F - H COS ‐ 1 stably expressing GFP ‐ lact ‐ C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h stained with recombinant 2xPH anti ‐ GM130 antibody . intensities of 2xPH GFP ‐ lact ‐ C2 in RE were measured . of 2xPH ( G ) of 2xPH to GFP ‐ lact ‐ C2 intensities ( H ) are shown . In ( H ) , data are normalized to that of control . Data represent mean ± SD were analyzed with two ‐ Student ' s t ‐ ( n > 26 from three independent experiments ) . * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant . Scale bars , 10 μm .

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F-H COS‐1 cells stably expressing GFP‐lact‐C2 were treated with ATP8A1 siRNA1 or EHD1 siRNA1 for 72 h and stained with recombinant 2xPH and anti‐GM130 antibody. The intensities of 2xPH staining and GFP‐lact‐C2 in RE area were measured. The intensity of 2xPH (G) and the ratio of 2xPH to GFP‐lact‐C2 intensities (H) are shown. In (H), data are normalized to that of control cells. Data represent mean ± SD and were analyzed with two‐tailed Student's t‐test (n > 26 cells from three independent experiments). **P 0.01; ***P 0.001; NS, not significant. Scale bars, 10 μm.


A COS ‐ 1 co ‐ transfected with GFP ‐ ATP8A2 ( WT ) myc ‐ CDC50A were stained for myc ‐ tag TfnR . Scale bar , 10 μm .

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A COS‐1 cells co‐transfected with GFP‐ATP8A2 (WT) and myc‐CDC50A were stained for myc‐tag and TfnR. Scale bar, 10 μm.


B effect of increasing of PS on ATPase activity of ATP8A2 ( WT I376M ) ‐ CDC50A .

B The effect of increasing levels of PS on the ATPase activity of ATP8A2 (WT and I376M)‐CDC50A complex.


C NBD ‐ labeled PS transport ( flippase ) activity of ATP8A2 ( WT I376M ) ‐ CDC50A . of NBD ‐ lipid flipped was shown .

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C NBD‐labeled PS transport (flippase) activity of ATP8A2 (WT and I376M)‐CDC50A complex. The percentage of NBD‐lipid flipped was shown.


D were treated with ATP8A1 siRNA1 for 72 h . ATP8A2 ‐ 1D4 ( WT or mutants ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . were then stained for EHD1 1D4 ‐ tag . Scale bars , 10 μm .

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D Cells were treated with ATP8A1 siRNA1 for 72 h. ATP8A2‐1D4 (WT or mutants) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for EHD1 and 1D4‐tag. Scale bars, 10 μm.


E of percentages of with perinuclear EHD1 localization . Data represent mean ± SD of three or four independent experiments . At least 20 were counted per experiment .

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E Quantification of percentages of cells with perinuclear EHD1 localization. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.


F were treated with ATP8A1 siRNA for 72 h . GFP ‐ ATP8A2 ( WT or mutant ) myc ‐ CDC50A were transfected 48 h after siRNA transfection . were then stained for TfnR . Data represent mean ± SD of three or four independent experiments . At least 20 were counted per experiment .

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F Cells were treated with ATP8A1 siRNA for 72 h. GFP‐ATP8A2 (WT or mutant) and myc‐CDC50A were transfected 48 h after siRNA transfection. The cells were then stained for TfnR. Data represent mean ± SD of three or four independent experiments. At least 20 cells were counted per experiment.


G , H Surface biotinylation with Atp8a2 + / + or Atp8a2 − / − cortical neurons . of surface to total TfnR was quantified normalized to Atp8a2 + / + . Data represent mean value ( 2 mice for each genotype ) .

