=10 per genotype). Plasma Leptin levels from male mice at 20wks of age (n=5 per genotype). Post-absorptive plasma insulin levels at 20wks of age (WT n=8, TG n=9, TG n=9). : Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Blood glucose ( I ) insulin ( J ) with total under curve ( AUC ) of insulin ( K ) during oral glucose tolerance ( oGTT ) at 17wks of ( WT n = 8 , TG n = 11 , TG n = 11 ) . Data are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice .

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Blood glucose (I) and insulin levels (J) with total area under the curve (AUC) of insulin (K) during oral glucose tolerance test (oGTT) at 17wks of age (WT n=8, TG n=11, TG n=11). Data are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice.


Physical activity of mice shown hourly over 24 - hrs as day night time . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

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Physical activity of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


energy expenditure of mice shown hourly over 24 - hrs as day night time . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

energy expenditure of male mice shown hourly over 24-hrs and as day and night time. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


, total assimilated energy of mice shown hourly over 24 - hrs as day night time . Table showing mean values of assimilated energy ( kJ ) at day / night time per 24 - hrs . Energy balance calculated as delta of assimilated energy ( AE ) energy expenditure ( EE ) energy balance ( J ) per 24 - hrs . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

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, total assimilated energy of male mice shown hourly over 24-hrs and as day and night time. Table showing mean values of assimilated energy (kJ) at day/night time and per 24-hrs. Energy balance calculated as delta of assimilated energy (AE) and energy expenditure (EE) and energy balance (J) per 24-hrs. Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


Respiratory quotient ( RQ ) shown hourly over 24 - hrs ( J ) , RQ amplitude ( K ) metabolic flexibility via relative cumulative frequency ( PRCF ) ( L ) . Data shown are from wild type ( WT ) , Ucp1 - TG ( TG ) , Ucp1 - TGxGdf15 - KO ( TGxKO ) mice at 17 - 18wks of ( WT n = 12 , TG n = 11 , TG n = 10 ) shown as means +

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Respiratory quotient (RQ) shown hourly over 24-hrs (J), RQ amplitude (K) and metabolic flexibility via percentage relative cumulative frequency (PRCF) (L). Data shown are from male wild type (WT), Ucp1-TG (TG), and Ucp1-TGxGdf15-KO (TGxKO) mice at 17-18wks of age (WT n=12, TG n=11, TG n=10) and shown as means + SEM


Relative quadriceps ( Quad ) mRNA expression of ISR components Atf4 , Atf5 , Atf6 Chop ( WT day n = 9 , WT night n = 5 , TG day n = 20 , TG night n = 12 ) . Data shown are from wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) .

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Relative quadriceps (Quad) mRNA expression of the ISR components Atf4, Atf5, Atf6 and Chop (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).


Representative immunoblots of ISR component eIF2α ( B ) of phospho - eIF2α ( p - eIF2aSer51 ) relative expression ( C ) in skeletal muscle ( Gastroc , WT day n = 4 , WT night n = 5 , TG day n = 9 , TG night n = 9 ) . Data shown are from wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) .

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Representative immunoblots of ISR component eIF2α (B) and quantification of phospho-eIF2α (p-eIF2aSer51) relative protein expression (C) in skeletal muscle (Gastroc, WT day n=4, WT night n=5, TG day n=9, TG night n=9). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).


Relative mRNA expression of Rev - erba ( D ) Gdf15 ( E ) in quadriceps , liver eWAT ( WT day n = 9 , WT night n = 5 , TG day n = 20 , TG night n = 12 ) . Data shown are from wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) .

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Relative mRNA expression of Rev-erba (D) and Gdf15 (E) in quadriceps, liver and eWAT (WT day n=9, WT night n=5, TG day n=20, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).


Skeletal muscle ( Quad ) GDF15 content normalized to total content ( WT day n = 4 , WT night n = 5 , TG day n = 14 , TG night n = 12 ) Circulating GDF15 plasma ( WT day n = 9 , WT night n = 5 , TG day n = 18 , TG night n = 12 ) . Data shown are from wild type ( WT ) vs . Ucp1 - TG ( TG ) mice sacrificed at day ( 10am ) or night ( 10pm ) .

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Skeletal muscle (Quad) GDF15 protein content normalized to total protein content (WT day n=4, WT night n=5, TG day n=14, TG night n=12) Circulating GDF15 plasma levels (WT day n=9, WT night n=5, TG day n=18, TG night n=12). Data shown are from male wild type (WT) vs. Ucp1-TG (TG) mice sacrificed at day (10am) or night (10pm).


( G ) Fold change in total modification upon loss of m5C . Fold changes were calculated by dividing peak of noNSUN samples by one of wild type samples . n = 3 independent biological replicates . Multiple t - . Data information : In G ) , data are presented as mean ± .

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(G) Fold change in total RNA modification levels upon loss of m5C. Fold changes were calculated by dividing the peak area ratio of noNSUN samples by the one of wild type samples. n = 3 independent biological replicates. Multiple t-tests. Data information: In G), data are presented as mean ± SEM.


( A , B ) Site - specific methylation by whole - transcriptome bisulfite sequencing . Scatter plots show cytosines their respective non - conversion rates in rRNAs ( A ) tRNAs ( B ) of wild type noNSUN strains ; pie chart showing most frequently methylated tRNA isoacceptors . Data information : In ( A , B n = 3 independent biological replicates .

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(A, B) Site-specific methylation analysis by whole-transcriptome bisulfite sequencing. Scatter plots show individual cytosines and their respective non-conversion rates in rRNAs (A) and tRNAs (B) of wild type and noNSUN strains; pie chart showing most frequently methylated tRNA isoacceptors. Data information: In (A, B n = 3 independent biological replicates.


( A ) bisulfite sequencing map for mitochondrial tRNA Met - CAU in wild type ( top ) noNSUN ( bottom ) strains . Each row represents one read each column one cytosine . Data information : In ( A ) , a representative map of replicates is shown , n = 3 independent biological replicates . Similar effects were observed in replicates analysed .

(A) RNA bisulfite sequencing map for mitochondrial tRNA Met-CAU in wild type (top) and noNSUN (bottom) strains. Each row represents one sequence read and each column one cytosine. Data information: In (A), a representative map of the replicates is shown, n = 3 independent biological replicates. Similar effects were observed in all replicates analysed.


D ) Viable progeny of wild - type noNSUN strains at 20 ° C 25 ° C ( D ) . Automatic counting was done using a Matlab script which processed plate in real - time . Data information : In ( D ) , data are presented as mean ± , unpaired two - Student ' s t - .

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D) Viable progeny counts of wild-type and noNSUN strains at 20°C and 25°C (D). Automatic counting was done using a Matlab script which processed plate images in real-time. Data information: In (D), data are presented as mean ± SEM, unpaired two-tailed Student's t-test.


( D ) Translation efficiency of UUG - enriched , leucine - enriched , proline - enriched random genes in each sample . A gene was considered enriched in a certain codon when proportion of this codon in gene was at least 3 - fold higher than proportion of same codon across transcriptome . Data information : In n = 3 biological replicates . In ( D ) , boxplots show median ( central band ) IQR ( boxes ) ± 1 . 5 x IQR ( whiskers ) , Welch ' s t - , p - value < 0 . 05 .

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(D) Translation efficiency of UUG-enriched, leucine-enriched, proline-enriched and random genes in each sample. A gene was considered enriched in a certain codon when the proportion of this codon in the gene was at least 3-fold higher than the proportion of the same codon across the transcriptome. Data information: In n = 3 biological replicates. In (D), boxplots show the median (central band) and IQR (boxes) ± 1.5 x IQR (whiskers), Welch's t-test, p-value < 0.05.


( A ) HeLa were fixed stained for p62 . different patterns of p62 distribution were scored in > 200 as with small bodies , with large intense bodies , equal , enriched . of in respective groups is indicated .

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(A) HeLa cells were fixed and stained for p62. The different patterns of p62 distribution were scored in >200 cells as cytoplasmic with small bodies, cytoplasmic with large and intense bodies, equal nuclear and cytoplasmic staining, and nuclear enriched. The percentage of cells in the respective groups is indicated.


( B ) Expression level of GFP - p62 in HeLa stably expressing GFP - p62 ( S - GFP - p62 ) compared with level of endogenous p62 in parent HeLa .

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(B) Expression level of GFP-p62 in HeLa cells stably expressing GFP-p62 (S-GFP-p62) compared with the level of endogenous p62 in the parent HeLa cells.