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G, H Surface biotinylation assay with Atp8a2+/+ or Atp8a2−/− cortical neurons. The ratio of surface to total TfnR was quantified and normalized to Atp8a2+/+. Data represent mean value (2 mice for each genotype).


c d . Representation of in epidermal ( e ) cortical ( c ) files . Optical , longitudinal sections of 5 DAG old Col - 0 roots 12 HAT to ammonium ( C ) or nitrate ( D ) supplemented media . first 20 epidermal ( e ) cortex ( c ) ( from quiescent center ( QC ) ) are highlighted in grey in red on ammonium ( C ) , in blue green on nitrate ( D ) , respectively . Scale bar = 30 µm . Column bars denote geometric mean of lengths at respective positions . Lines represent a polynomial regression fit , with calculated slopes between 10 20 of 3 . 32639 + 0 . 17172 ( ammonium , epidermis ) , 1 . 22033 + 0 . 08754 ( ammonium , cortex ) 1 . 70502 + 0 . 09532 ( nitrate , epidermis ) , 0 . 82342 + 0 . 06973 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 18 roots in each case .

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c and d. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG old Col-0 roots 12 HAT to ammonium (C) or nitrate (D) supplemented media. The first 20 epidermal (e) and cortex (c) cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (C), and in blue and green on nitrate (D), respectively. Scale bar=30 µm. Column bars denote the geometric mean of the cell lengths at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 3.32639+0.17172 (ammonium, epidermis), 1.22033+0.08754 (ammonium, cortex) and 1.70502+0.09532 (nitrate, epidermis), 0.82342+0.06973 (nitrate, cortex). Data are derived from 3 biological replicates, n=18 roots in each case.


a b . Maximum Z - stack projection of 5 DAG old roots expressing R2D2 auxin signaling reporter 12 HAT to ammonium ( a ) or nitrate ( b ) supplemented media . arrows mark position of 10th 20th from QC ; " e " " c " mark epidermis cortex , respectively . Scale bar = 50 µm . Graphs denote normalized relative auxin at respective positions . Lines represent polynomial regression fit with 95 % confidence band . Data are derived from 5 roots per condition from 3 biological replicates .

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a and b. Maximum intensity Z-stack projection images of 5 DAG old roots expressing the R2D2 auxin signaling reporter 12 HAT to ammonium (a) or nitrate (b) supplemented media. White arrows mark the position of the 10th and 20th cells from QC; "e" and "c" mark epidermis and cortex, respectively. Scale bar=50 µm. Graphs denote normalized relative auxin levels at the respective positions. Lines represent polynomial regression fit with 95% confidence band. Data are derived from 5 roots per condition from 3 biological replicates.


b c . Representation of in epidermal ( e ) cortical ( c ) files . Optical , longitudinal sections of 5 DAG eir1 - 4 roots 12 HAT to ammonium ( B ) or nitrate ( C ) supplemented media . first 20 - 20 epidermal cortex ( from quiescent center ( QC ) ) are highlighted in grey in red on ammonium ( B ) in blue green on nitrate ( C ) , respectively . Scale bar = 30 µm . Column bars denote geometric mean of at respective positions . Lines represent a polynomial regression fit , with calculated slopes between 10 20 of 0 . 75884 + 0 . 02624 ( ammonium , epidermis ) , 1 . 13088 + 0 . 08446 ( ammonium , cortex ) 2 . 06912 + 0 . 10341 ( nitrate , epidermis ) , 0 . 99878 + 0 . 07278 ( nitrate , cortex ) . Data are derived from 3 biological replicates , n = 9 ( ammonium ) 8 ( nitrate ) roots .

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

b and c. Representation and quantification of cell length in epidermal (e) and cortical (c) cell files. Optical, longitudinal sections of 5 DAG eir1-4 roots 12 HAT to ammonium (B) or nitrate (C) supplemented media. The first 20-20 epidermal and cortex cells (from quiescent center (QC)) are highlighted in grey and in red on ammonium (B) and in blue and green on nitrate (C), respectively. Scale bar=30 µm. Column bars denote the geometric mean of cell length at the respective positions. Lines represent a polynomial regression fit, with calculated slopes between cells 10 and 20 of 0.75884+0.02624 (ammonium, epidermis), 1.13088+0.08446 (ammonium, cortex) and 2.06912+0.10341 (nitrate, epidermis), 0.99878+0.07278 (nitrate, cortex). Data are derived from 3 biological replicates, n=9 (ammonium) and 8 (nitrate) roots.


b . Higher magnification of pseudo - colored confocal of 5 DAG old roots expressing PIN2 - GFP 12 HAT to ammonium or nitrate supplemented media . " e " denotes epidermis " c " cortex , respectively . Color code represents GFP from low ( blue ) to high ( ) values . Scale bar = 12 µm . stars mark PIN2 - GFP localization on lateral membranes . Box plots display lateralization index ( signal detected on apical / basal membranes divided by signal value at inner / outer membranes ) of roots on ammonium ( n = 31 from 6 roots ) or nitrate ( n = 24 from 6 roots ) supplemented medium . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

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

b. Higher magnification of pseudo-colored confocal images of 5 DAG old roots expressing PIN2-GFP 12 HAT to ammonium or nitrate supplemented media. "e" denotes epidermis and "c" cortex, respectively. Color code represents GFP intensity from low (blue) to high (white) values. Scale bar=12 µm. White stars mark PIN2-GFP protein localization on the lateral membranes. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes divided by the signal value at inner/outer membranes) of roots on ammonium (n=31 cells from 6 roots) or nitrate (n=24 cells from 6 roots) supplemented medium. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


d . Microscopic showing PIN2 - Dendra signal five hours after photoconversion of PIN2 - Dendra into its red form . Depletion of red signal recovery of green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media . Note increase in of green signal in roots transferred to nitrate . Graph represents mean signal + SD ( n = 6 roots per condition , 20 per root analyzed ) . experiment was repeated 3 times . Scale bar = 20 µm .

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d. Microscopic images showing PIN2-Dendra fluorescent signal five hours after photoconversion of PIN2-Dendra into its red form. Depletion of the red signal and recovery of the green signal over a 6 hours period was followed in parallel in 5 DAG old roots12 HAT to ammonium or nitrate supplemented media. Note the increase in the intensity of the green signal in roots transferred to nitrate. Graph represents the mean signal + SD (n=6 roots per condition, 20 cells per root analyzed). The experiment was repeated 3 times. Scale bar=20 µm.


d . Box plots display distribution of derived PIN2 - GFP ( FI ) values ( in arbitrary units , a . u . ) in roots transferred to ammonium ( ( grey , epidermis ( ep ) red , cortex ( co ) to nitrate ( blue , epidermis ( ep ) green , cortex ( co ) ) . 5 per roots were analyzed in at least 9 roots per genotype per treatment . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

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d. Box plots display the distribution of the cell membrane derived PIN2-GFP fluorescence intensity (FI) values (in arbitrary units, a.u.) in roots transferred to ammonium ((grey, epidermis (ep) and red, cortex (co) and to nitrate (blue, epidermis (ep) and green, cortex (co)). 5 cells per roots were analyzed in at least 9 roots per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


g . Box plots display lateralization index ( signal detected on apical / basal membranes vs inner / outer membranes ) of P2wt , P2D P2A roots transferred to ammonium ( grey ) or nitrate ( red ) supplemented medium . At least 24 from 5 roots were analyzed per genotype per treatment . statistical significance was evaluated with ANOVA at p < 0 . 05 . box chart components are defined as ; box ( 25 % - 75 % ) , central band ( median ) , central box ( mean ) range is within 1 . 5IQR .

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g. Box plots display lateralization index (fluorescent signal detected on apical/basal membranes vs inner/outer membranes) of P2wt, P2D and P2A roots transferred to ammonium (grey) or nitrate (red) supplemented medium. At least 24 cells from 5 roots were analyzed per genotype per treatment. The statistical significance was evaluated with ANOVA at p<0.05. The box chart components are defined as; box (25%-75%), central band (median line), central box (mean) and the range is within 1.5IQR.