( C ) stably expressing GFP - p62 display a punctuate localization of GFP - tagged p62 similar to endogenous p62 . Video microscopy of live demonstrated that small , faint bodies ( thin arrows ) displayed directed migration , whereas majority of larger , intense bodies ( thick arrows ) were nonmigratory ( Videos 1 2 , available at http : / / www . jcb . org / cgi / content / full / jcb . 200507002 / DC1 ) . A still image from Video 1 is shown here .

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(C) Cells stably expressing GFP-p62 display a cytoplasmic punctuate localization of GFP-tagged p62 similar to endogenous p62. Video microscopy of live cells demonstrated that the small, faint bodies (thin arrows) displayed directed migration, whereas the majority of the larger, intense bodies (thick arrows) were nonmigratory (Videos 1 and 2, available at http://www.jcb.org/cgi/content/full/jcb.200507002/DC1). A still image from Video 1 is shown here.


( D ) Bodies containing either endogenous p62 or transiently or stably transfected GFP - p62 contain polyubiquitin . HeLa were fixed stained with p62 polyubiquitin ( clone FK1 ) mAbs directly coupled with AlexaFluor555 ( red ) AlexaFluor488 ( green ) , respectively . expressing GFP - p62 were only stained for polyubiquitin ( red ) . Bars , 20 μm .

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(D) Bodies containing either endogenous p62 or transiently or stably transfected GFP-p62 all contain polyubiquitin. HeLa cells were fixed and stained with p62 and polyubiquitin (clone FK1) mAbs directly coupled with AlexaFluor555 (red) and AlexaFluor488 (green), respectively. Cells expressing GFP-p62 were only stained for polyubiquitin (red). Bars, 20 μm.


( A ) indicated deletion constructs were fused COOH terminal to GFP expressed in NIH3T3 fibroblasts . Asterisks indicate point mutations in PB1 ( D69A ) UBA ( I431A ) domains , respectively .

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(A) The indicated deletion constructs were fused COOH terminal to GFP and expressed in NIH3T3 fibroblasts. Asterisks indicate point mutations in the PB1 (D69A) and UBA (I431A) domains, respectively.


( B ) S - GFP - p62 were transiently transfected with indicated myc - tagged p62 constructs , fixed , stained for myc tag . Bars , 20 μm .

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(B) S-GFP-p62 cells were transiently transfected with the indicated myc-tagged p62 constructs, fixed, and stained for the myc tag. Bars, 20 μm.


( A ) Localization of bodies containing endogenous p62 or transiently expressed GFP - p62 relative to EEA1 - positive early endosomes . Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488 , EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555 . Alternatively , transiently expressed GFP - p62 was expressed in HeLa , EEA1 was stained red with EEA1 mAb ( bottom ) . boxed is shown to right at a higher magnification .

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(A) Localization of bodies containing endogenous p62 or transiently expressed GFP-p62 relative to EEA1-positive early endosomes. Endogenous p62 were stained green using p62 antibodies directly labeled with AlexaFluor488, and EEA1 was stained red with EEA1 mAbs directly labeled with AlexaFluor555. Alternatively, transiently expressed GFP-p62 was expressed in HeLa cells, and EEA1 was stained red with EEA1 mAb (bottom). The boxed area is shown to the right at a higher magnification.


( B ) Colocalization of GFP - p62 CD63 ( stained red using a mAb ) in HeLa stably expressing GFP - p62 .

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(B) Colocalization of GFP-p62 and CD63 (stained red using a mAb) in HeLa cells stably expressing GFP-p62.


( C ) Immunoelectron micrograph of S - GFP - p62 stained with a GFP pAb ( 10 - nm gold particle , arrows ) monoclonal CD63 ( 15 - nm gold particle , arrowheads ) .

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(C) Immunoelectron micrograph of S-GFP-p62 stained with a GFP pAb (10-nm gold particle, arrows) and monoclonal CD63 (15-nm gold particle, arrowheads).


( D ) Rapid detergent extraction of GFP - p62 from LysoTracker - positive acidic organelles . HeLa transiently expressing GFP - p62 were labeled with LysoTracker for 60 min . detergent extractions were imaged in a time series with 15 - s time intervals after adding 1 % Triton X - 100 . boxed is shown in bottom two at a higher magnification before after detergent extraction . Bars ( A , B , D ) , 20 μm .

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(D) Rapid detergent extraction of GFP-p62 from LysoTracker-positive acidic organelles. HeLa cells transiently expressing GFP-p62 were labeled with LysoTracker for 60 min. The detergent extractions were imaged in a time series with 15-s time intervals after adding 1% Triton X-100. The boxed area is shown in the bottom two images at a higher magnification before and after detergent extraction. Bars (A, B, and D), 20 μm.


( A ) Immuno - EM of GFP - p62 . S - GFP - p62 were labeled with rabbit anti - GFP ( Abcam ) followed by A - gold ( 15 nm ) . We observed labeling in - free structures sequestosomes ( arrows ) as well as in endosomes ( Endo ) .

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(A) Immuno-EM of GFP-p62. S-GFP-p62 cells were labeled with rabbit anti-GFP (Abcam) followed by protein A-gold (15 nm). We observed labeling in membrane-free cytosolic structures and sequestosomes (arrows) as well as in endosomes (Endo).


( B ) Correlative immunofluorescence / EM of HeLa treated with 10 μM PSI for 5 h displaying typical sequestosomes . insets show two magnifications of sequestosome , which is labeled 1 .

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(B) Correlative immunofluorescence/EM of HeLa cells treated with 10 μM PSI for 5 h displaying typical sequestosomes. The insets show two magnifications of the sequestosome, which is labeled 1.


( C ) Representative image of a p62 - containing autophagosome . HeLa transfected with GFP - p62 were immunogold labeled as in A . Note cisternal - like ( arrowheads ) surrounding GFP - positive material . arrow indicates a fused endosome .

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(C) Representative image of a p62-containing autophagosome. HeLa cells transfected with GFP-p62 were immunogold labeled as in A. Note the cisternal-like membrane (arrowheads) surrounding the GFP-positive material. The arrow indicates a fused endosome.


( A ) Comparison of p62 degradation using - chase labeling with 35S - methionine immunoblotting after cycloheximide ( CHX ) treatment . HeLa were pulsed with 35S - methionine incubated for indicated times in nonradioactive medium . After immunopurification , amount of radioactive p62 was determined by autoradiography , total amount of p62 was determined by an immunoblot of same . p62 level in total lysates after different times of 10 μg / ml cycloheximide treatment determined by immunoblotting ( bottom ) .

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(A) Comparison of p62 degradation using pulse-chase labeling with 35S-methionine and immunoblotting after cycloheximide (CHX) treatment. HeLa cells were pulsed with 35S-methionine and incubated for the indicated times in nonradioactive medium. After immunopurification, the amount of radioactive p62 was determined by autoradiography, and the total amount of p62 was determined by an immunoblot of the same membrane. The p62 level in total cellular lysates after different times of 10 μg/ml cycloheximide treatment determined by immunoblotting (bottom).


( B ) of p62 LC3 - II change with autophagic activity . HeLa or S - GFP - p62 were either left untreated or rapamycin ( 10 μg / ml ) or bafilomycin A1 ( Baf . A1 ; 10 μg / ml ) was added for 18 h . Immunoblots were sequentially probed using LC3 , p62 , actin antibodies .

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(B) The levels of p62 and LC3-II change with autophagic activity. HeLa or S-GFP-p62 cells were either left untreated or rapamycin (10 μg/ml) or bafilomycin A1 (Baf. A1; 10 μg/ml) was added for 18 h. Immunoblots were sequentially probed using LC3, p62, and actin antibodies.


( C D ) amount of p62 located to bodies increases upon inhibition of autophagy . HeLa or S - GFP - p62 HeLa were left untreated or bafilomycin A1 was added for 18 h , were fixed , p62 was either stained red using a p62 mAb ( C ) or imaged directly ( D ) . Nuclei were visualized using Draq5 stain . settings for imaging were identical for treated untreated control .

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(C and D) The amount of p62 located to cytoplasmic bodies increases upon inhibition of autophagy. HeLa cells or S-GFP-p62 HeLa cells were left untreated or bafilomycin A1 was added for 18 h, the cells were fixed, and p62 was either stained red using a p62 mAb (C) or imaged directly (D). Nuclei were visualized using the Draq5 DNA stain. The settings for imaging were identical for the treated cells and the untreated control.


( E ) majority of S - GFP - p62 bodies are stained with antibodies recognizing transiently expressed LC3 . S - GFP - p62 were transiently transfected with myc - LC3 . Myc - LC3 was stained red using an anti - myc tag mAb . boxed indicates part of that is shown to left at a higher magnification . Bars , 5 μm .

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(E) The majority of S-GFP-p62 cytoplasmic bodies are stained with antibodies recognizing transiently expressed LC3. S-GFP-p62 cells were transiently transfected with myc-LC3. Myc-LC3 was stained red using an anti-myc tag mAb. The boxed area indicates the part of the cell that is shown to the left at a higher magnification. Bars, 5 μm.


( A ) HeLa transiently transfected with GFP - LC3 alone or cotransfected with siRNA against p62 , HA - p62 , or HA - p62 D69A were either left in normal medium or starved for for 1 h . were fixed , p62 was stained using a p62 mAb . More than 200 randomly selected for each condition were scored for pattern of GFP - LC3 as either diffuse or punctuate . frequency of GFP - LC3 - positive with punctuate localization are indicated to right .

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(A) HeLa cells transiently transfected with GFP-LC3 alone or cotransfected with siRNA against p62, HA-p62, or HA-p62 D69A were either left in normal medium or starved for amino acids for 1 h. The cells were fixed, and p62 was stained using a p62 mAb. More than 200 randomly selected cells for each condition were scored for the cytoplasmic pattern of GFP-LC3 as either diffuse or punctuate. The frequency of GFP-LC3-positive cells with punctuate localization are indicated to the right.


( B ) Endogenous p62 as well as coexpressed myc - tagged wild - type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP - LC3 from HeLa extracts . GFP or GFP - LC3 was immunoprecipitated from total extracts after cotransfecting indicated constructs . cultures were either left untreated or starved for for 1 h as indicated . Copurified endogenous or ectopically expressed myc - tagged p62 constructs were detected using p62 mAb . Bars , 20 μm .

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(B) Endogenous p62 as well as coexpressed myc-tagged wild-type p62 or a UBA deletion mutant of p62 coimmunoprecipitated with GFP-LC3 from HeLa cell extracts. GFP or GFP-LC3 was immunoprecipitated from total cellular extracts after cotransfecting the indicated constructs. The cell cultures were either left untreated or starved for amino acids for 1 h as indicated. Copurified endogenous or ectopically expressed myc-tagged p62 constructs were detected using the p62 mAb. Bars, 20 μm.


( A ) HeLa transiently expressing a Flag - tagged NH2 - terminal fragment ( 1 - 171 ) of huntingtin containing a 68 - polyglutamine expansion , Flag - N - HttQ68 . After 48 h , were fixed , Flag - N - HttQ68 was stained green using a Flag mAb . Endogenous p62 was stained red using p62 pAb .

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(A) HeLa cells transiently expressing a Flag-tagged NH2-terminal fragment (amino acids 1-171) of huntingtin containing a 68-polyglutamine expansion, Flag-N-HttQ68. After 48 h, the cells were fixed, and Flag-N-HttQ68 was stained green using a Flag mAb. Endogenous p62 was stained red using the p62 pAb.


( B ) Endogenous p62 forms a shell around aggregated GFP - N - HttQ68 . Transiently transfected HeLa were fixed 48 h after transfection , endogenous p62 was detected using a p62 mAb ( red ) .

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(B) Endogenous p62 forms a shell around aggregated GFP-N-HttQ68. Transiently transfected HeLa cells were fixed 48 h after transfection, and endogenous p62 was detected using a p62 mAb (red).


( C D ) p62 shell surrounding GFP - N - HttQ68 was detected independently of use of antibodies . ( C ) GFP - N - HttQ68 DsRed - tagged p62 were cotransfected into HeLa , of live were obtained after 48 h of expression . left panel shows one confocal plane of a representative GFP - N - HttQ68 DsRed - p62 double - positive structure , right panel shows Z - stack of planes with side views of aggregate at side at top . N , nucleus . ( A - C ) Nuclei were detected using Draq5 . ( D ) GFP - N - HttQ68 tdTomato - tagged p62 were cotransfected into HeLa , of live were obtained after 24 h of expression .

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(C and D) The p62 shell surrounding GFP-N-HttQ68 was detected independently of the use of antibodies. (C) GFP-N-HttQ68 and DsRed-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 48 h of expression. The left panel shows one confocal plane of a representative GFP-N-HttQ68 and DsRed-p62 double-positive structure, and the right panel shows the Z-stack of planes with side views of the aggregate at the side and at the top. N, nucleus. (A-C) Nuclei were detected using Draq5. (D) GFP-N-HttQ68 and tdTomato-tagged p62 were cotransfected into HeLa cells, and images of live cells were obtained after 24 h of expression.


( E ) Transiently expressed Flag - N - HttQ68 myc - LC3 colocalize in GFP - p62 - positive structures . S - GFP - p62 were fixed 48 h after transfection , Flag - N - HttQ68 was stained red using a Flag tag mAb , myc - LC3 was stained far red ( blue ) using a chicken myc tag pAb ( top ) . bottom panel shows a live image of a GFP - N - HttQ68 aggregate surrounded by a shell containing tdTomato - LC3 . Bars , 5 μm .

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(E) Transiently expressed Flag-N-HttQ68 and myc-LC3 colocalize in GFP-p62-positive structures. S-GFP-p62 cells were fixed 48 h after transfection, Flag-N-HttQ68 was stained red using a Flag tag mAb, and myc-LC3 was stained far red (blue) using a chicken myc tag pAb (top). The bottom panel shows a live cell image of a GFP-N-HttQ68 aggregate surrounded by a shell containing tdTomato-LC3. Bars, 5 μm.


( A ) GFP - positive were scored as live or apoptotic by appearance of Draq5 . After 48 h of expression , GFP - positive attached to surface with a normal appearance ( open arrowheads ) were scored as live , whereas rounded , partially detached with a condensed or fragmented nuclei ( arrows ) were scored as dead . More than 200 GFP - positive in randomly selected microscope views were scored . Bars , 20 μm .

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(A) GFP-positive cells were scored as live or apoptotic by the appearance of nuclear Draq5 staining. After 48 h of expression, GFP-positive cells attached to the surface with a normal nuclear appearance (open arrowheads) and were scored as live, whereas rounded, partially detached cells with a condensed or fragmented nuclei (arrows) were scored as dead. More than 200 GFP-positive cells in randomly selected microscope views were scored. Bars, 20 μm.


( B ) effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking UBA domain on N - HttQ68 - induced in HeLa SHSY - 5Y neuroblastoma .

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(B) The effect of coexpressing either a p62 antisense construct or a deletion construct of p62 lacking the UBA domain on N-HttQ68-induced cell death in HeLa and SHSY-5Y neuroblastoma cells.


G ) Representative of EdU in freshly isolated YC OC FAPs following EdU in vivo exposure for 3 days ( EdU in red / DAPI in blue ) . Scale bar = 25 μm . H ) Graph showing of EdU positive for shown in ( G ) .

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G) Representative images of the EdU staining in freshly isolated YC and OC FAPs following EdU in vivo exposure for 3 days (EdU in red/DAPI in blue). Scale bar = 25 μm. H) Graph showing the percentage of EdU positive cells for the staining shown in (G).


I ) Representative of LMNB1 in freshly isolated YC OC FAPs ( LMNB1 in green / DAPI in blue ) . Scale bar = 25 μm . J ) Graph showing relative of LMNB1 normalized for nuclei number , in shown in ( I ) .

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I) Representative images of the LMNB1 staining in freshly isolated YC and OC FAPs (LMNB1 in green/DAPI in blue). Scale bar = 25 μm. J) Graph showing the relative quantification of LMNB1 fluorescence intensity normalized for nuclei number,in the staining shown in (I).


L ) Representative of γH2AX KI - 67 in freshly isolated YC OC FAPs ( γH2AX in green / KI - 67 in red / DAPI in blue ) . Scale bar = 10 μm . Magnification in insert ( scale bar = 3 μm ) shows γH2AX positive CCF in OC FAPs . M ) Graph showing of positive for γH2AX positive CCF , for shown in ( L ) . N ) Graph showing of positive for KI - 67 for shown in ( L ) .