Immunofluorescence of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre ; ranklfl / fl mice ( Rankl - / - ) ranklfl / fl mice ( Rankl + / + ) . Scale bar = 50 μm .

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Immunofluorescence staining of RANKL during in vitro adipogenesis of BMSCs from AdipoqCre; ranklfl/fl mice (Rankl-/-) and ranklfl/fl mice (Rankl+/+). Scale bar=50 μm.


Micro - CT statistical analyses of BMD , BV / TV , Tb . N Tb . Sp in trabecular bone from AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice at 4 , 8 16 ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t - ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Micro-CT and statistical analyses of BMD, BV/TV, Tb.N and Tb.Sp in trabecular bone from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 4 weeks, 8 weeks and 16 weeks (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t-test (** indicates P < 0.01), error bars are standard deviations.


Representative of TRAP of femur from AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice at 8 . Scale bar = 200 μm . Panel a1 a4 indicate trabecular bone , a2 a5 stand for growth plate , a3 a6 denote cortical bone .

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Representative images of TRAP staining of femur from AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice at 8 weeks. Scale bar=200 μm. Panel a1 and a4 indicate trabecular bone, a2 and a5 stand for growth plate, a3 and a6 denote cortical bone.


Immunofluorescence of osteocalcin from 8 - week - old mice ( n = 6 mice in Rankl - / - group 4 mice in Rankl + / + group ) . Scale bar = 50 μm . TB : trabecular bone . Statistical analyses of Ocn + ( n = 6 independent microscopic vision fields from random mice samples ) . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Immunofluorescence staining of osteocalcin from 8-week-old mice (n=6 mice in Rankl-/- group and 4 mice in Rankl+/+ group). Scale bar=50 μm. TB: trabecular bone. Statistical analyses of Ocn+ cells (n=6 independent microscopic vision fields from random mice samples). Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Calcein of trabecular bone at 8 . Scale bar = 50 μm . TB : trabecular bone . are representative of 6 independent biological replicates in Rankl - / - 5 replicates in Rankl + / + group . Mineral apposition rate ( MAR ) of trabecular bone at 8 . ( n = 6 mice in Rankl - / - group 5 mice in Rankl + / + group ) . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Calcein staining of trabecular bone at 8 weeks. Scale bar=50 μm. TB: trabecular bone. Images are representative of 6 independent biological replicates in Rankl-/- and 5 replicates in Rankl+/+ group. Mineral apposition rate (MAR) of trabecular bone at 8 weeks.(n=6 mice in Rankl-/- group and 5 mice in Rankl+/+ group). Data were compared using an unpaired t‐test ( ** indicates P < 0.01), error bars are standard deviations.


Serum CTX - 1 , OCN bone marrow RANKL from Rankl - / - Rankl + / + mice at 4 ( n = 5 5 ) , 8 ( n = 5 6 ) 16 ( n = 5 5 ) . Data were compared using an unpaired t ‐ ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations . .

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Serum CTX-1, OCN and bone marrow RANKL levels from Rankl-/- and Rankl+/+ mice at 4 weeks (n=5 and 5), 8 weeks (n=5 and 6) and 16 weeks (n=5 and 5). Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations..


Micro - CT analyses of trabecular bone from Grounded Tail suspended model ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * indicates P < 0 . 05 , * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Micro-CT analyses of trabecular bone from Grounded and Tail suspended model (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (* indicates P < 0.05, ** indicates P < 0.01), error bars are standard deviations.