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L) Representative images of the γH2AX and KI-67 staining in freshly isolated YC and OC FAPs (γH2AX in green/KI-67 in red/DAPI in blue). Scale bar = 10 μm. Magnification in the insert (scale bar = 3 μm) shows γH2AX positive CCF in OC FAPs. M) Graph showing the percentage of cells positive for γH2AX positive CCF, for the staining shown in (L). N) Graph showing the percentage of cells positive for KI-67 for the staining shown in (L).


D ) Representative of phospho - NFkB in FAPs ( pNFkB - p65 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal section of YC , YT , OC OT mice ; Scale bar = 25 μm ; E ) Box plot showing of pNFkB - p65 positive FAPs in shown in ( D )

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D) Representative images of the phospho-NFkB staining in FAPs (pNFkB-p65 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal section of YC, YT, OC and OT mice; Scale bar = 25 μm; E) Box plot showing the percentage of pNFkB-p65 positive FAPs in the staining shown in (D)


F ) Representative of phospho - p38 in FAPs ( pP38 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC OT mice . Scale bar = 25 μm ; G ) Box plot showing of pP38 positive FAPs in shown

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F) Representative images of the phospho-p38 staining in FAPs (pP38 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; G) Box plot showing the percentage of pP38 positive FAPs in the staining shown in


H ) Representative of phospho - SMAD2 / 3 in FAPs ( pSMAD2 / 3 in red / CD90 in green / Laminin in grey / DAPI in blue ) on tibialis anterior muscle transversal sections of YC , YT , OC OT mice . Scale bar = 25 μm ; I ) Box plot showing of pSMAD2 / 3 positive FAPs in shown in ( H ) .

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H) Representative images of the phospho-SMAD2/3 staining in FAPs (pSMAD2/3 in red/CD90 in green/Laminin in grey/DAPI in blue) on tibialis anterior muscle transversal sections of YC, YT, OC and OT mice. Scale bar = 25 μm; I) Box plot showing the percentage of pSMAD2/3 positive FAPs in the staining shown in (H).


Pulldowns of pointed end site 1 - 4 mutants using Strep - Hook3 ( D ) or Strep - BICD2 ( E ) . Binding is shown relative to binding of wildtype pointed end construct ( dashed lines ) . * shows p < 0 . 05 . For BICD2 , p = 0 . 06 for Site 1 mutant , p = 0 . 1 for Site 4 mutant . Data presented as mean ± , n = 3 . Statistical significance calculated using ANOVA with Tukey ' s for multiple comparisons .

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Pulldowns of pointed end site 1-4 mutants using Strep-Hook3 (D) or Strep-BICD2 (E). Binding is shown relative to the binding of the wildtype pointed end construct (dashed lines). * shows p<0.05. For BICD2, p=0.06 for Site 1 mutant, and p=0.1 for Site 4 mutant. Data presented as mean ±SEM, n=3. Statistical significance calculated using ANOVA with Tukey's test for multiple comparisons.


( A ) Representative ( details from boxed regions ) of WT MEF transfected with YFPParkin ( green ) treated with FCCP ( 10 μM ) to induce mitophagy . were immunostained with anti - Tom20 ( magenta ) to reveal mitochondrial network . Each row represents one stage in process of mitophagy that has been used to score progression of mitophagy at different time - points for different conditions tested ( specific time - point of each example is indicated in image ) . From top to bottom : i ) " Diffuse Parkin " defines in where Parkin expression is homogenously distributed throughout cytoplasm ( quantified in B ) . ii ) Soon after mitochondrial damage Parkin appears enriched in small " " throughout cytoplasm localizing within isolated mitochondria ( quantified in C ) . iii ) Later in process these structures aggregate in bigger structures accumulating in perinuclear regions ( quantified in D ) . iv ) process of Parkin translocation eventually affects mitochondrial population usually collapses in few large structures possibly forming part of big autolysosomes in perinuclear region ( quantified in D ) . Scoring was performed from 3 independent experiments ( n = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 * * p < 0 .

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(A) Representative images (and details from boxed regions) of WT MEF cells transfected with YFPParkin (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one stage in the process of mitophagy that has been used to score the progression of mitophagy at different time-points for the different conditions tested (the specific time-point of each example is indicated in the image). From top to bottom: i) "Diffuse Parkin" defines cells in where Parkin expression is homogenously distributed throughout the cytoplasm (quantified in B). ii) Soon after mitochondrial damage Parkin appears enriched in small "puncta" throughout the cytoplasm localizing within isolated mitochondria (quantified in C). iii) Later in the process these puncta structures aggregate in bigger structures accumulating in perinuclear regions (quantified in D). iv) The process of Parkin translocation eventually affects all the mitochondrial population and usually collapses in few large structures possibly forming part of big autolysosomes in the perinuclear region (quantified in D). Scoring was performed from 3 independent experiments (n=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


( F ) Representative ( details from boxed regions ) of WT MEFs transfected with GFPPINK1 ( green ) treated with FCCP ( 10 μM ) to induce mitophagy . were immunostained with anti - Tom20 ( magenta ) to reveal mitochondrial network . Each row represents one of three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage ( , intermediate mitochondrial ) . scoring of these categories was used to describe PINK1 translocation to mitochondria in WT MiroDKO at different time - points shortly after mitochondrial insult ( G ) . Data collected from 3 independent experiments ( n = 3 ) . Error bars represent s . e . m . Significance : * p < 0 . 05 * * p < 0 . 01

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(F) Representative images (and details from boxed regions) of WT MEFs cells transfected with GFPPINK1 (green) and treated with FCCP (10 μM) to induce mitophagy. Cells were immunostained with anti-Tom20 (magenta) to reveal the mitochondrial network. Each row represents one of the three categories of GFP signal used to describe PINK1 translocation to mitochondria after mitochondrial damage (cytoplasmic, intermediate and mitochondrial). The scoring of these categories was used to describe PINK1 translocation to mitochondria in WT and MiroDKO cells at different time-points shortly after mitochondrial insult (G). Data collected from 3 independent experiments (n=3). Error bars represent s.e.m. Significance: *p<0.05 and **p<0.01


B ) Representative showing full control selected Miro1 lysine mutants ( Miro15R , Miro1allR Miro1R572K ) expressing in MiroDKO MEF . constructs ( myc - tag , green ) localise to mitochondria ( Tom20 , red ) .

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B) Representative images showing full length control and selected Miro1 lysine mutants (Miro15R, Miro1allR and Miro1R572K) expressing in MiroDKO MEF cells. All constructs (myc-tag, green) localise to mitochondria (Tom20, red).


C ) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH - SY5Y .

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C) Immunoblot showing efficient expression of Miro1 lysine mutants in FlagParkin expressing SH-SY5Y cells.


D ) Ubiquitination showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment ( 1h , 10 μM ) in FlagParkin overexpressing SH - SY5Y . effect is quantified in ( E ) ( n = 4 independent experiments for Miro1WT Miro15R n = 3 for Miro1allR Miro1R572K , ANOVA with Sidak ' s post hoc ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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D) Ubiquitination assay showing that Miro1 lysine mutants have reduced ubiquitination upon FCCP treatment (1h, 10 μM) in FlagParkin overexpressing SH-SY5Y. The effect is quantified in (E) (n=4 independent experiments for Miro1WT and Miro15R and n=3 for Miro1allR and Miro1R572K, ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


F ) Representative western blot showing a degradation in FlagParkin overexpressing SH - SY5Y transfected with Miro1WT , Miro15R or Miro1allR constructs treated with FCCP ( 10 μM ) for 3 or 6 hours . of Miro1 ( G ) matrix PDH E1α in ( H ) Parkin ( I ) ( n = 4 independent experiments ; ANOVA with Sidak ' s post hoc ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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F) Representative western blot showing a degradation assay in FlagParkin overexpressing SH-SY5Y cells transfected with Miro1WT, Miro15R or Miro1allR constructs and treated with FCCP (10 μM) for 3 or 6 hours. Quantification of Miro1 levels (G) the matrix protein PDH E1α in (H) and Parkin levels (I) (n=4 independent experiments; ANOVA with Sidak's post hoc test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative at 3 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO co - transfected with specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal mitochondrial network . A detail of YFPParkin translocation onto mitochondrial network is also shown for each condition .

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(A) Representative images at 3 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. A detail of YFPParkin translocation onto the mitochondrial network is also shown for each condition.


( B C ) of fraction of showing advanced stages of mitophagic process ( showing mitochondrial aggregation due to advanced Parkin translocation or complete translocation of Parkin onto mitochondrial network ) at 1 hour ( B ) or 3 hours ( C ) after FCCP treatment ( data collected from at least 3 independent experiments : WT n = 7 , MiroDKO n = 8 , + mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; mycMiro2 = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(B and C) Quantification of the fraction of cells showing advanced stages of the mitophagic process (cells showing mitochondrial aggregation due to advanced Parkin translocation or the complete translocation of Parkin onto all the mitochondrial network) at 1 hour (B) or 3 hours (C) after FCCP treatment (data collected from at least 3 independent experiments: WT n=7, MiroDKO n=8, +mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; mycMiro2=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


D ) Western blots of Miro1 degradation in MiroDKO co - expressing YFPParkin indicated myc - tagged Miro1 constructs treated with FCCP ( 10 μM ) for indicated times ( n = 3 different experiments ; 2 - way - ANOVA with Dunnett post ; comparisons between Miro1WT Miro1allR are represented with yellow asteriscs , comparisons between Miro1WT Miro15R are represented in green comparisons between Miro15R Miro1allR are represented in black ) .

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D) Western blots and quantification of Miro1 degradation in MiroDKO cells co-expressing YFPParkin and the indicated myc-tagged Miro1 constructs and treated with FCCP (10 μM) for the indicated times (n=3 different experiments; 2-way-ANOVA with Dunnett post test; comparisons between Miro1WT and Miro1allR are represented with yellow asteriscs, comparisons between Miro1WT and Miro15R are represented in green and comparisons between Miro15R and Miro1allR are represented in black).


E ) . Representative at 24 hours of FCCP treatment ( 10 μM ) of YFPParkin ( green ) expressing MiroDKO co - transfected with specified myc - tagged versions of Miro1 ( cyan ) . Tom20 ( red ) was used to reveal mitochondrial network . Expression of both ubiquitin mutants ( Miro15R Miro1allR - arrowheads ) protects from mitophagic clearance of mitochondria ( arrows ) after damage . ( F ) of fraction of showing complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment ( data collected from at least 3 independent experiments : MiroDKO n = 8 , mycMiro1WT = 7 , mycMiro15R = 3 , mycMiro1allR = 3 ; one way ANOVA with Dunnett post ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001

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E). Representative images at 24 hours of FCCP treatment (10 μM) of YFPParkin (green) expressing MiroDKO cells co-transfected with the specified myc-tagged versions of Miro1 (cyan). Tom20 (red) was used to reveal the mitochondrial network. Expression of both ubiquitin mutants (Miro15R and Miro1allR - arrowheads) protects from the mitophagic clearance of mitochondria (arrows) after damage. (F) Quantification of the fraction of cells showing the complete loss of Tom20 signal of mitochondria after 24 hours of FCCP treatment (data collected from at least 3 independent experiments: MiroDKO n=8, mycMiro1WT=7, mycMiro15R=3, mycMiro1allR=3; one way ANOVA with Dunnett post test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative confocal of soma of WT Miro1KO cortical neurons expressing YFPParkin MtDsRed after valinomycin treatment at indicated time points ( scale bars = 5 µm ) .

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(A) Representative confocal images of the soma of WT and Miro1KO cortical neurons expressing YFPParkin and MtDsRed after valinomycin treatment at the indicated time points (scale bars = 5 µm).


( B ) Airyscan confocal of Parkin recruitment after 5 hours of valinomycin treatment in WT Miro1KO neuronal processes ( scale bars = 2 . 5 µm ) .

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(B) Airyscan confocal images of Parkin recruitment after 5 hours of valinomycin treatment in WT and Miro1KO neuronal processes (scale bars = 2.5 µm).


( C ) linescans of YFPParkin MtDsRed signal in WT Miro1KO neurons after 5 hours of valinomycin treatment ( lines shown in A ) .

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(C) Fluorescent linescans of YFPParkin and MtDsRed signal in WT and Miro1KO neurons after 5 hours of valinomycin treatment (lines shown in A).


( D ) of Parkin recruitment to mitochondria ( YFPParkin signal overlapping MtDsRed signal - - adjusted normalised to t = 0 ) in WT Miro1KO neurons following valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(D) Quantification of Parkin recruitment to mitochondria (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted and normalised to t=0) in WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( E ) of mitochondrial occupancy ( of MtDsRed signal in soma / entire of soma ) in somas of WT Miro1KO neurons following valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; two - way ANOVA ) .

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(E) Quantification of mitochondrial occupancy (area of MtDsRed signal in soma / entire area of soma) in the somas of WT and Miro1KO neurons following valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; two-way ANOVA).


( F ) of mitochondrial clearance in somas of WT Miro1KO neurons between 2 5 hours of valinomycin treatment ( n = 15 conditions per genotype over 3 neuronal preparations ; Unpaired t - ) .

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(F) Quantification of mitochondrial clearance in somas of WT and Miro1KO neurons between 2 and 5 hours of valinomycin treatment (n=15 cells all conditions per genotype over 3 neuronal preparations; Unpaired t-test).


( G ) Representative confocal of WT , Miro1KO PINK1KO cortical neurons immunostained with MAP2 ( cyan ) s65 - phospho - ubiquitin ( green ) after valinomycin treatment ( Scale bars = 20µm ) . ( H ) of s65 - phospho - ubiquitin signal within MAP2 signal ( normalised to MAP2 t = 0 ) in Miro1WT , Miro1KO PINK1KO neurons following valinomycin treatment n = 3 , 4 3 from WT , Miro1KO PINK1KO , respectively ( 6 ROIs per condition ; two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(G) Representative confocal images of WT, Miro1KO and PINK1KO cortical neurons immunostained with MAP2 (cyan) and s65-phospho-ubiquitin (green) after valinomycin treatment (Scale bars = 20µm). (H) Quantification of s65-phospho-ubiquitin signal intensity within MAP2 signal (normalised to MAP2 area and t=0) in Miro1WT, Miro1KO and PINK1KO neurons following valinomycin treatment n= 3, 4 and 3 embryos from WT, Miro1KO and PINK1KO embryos, respectively (6 ROIs per condition; two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( I ) Representative confocal of soma of Miro1KO cortical neurons expressing Miro1WT mutant forms of Miro1 , YFPParkin MtDsRed without valinomycin treatment ( Scale bars = 10 µm ) . of parkin colocalization with MtDsRed ( YFPParkin signal overlapping MtDsRed signal - - adjusted ) in soma of Miro1KO PINK1KO cortical neurons ( n = 12 conditions per genotype over 3 neuronal preparations ; one - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 .

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(I) Representative confocal images of the soma of Miro1KO cortical neurons expressing Miro1WT and mutant forms of Miro1, YFPParkin and MtDsRed without valinomycin treatment (Scale bars = 10 µm). Quantification of parkin colocalization with MtDsRed (YFPParkin signal overlapping MtDsRed signal - intensity-adjusted) in the soma of Miro1KO and PINK1KO cortical neurons (n=12 cells all conditions per genotype over 3 neuronal preparations; one-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( J ) Representative confocal of soma of Miro1KO cortical neurons expressing WT mutant forms of Miro1 , YFPParkin MtDsRed after 5 hours of valinomycin treatment ( Scale bars = 10 µm ) . of Parkin recruitment to mitochondria ( Normalised to t = 0 ) in Miro1KO PINK1KO cortical neurons following valinomycin treatment ( n = 12 conditions per genotype over 3 neuronal preparations , two - way ANOVA ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001

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(J) Representative confocal images of the soma of Miro1KO cortical neurons expressing WT and mutant forms of Miro1, YFPParkin and MtDsRed after 5 hours of valinomycin treatment (Scale bars = 10 µm). Quantification of Parkin recruitment to mitochondria (Normalised to t=0) in Miro1KO and PINK1KO cortical neurons following valinomycin treatment (n=12 cells all conditions per genotype over 3 neuronal preparations, two-way ANOVA). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001


( A ) Representative blots of 4 12 months old hippocampal lysates from control , Miro1CKO Miro2KO . B ) of in 4 month lysates 12 month lysates ( n = 3 / genotype at 4 months n = 4 / genotype at 12 months ; one - way ANOVA with post - hoc Dunnett ' s ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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(A) Representative blots of 4 and 12 months old hippocampal lysates from control, Miro1CKO and Miro2KO. B) Quantification of protein levels in 4 month lysates and 12 month lysates (n=3 animals / genotype at 4 months and n=4 animals / genotype at 12 months; one-way ANOVA with post-hoc Dunnett's test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


C ) Representative of hippocampal regions from control Miro1CKO crossed with MitoDendra .