Micro - CT analyses of sham OVX model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice ( n = 5 independent biological replicates ) . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Micro-CT analyses of sham and OVX model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice (n=5 independent biological replicates). Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP statistical analyses of numbers of osteoclasts in Sham OVX model ( are representative of 5 independent biological replicates in ( C ) , n = 5 independent microscopic vision fields in ( D ) ) . Scale bar = 100 µm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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TRAP staining and statistical analyses of numbers of osteoclasts in Sham and OVX model (Images are representative of 5 independent biological replicates in (C), and n=5 independent microscopic vision fields in (D)). Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Immunofluorescence of OCN statistical analyses of number of osteoblasts ( are representative of 5 independent biological replicates in ( E ) , n = 5 independent microscopic vision fields in ( F ) ) . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Immunofluorescence staining of OCN and statistical analyses of the number of osteoblasts (Images are representative of 5 independent biological replicates in (E), and n=5 independent microscopic vision fields in (F)). Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


Three - dimensional CT analyses of BMD , BV / TV , Tb . N , Tb . Sp in trabecular bone from Vehicle ( n = 5 independent biological replicates ) ROS ( n = 6 independent biological replicates ) model of AdipoqCre ; ranklfl / fl ( Rankl - / - ) ranklfl / fl ( Rankl + / + ) mice . Scale bar = 1 mm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations .

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Three-dimensional CT analyses of BMD, BV/TV, Tb.N, and Tb.Sp in trabecular bone from Vehicle (n=5 independent biological replicates) and ROS (n=6 independent biological replicates) model of AdipoqCre; ranklfl/fl (Rankl-/-) and ranklfl/fl (Rankl+/+) mice. Scale bar=1 mm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations.


TRAP statistical analyses of numbers of osteoclasts in trabecular bone from Vehicle ( n = 6 5 independent biological replicates ) ROS ( n = 6 6 independent biological replicates ) treatment . Scale bar = 100 µm . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( D ) .

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TRAP staining and statistical analyses of numbers of osteoclasts in the trabecular bone from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=100 µm. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (D).


OCN statistical analyses of number of osteoblasts in femurs from Vehicle ( n = 6 5 independent biological replicates ) ROS ( n = 6 6 independent biological replicates ) treatment . Scale bar = 50 µm . TB : trabecular bone . Data were compared using an unpaired t ‐ ( * * indicates P < 0 . 01 ) , error bars are standard deviations . n = 5 independent microscopic vision fields in ( F ) .

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OCN staining and statistical analyses of the number of osteoblasts in femurs from Vehicle (n=6 and 5 independent biological replicates) and ROS (n=6 and 6 independent biological replicates) treatment. Scale bar=50 µm. TB: trabecular bone. Data were compared using an unpaired t‐test (** indicates P < 0.01), error bars are standard deviations. n=5 independent microscopic vision fields in (F).


( B ) Numbers of surface tumors per liver were counted . ( C ) cumulative diameter of surface tumors per liver per mouse reflects overall load .

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(B) Numbers of surface tumors per liver were counted. (C) The cumulative diameter of all surface tumors per liver per mouse reflects the overall tumor load.


( D ) of livers including mass relative to ( LW / BW ) was recorded .

(D) Weight of the livers including tumor mass relative to the body weight (LW/BW ratio) was recorded.


( E ) Representative pictures of total livers with tumors of indicated genotypes .

(E) Representative pictures of total livers with tumors of the indicated genotypes.


( A ) Immunoblots of murine wt non - tumorous tissue lysates were probed for CASP2 , RAIDD , p53 . HSP90 PonceauS served as loading controls .

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(A) Immunoblots of murine wt non-tumorous and tumor tissue lysates were probed for CASP2, RAIDD, and p53. HSP90 and PonceauS protein staining served as loading controls.


( B ) Proliferation of tissue was determined by of Ki67 - positive nuclei analyzed by flow cytometry . T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 . N - numbers refer to biological replicates .

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(B) Proliferation of tumor tissue was determined by the percentage of Ki67-positive nuclei analyzed by flow cytometry. T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5. N-numbers refer to biological replicates.


( D ) Immunoblots of experiment described in ( C ) at t ( 8h ) , t ( 48h ) , t ( weaning + 5d ) for CASP2 , RAIDD , p53 , γH2AX , HSP90 as loading control .