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C) Representative images of hippocampal regions from control and Miro1CKO crossed with MitoDendra animals.


( D ) Details from cortical regions of control , Miro1CKO Miro2KO at 12 months of showing large mitochondrial structures occurring only in case of Miro1CKO .

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(D) Details from cortical regions of control, Miro1CKO and Miro2KO animals at 12 months of age showing large mitochondrial structures occurring only in the case of Miro1CKO animals.


( E ) Electron microcopy of control Miro1CKO brains showing ultrastructure of mitochondrial compartment . mitochondrial units in Miro1CKO are enlarged show altered cristae structure at 4 months of . This process is progressive as mitochondrial structures appear larger with reduced intramitochondrial complexity at 12 months of .

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(E) Electron microcopy images of control and Miro1CKO brains showing the ultrastructure of the mitochondrial compartment. The mitochondrial units in Miro1CKO cells are enlarged and show altered cristae structure at 4 months of age. This process is progressive as mitochondrial structures appear larger and with reduced intramitochondrial complexity at 12 months of age.


Representative ( F ) ( G ) of Mfn1 Mfn2 in cortical slices from 12 month WT Miro1CKO mice ( n = 4 / genotype ; student ' s t - ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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Representative images (F) and quantification (G) of Mfn1 and Mfn2 staining in cortical slices from 12 month WT and Miro1CKO mice (n=4 animals / genotype; student's t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( A ) Representative western blot of brain lysates from 12 - month WT , Miro1CKO Miro2KO mice immunoblotted with antibodies stated . ( B ) of eIF2α P - eIF2α band ( normalised to actin band ) from 12 - month WT , Miro1CKO Miro2KO brain lysates . Phosphorylation of eIF2α is defined as P - eIF2α / eIF2α ( n = 4 mice per genotype , unpaired t - ) . Error bars represent s . e . m . Significance : * p < 0 . 05 , * * p < 0 . 01 * * * p < 0 . 001 .

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(A) Representative western blot images of brain lysates from 12-month WT, Miro1CKO and Miro2KO mice immunoblotted with the antibodies stated. (B) Quantification of eIF2α and P-eIF2α band intensity (normalised to actin band intensity) from 12-month WT, Miro1CKO and Miro2KO brain lysates. Phosphorylation of eIF2α is defined as P-eIF2α/eIF2α (n=4 mice per genotype, unpaired t-test). Error bars represent s.e.m. Significance: *p<0.05, **p<0.01 and ***p<0.001.


( C ) Representative confocal of cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice stained with MAP2 P - eIF2α . ( D ) of P - eIF2α signal from cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice ( n = 4 mice per genotype ; unpaired t - ) .

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(C) Representative confocal images of cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. (D) Quantification of P-eIF2α signal intensity from cortical regions of 12-month mitoDendra-crossed WT and Miro1CKO mice (n=4 mice per genotype; unpaired t-test).


( E ) Representative confocal ( 63x ) of cortical regions of 12 - month mitoDendra - crossed WT Miro1CKO mice stained with MAP2 P - eIF2α . Arrows indicate presence of megamitochondria ( > 2 . 5 µm2 ) , stars indicate neuronal somas without megamitochondria .

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(E) Representative confocal images (63x) of cortical regions of 12-month mitoDendra -crossed WT and Miro1CKO mice stained with MAP2 and P-eIF2α. Arrows indicate the presence of megamitochondria (>2.5 µm2), stars indicate neuronal somas without megamitochondria.


( F ) Example zoom of neuronal somas with without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO mice stained with P - eIF2α . ( G ) P - eIF2α signal ( a . u . ) from neuronal somas with without megamitochondria from cortical regions of 12 - month mitoDendra - crossed Miro1CKO ( n = 16 sections from 4 mice ; paired t - ) .

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(F) Example zoom images of neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra -crossed Miro1CKO mice stained with P-eIF2α. (G) Quantification P-eIF2α signal intensity (a.u.) from neuronal somas with and without megamitochondria from cortical regions of 12-month mitoDendra-crossed Miro1CKO (n=16 sections from 4 mice; paired t-test).


A Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 54 ) close contacts ( n = 86 ) were measured by surrogate neutralization ( sVNT ) . inhibition of ACE2 binding to RBD at plasma dilution of 1 : 800 is given . wild type ( Wuhan ) or Delta ( B . 1 . 617 . 2 ) RBD was used respectively .

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A Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=54) and close contacts (n=86) were measured by surrogate virus neutralization test (sVNT). The percentage inhibition of ACE2 binding to RBD at plasma dilution of 1:800 is given. The wild type (Wuhan) strain or Delta (B.1.617.2) strain RBD was used respectively.


B Anti - SARS - CoV - 2 neutralizing antibodies in plasma of vaccine breakthrough cases ( n = 29 ) close contacts ( n = 86 ) were measured by pseudovirus neutralization .

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B Anti-SARS-CoV-2 neutralizing antibodies in plasma of vaccine breakthrough cases (n=29) and close contacts (n=86) were measured by pseudovirus neutralization assay.


C Anti - SARS - CoV - 2 S antibodies in plasma of vaccine breakthrough cases ( n = 48 ) close contacts ( n = 86 ) was determined by S flow cytometry - ( SFB ) . of plasma antibody - bound out of total population of transfected with - anchored SARS - CoV - 2 S is given . In graphs error bars denote median interquartile range . Red solid circles indicate Delta vaccine breakthrough , red open circles indicate non - Delta or unknown .

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C Anti-SARS-CoV-2 S protein antibodies in plasma of vaccine breakthrough cases (n=48) and close contacts (n=86) was determined by S protein flow cytometry-based assay (SFB). The percentage of plasma antibody-bound cells out of the total population of cells transfected with membrane-anchored SARS-CoV-2 S protein is given. In all graphs error bars denote median and interquartile range. Red solid circles indicate Delta strain vaccine breakthrough, red open circles indicate non-Delta or unknown strain.


B , C of anti - SARS - CoV - 2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate neutralization ( sVNT ) , in supernatant from 5 - day culture of activated memory B in plasma . difference in sVNT % inhibition between WT Delta is taken , this difference is compared between plasma memory B supernatant for vaccine breakthrough cases ( n = 48 pairs ) ( B ) , for close contacts ( n = 86 pairs ) ( C ) .

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B, C Levels of anti-SARS-CoV-2 neutralizing antibodies inhibiting ACE2 binding to WT or Delta are determined by surrogate virus neutralization test (sVNT), in supernatant from 5-day culture of activated memory B cells and in plasma. The difference in sVNT % inhibition between WT and Delta is taken, and this difference is compared between plasma and memory B cell supernatant for vaccine breakthrough cases (n=48 pairs) (B), and for close contacts (n=86 pairs) (C).


A - D PBMCs from vaccine breakthrough cases ( n = 15 ) close contacts ( n = 26 ) were examined for T responses . PBMCs were left unstimulated ( Unstim ) , were stimulated with pooled SARS - CoV - 2 PepTivator ® S , S1 , M N peptides for 6 hours ( SARS - CoV - 2 Peptide Stim ) , or non - specifically stimulated with phorbol 12 - myristate 13 - acetate ( PMA ) , then assessed by high - dimensional flow cytometry . CD4 + T positive for intracellular of IFNγ ( a ) , IL - 2 ( b ) , TNF ( c ) , or CD8 + T positive for intracellular of granzyme B ( d ) are shown . Error bars indicate median interquartile range .

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A-D PBMCs from vaccine breakthrough cases (n=15) and close contacts (n=26) were examined for T cell responses. PBMCs were left unstimulated (Unstim), were stimulated with pooled SARS-CoV-2 PepTivator® S, S1, M and N peptides for 6 hours (SARS-CoV-2 Peptide Stim), or non-specifically stimulated with phorbol 12-myristate 13-acetate (PMA), then assessed by high-dimensional flow cytometry. CD4+ T cells positive for intracellular staining of IFNγ (a), IL-2 (b), TNF (c), or CD8+ T cells positive for intracellular staining of granzyme B (d) are shown. Error bars indicate median and interquartile range.


F differentiation status of SARS - CoV - 2 peptide - stimulated CD4 + T ( left ) CD8 + T ( right ) were compared on CD27 CD45RA expression ( Naïve : CD27 + CD45RA + ; T central memory ( TCM ) : CD27 + CD45RA - ; T effector memory ( ) : CD27 - CD45RA - ; TEMRA : CD27 - CD45RA + ) . average of vaccine breakthrough cases or close contacts are plotted respectively .

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F The differentiation status of SARS-CoV-2 peptide-stimulated CD4+ T cells (left) and CD8+ T cells (right) were compared based on CD27 and CD45RA expression (Naïve: CD27+ CD45RA+; T central memory (TCM): CD27+ CD45RA-; T effector memory (TEM): CD27- CD45RA-; TEMRA: CD27- CD45RA+). The average of all vaccine breakthrough cases or close contacts are plotted respectively.


B of selected immune mediator in plasma samples of healthy controls , vaccinated close contacts , COVID - 19 vaccine breakthrough cases , unvaccinated COVID - 19 with infection . Each color represents relative of a particular analyte . Blue red indicate low high , respectively .

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B Heatmap of selected immune mediator levels in plasma samples of healthy controls, vaccinated close contacts, COVID-19 vaccine breakthrough cases, and unvaccinated COVID-19 patients with primary infection. Each color represents the relative concentration of a particular analyte. Blue and red indicate low and high concentration, respectively.


( B ) upper panel showing qRT - PCR of four identified LUAD - associated RBPs ( ADARB1 , CELF2 , QKI ZFP36 ) mRNA in 61 LUAD tissues matched paracarcinoma tissues . lower panel showing relative mRNA expression ( T / N ) of four RBPs in metastatic ( n = 28 ) non - metastatic ( n = 33 ) LUAD tissues . LUAD tissues were classified into metastatic non - metastatic tissues as described in Materials Methods . T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents log10 transformed fold change of mRNA expression ( T / N ) of four RBPs .

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(B) The upper panel showing qRT-PCR analysis of four identified LUAD-associated RBPs (ADARB1, CELF2, QKI and ZFP36) mRNA levels in 61 LUAD tissues and matched paracarcinoma tissues. The lower panel showing relative mRNA expression (T/N) of the four RBPs in metastatic (n = 28) and non-metastatic (n = 33) LUAD tissues. LUAD tissues were classified into metastatic and non-metastatic tissues as described in Materials and Methods. T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression (T/N) of four RBPs.


( E - G ) Kaplan - Meier survival curves of LC , LUAD LUSC ( n = 1540 , n = 865 n = 675 , respectively ) with high or low expression of QKI . log - rank was used to analyze difference between two groups .

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(E-G) Kaplan-Meier survival curves of LC, LUAD and LUSC patients (n=1540, n=865 and n=675, respectively) with high or low expression levels of QKI. The log-rank test was used to analyze the difference between two groups.


( H ) qRT - PCR of endogenous mRNA of QKI - 5 , QKI - 6 , QKI - 7 in human bronchial epithelial BEAS - 2B , LUAD lines A549 H1299 .

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(H) qRT-PCR analysis of endogenous mRNA levels of QKI-5, QKI-6, and QKI-7 in human bronchial epithelial cell line BEAS-2B, LUAD cell lines A549 and H1299.


( J ) In vitro pull - down assays were performed using biotin - labeled TGFβR1 3 ' UTR segments in A549 H1299 . After pull - down , endogenous QKI - 5 enrichments were detected by western blot analyses . β - actin was used as control .

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(J) In vitro RNA pull-down assays were performed using biotin-labeled TGFβR1 3' UTR segments in A549 and H1299 cells. After pull-down, endogenous QKI-5 enrichments were detected by western blot analyses. β-actin was used as the protein control.


( A B ) qRT - PCR western blot analyses of TGFβR1 mRNA in QKI - 5 - overexpressing A549 H1299 . ( C D ) Expression of TGFβR1 mRNA in QKI - 5 - silenced A549 H1299 .

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(A and B) qRT-PCR and western blot analyses of TGFβR1 mRNA and protein levels in QKI-5-overexpressing A549 and H1299 cells. (C and D) Expression of TGFβR1 mRNA and protein in QKI-5-silenced A549 and H1299 cells.


( K ) QKI - 5 - overexpressing A549 H1299 were treated subjected to qRT - PCR assays to detect mRNA expression of downstream genes of TGF - β / SMAD signaling , including PAI - 1 , Slug , Snail , E - cadherin , / or N - cadherin .

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(K) QKI-5-overexpressing A549 and H1299 cells were treated and subjected to qRT-PCR assays to detect the mRNA expression of downstream genes of the TGF-β/SMAD signaling, including PAI-1, Slug, Snail, E-cadherin, and/or N-cadherin.


( C - F ) Wound - healing migration assays were performed on QKI - 5 - overexpressing A549 H1299 as well as control wound healing was recorded ( C E ) quantitatively measured ( D F ) at least six times .

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(C-F) Wound-healing migration assays were performed on QKI-5-overexpressing A549 and H1299 cells as well as control cells The wound healing was recorded (C and E) and quantitatively measured (D and F) at least six times.


( G H ) In presence or absence of TGF - β1 , QKI - 5 - overexpressing A549 H1299 were allowed to migrate through a polycarbonate in Transwells . After 24 h , migrated were stained , photographed counted in at least four random fields under a microscope . Representative ( left ) migrated numbers ( right ) were presented . ( I J ) QKI - 5 - overexpressing A549 H1299 were treated allowed to invade through Matrigel - coated in Transwells . Invasive were

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(G and H) In the presence or absence of TGF-β1, QKI-5-overexpressing A549 and H1299 cells were allowed to migrate through a polycarbonate membrane in Transwells. After 24 h, migrated cells were stained, photographed and counted in at least four random fields under a light microscope. Representative images (left) and the migrated cell numbers (right) were presented. (I and J) QKI-5-overexpressing A549 and H1299 cells were treated and allowed to invade through Matrigel-coated membrane in Transwells. Invasive cells were determined


( E ) H & E was performed for evaluation of lung micrometastases . Representative showing micrometastases of lung tissues from a pair of mice Blue red arrowheads indicate lung micrometastases of vector group QKI - 5 - overexpressing group , respectively . ( F ) Dot plots showing difference of micrometastases in lung tissues between QKI - 5 - overexpressing group vector control group ( n = 10 mice per group ) . ( G ) Representative microscopic of H & E for liver micrometastases in a pair of mice Blue red arrowheads indicate liver micrometastases . ( H ) Dot plots showing difference of micrometastases in liver tissues between QKI - 5 - overexpressing group control vector group ( n = 10 mice per group ) .

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(E) H&E staining was performed for the evaluation of lung micrometastases. Representative images showing micrometastases of lung tissues from a pair of mice Blue and red arrowheads indicate lung micrometastases of vector group and QKI-5-overexpressing group, respectively. (F) Dot plots showing the difference of the micrometastases counts in lung tissues between QKI-5-overexpressing group and vector control group (n = 10 mice per group). (G) Representative microscopic photographs of H&E staining for liver micrometastases in a pair of mice Blue and red arrowheads indicate liver micrometastases. (H) Dot plots showing the difference of the micrometastases counts in liver tissues between QKI-5-overexpressing group and control vector group (n = 10 mice per group).


( C - F ) In presence or absence of TGF - β1 , migratory invasive capabilities of above - treated A549 H1299 were evaluated by Transwell assays Migrated invasive were stained counted under a microscope .

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(C-F) In the presence or absence of TGF-β1, the migratory and invasive capabilities of above-treated A549 and H1299 cells were evaluated by Transwell assays Migrated and invasive cells were stained and counted under a light microscope.


( J ) Kaplan - Meier survival curves of LUAD ( n = 865 ) with high or low expression of TGFβR1 . log - rank was used to compare difference between two groups .

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(J) Kaplan-Meier survival curves of LUAD patients (n=865) with high or low expression levels of TGFβR1. The log-rank test was used to compare the difference between two groups.


qRT - PCR analyses of mRNA expression of QKI - 5 in A549 transfected with siRNAs of TFs , which have potential binding sites in QKI - 5 promoter .

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qRT-PCR analyses of mRNA expression of QKI-5 in A549 cells transfected with siRNAs of TFs, which have potential binding sites in QKI-5 promoter.


( E ) Western blot of QKI - 5 in KLF6 - silenced A549 .

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(E) Western blot analysis of QKI-5 protein levels in KLF6-silenced A549 cells.


( F ) An overview of KLF6 - ChIP - seq data ( GSE96355 ) is illustrated using Integrative Genomics Viewer ( IGV ) software . An obvious peak was detected at position - 100 to + 100 in QKI - 5 promoter .

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(F) An overview of KLF6-ChIP-seq data (GSE96355) is illustrated using Integrative Genomics Viewer (IGV) software. An obvious peak was detected at position -100 to +100 in the QKI-5 promoter.


qRT - PCR of KLF6 mRNA in 61 LUAD tissues matched paracarcinoma tissues ( I ) T , LUAD tissues ; N , paracarcinoma tissues . Y axis represents log10 transformed fold change of mRNA expression of KLF6 . Central dotted of violin plot represents median , upper lower dotted represents quartile ( I ) .

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qRT-PCR analysis of KLF6 mRNA in 61 LUAD tissues and matched paracarcinoma tissues (I) T, LUAD tissues; N, paracarcinoma tissues. Y axis represents the log10 transformed fold change of mRNA expression of KLF6. Central dotted line of the violin plot represents median, and upper and lower dotted line represents quartile (I).


( A ) Western blot of supernatants containing long short mouse human DNGR - 1 ECD under reducing non - reducing conditions .

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(A) Western blot analysis of supernatants containing long and short mouse and human DNGR-1 ECD proteins under reducing and non-reducing conditions.


( B ) Western blot of supernatants after production of WT C94S mutant long mouse DNGR - 1 ECD under reducing non - reducing conditions . C94S mutant - containing supernatant was 200x concentrated before .

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(B) Western blot analysis of supernatants after protein production of WT and C94S mutant long mouse DNGR-1 ECD proteins under reducing and non-reducing conditions. C94S mutant-containing supernatant was 200x concentrated before analysis.


( C ) Western blot of supernatants containing indicated DNGR - 1 ECD after treatment with PNGase F overnight . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers .

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(C) Western blot analysis of supernatants containing the indicated DNGR-1 ECD proteins after treatment with PNGase F overnight. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers.


( A ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into 10 mM Tris buffer of indicated pH ( left panel ) or into 10 mM Tris pH 7 . 4 buffer supplemented with increasing amounts of NaCl ( 1 - 250 mM ; right panel ) , reduction sensitivity of DNGR - 1 was assessed by reducing SDS - PAGE Western blot . ( B ) 0 . 25 μg of purified DNGR - 1 long mouse ECD was diluted into buffers of different pH ionic strength , its reduction sensitivity was assessed by reducing SDS - PAGE Western blot . of bands corresponding to dimer monomer was determined densitometrically , was plotted as a function of buffer ionic strength pH .

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(A) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into 10 mM Tris buffer of the indicated pH (left panel) or into 10 mM Tris pH 7.4 buffer supplemented with increasing amounts of NaCl (1 - 250 mM; right panel), and reduction sensitivity of DNGR-1 was assessed by reducing SDS-PAGE and Western blot. (B) 0.25 μg of purified DNGR-1 long mouse ECD was diluted into buffers of different pH and ionic strength, and its reduction sensitivity was assessed by reducing SDS-PAGE and Western blot. The intensity of bands corresponding to dimer and monomer was determined densitometrically, and the ratio was plotted as a function of buffer ionic strength and pH.


( C ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffer , its reduction sensitivity was tested under mildly ( Laemmli buffer ) or strongly denaturing ( 8 M urea ) conditions by reducing SDS - PAGE Western blot .

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(C) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffer, and its reduction sensitivity was tested under mildly (Laemmli buffer) or strongly denaturing (8 M urea) conditions by reducing SDS-PAGE and Western blot.


( D ) 0 . 25 μg of purified long mouse DNGR - 1 ECD was diluted into PBS or 10 mM MES pH 6 . 1 buffers , its reduction sensitivity after different lengths of heat - denaturation in Laemmli buffer was tested by reducing SDS - PAGE Western blot . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(D) 0.25 μg of purified long mouse DNGR-1 ECD was diluted into PBS or 10 mM MES pH 6.1 buffers, and its reduction sensitivity after different lengths of heat-denaturation in Laemmli buffer was tested by reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) 1 μg of purified long mouse DNGR - 1 ECD was transferred into 10mM MES pH 6 . 1 0 . 5 μg into PBS . Half of MES sample whole PBS sample were dialyzed against 2 l of PBS overnight at 4 ° C . After dialysis , samples were prepared for reducing SDS - PAGE Western blot .

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(A) 1 μg of purified long mouse DNGR-1 ECD was transferred into 10mM MES pH 6.1 and 0.5 μg into PBS. Half of the MES sample and the whole PBS sample were dialyzed against 2 l of PBS overnight at 4°C. After dialysis, all samples were prepared for reducing SDS-PAGE and Western blot.


( B ) Supernatants after production of indicated in 293F were harvested diluted into indicated buffers at a 2 : 3 analyzed using reducing SDS - PAGE Western blot . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(B) Supernatants after production of indicated proteins in 293F cells were harvested and diluted into indicated buffers at a 2:3 ratio and analyzed using reducing SDS-PAGE and Western blot. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( B ) Supernatants containing ECD of long mouse DNGR - 1 , Dectin - 1 chimeric receptor were prepared for SDS - PAGE Western blot under non - reducing conditions .

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(B) Supernatants containing ECD of long mouse DNGR-1, Dectin-1 and the chimeric receptor were prepared for SDS-PAGE and Western blot under non-reducing conditions.


( C ) Supernatants containing indicated were diluted into PBS or pH 6 . 1 MES buffer analyzed using SDS - PAGE Western blot under reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction sensitive with a + .

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(C) Supernatants containing indicated proteins were diluted into PBS or pH 6.1 MES buffer and analyzed using SDS-PAGE and Western blot under reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction sensitive protein with a +.


( A ) Long mouse human DNGR - 1 ECD were diluted in PBS or 10 mM MES pH 6 . 1 buffer 20 independent far - UV spectra were acquired for each condition ( red black lines depict composite curve for each condition )

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(A) Long mouse and human DNGR-1 ECD proteins were diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent far-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition)


( B ) Long mouse DNGR - 1 ECD was diluted in PBS or 10 mM MES pH 6 . 1 buffer 20 independent near - UV spectra were acquired for each condition ( red black lines depict composite curve for each condition blue shows difference between two ) .

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(B) Long mouse DNGR-1 ECD was diluted in PBS or 10 mM MES pH 6.1 buffer and 20 independent near-UV spectra were acquired for each condition (red and black lines depict the composite curve for each condition and the blue line shows the difference between the two).


( B ) ( C ) Supernatants containing indicated ECD ( mouse , long isoform ) were diluted 1 : 2 into indicated buffers analyzed by SDS - PAGE Western blot under reducing or non reducing conditions . HRP - conjugated anti - FLAG antibody was used for detection of . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(B) and (C) Supernatants containing the indicated ECD proteins (mouse, long isoform) were diluted 1:2 into the indicated buffers and analyzed by SDS-PAGE and Western blot under reducing or non reducing conditions. HRP-conjugated anti-FLAG antibody was used for detection of all proteins. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( D ) Phoenix expressing indicated were lysed in 1 % SDS in PBS or 10mM MES pH 6 . 1 buffers lysates were analyzed by SDS - PAGE Western blot under reducing conditions . Anti - DNGR - 1 antibody ( clone 397 ) followed by HRP - conjugated secondary anti - rat antibody was used for detection . Numbers on side of blots indicate positions of molecular markers . Reduction - resistant dimers are indicated with an * reduction - sensitive with a + .

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(D) Phoenix cells expressing the indicated proteins were lysed in 1% SDS in PBS or 10mM MES pH 6.1 buffers and the lysates were analyzed by SDS-PAGE and Western blot under reducing conditions. Anti-DNGR-1 antibody (clone 397) followed by HRP-conjugated secondary anti-rat antibody was used for detection. Numbers on the side of blots indicate positions of molecular weight markers. Reduction-resistant dimers are indicated with an * and reduction-sensitive protein with a +.


( A ) Decreasing amounts of F - actin were spotted onto a nitrocellulose binding of indicated DNGR - 1 ECD ( mouse , long isoform ) was tested in PBS or MES pH 6 . 1 buffers . HRP - conjugated anti - FLAG antibody was used for detection . signal was quantified by densitometry quantitation of four independent experiments is shown .

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(A) Decreasing amounts of F-actin were spotted onto a nitrocellulose membrane and binding of indicated DNGR-1 ECD (mouse, long isoform) proteins was tested in PBS or MES pH 6.1 buffers. HRP-conjugated anti-FLAG antibody was used for detection. The signal was quantified by densitometry and quantitation of four independent experiments is shown.