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(D) Immunoblots of the experiment described in (C) at t(8h), t(48h), and t(weaning+5d) for CASP2, RAIDD, p53, γH2AX, and HSP90 as loading control.


( E ) number of binucleated hepatocytes per field was counted on H & E stained paraffin sections of PBS treated wt Pidd1 - / - mice at t ( 0h ) at t ( 48h ) after PBS or DEN treatment . N - numbers refer to biological replicates .

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(E) The number of binucleated hepatocytes per field was counted on H&E stained paraffin sections of PBS treated wt and Pidd1-/- mice at t(0h) and at t(48h) after PBS or DEN treatment. N-numbers refer to biological replicates.


( F ) Immunofluorescence of same livers was perfomed for distal centriolar CP110 to stain centrioles , β - catenin for borders , DAPI to stain . Representative of a mononucleated hepatocyte with 2 centrioles a binucleated hepatocyte with 4 centrioles is shown .

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(F) Immunofluorescence of the same livers was perfomed for the distal centriolar protein CP110 to stain centrioles, β-catenin for cell borders, and DAPI to stain DNA. Representative images of a mononucleated hepatocyte with 2 centrioles and a binucleated hepatocyte with 4 centrioles is shown.


( G ) Centriole number was quantified with respect to number of nuclei per in wt Pidd1 - / - livers at time points described in ( E ) ; n = 3 biological replicates .

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(G) Centriole number was quantified with respect to the number of nuclei per cell in wt and Pidd1-/- livers at the time points described in (E); n=3 biological replicates.


( A ) Binucleation was assessed blinded in H & E stained sections of murine non - ( NT ) tissue ( T ) T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates .

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(A) Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.


Binucleation was assessed blinded in H & E stained sections of murine non - ( NT ) tissue ( T ) also shown in ( B ) . T : wt n = 14 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 10 ; NT : wt n = 8 , Casp - 2 - / - n = 10 , Raidd - / - n = 8 , Pidd1 - / - n = 9 . N - numbers refer to biological replicates .

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Binucleation was assessed blinded in H&E stained sections of murine non-tumor (NT) and tumor tissue (T) also shown in (B). T: wt n=14, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=10; NT: wt n=8, Casp-2-/- n=10, Raidd-/- n=8, Pidd1-/- n=9. N-numbers refer to biological replicates.


C , D ( C ) Representative histograms of nuclei stained for , which were isolated from non - tissue of wt Casp2 - / - mice . These are marked as red " x " in shown in ( D ) were degree of polyploidy is reflected as of octaploid nuclei ( T : wt n = 17 , Casp - 2 - / - n = 11 , Raidd - / - n = 10 , Pidd1 - / - n = 8 ; NT : wt n = 12 , Casp - 2 - / - n = 11 , Raidd - / - n = 7 , Pidd1 - / - n = 5 ) . N - numbers refer to biological replicates .

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C, D (C) Representative histograms of nuclei stained for DNA, which were isolated from non-tumor and tumor tissue of wt and Casp2-/- mice. These animals are marked as red "x" in the quantification shown in (D) were the degree of polyploidy is reflected as percentage of octaploid nuclei (T: wt n=17, Casp-2-/- n=11, Raidd-/- n=10, Pidd1-/- n=8; NT: wt n=12, Casp-2-/- n=11, Raidd-/- n=7, Pidd1-/- n=5). N-numbers refer to biological replicates.


Copy number variations were determined using whole genome sequencing of 30 nuclei " mini - bulks " isolated from Casp2 - / - or wt tumors ( n = 11 per genotype ) . Diploid tetraploid pools of nuclei were analyzed separately . A , B ( A ) Representative dot plots show degree of aneuploidy heterogeneity are quantified in ( B , empty symbols ) . Data are represented as median , statistical significance determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 . N - numbers refer to biological replicates .

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Copy number variations were determined using whole genome sequencing of 30 nuclei "mini-bulks" isolated from Casp2-/- or wt tumors (n=11 per genotype). Diploid and tetraploid pools of tumor cell nuclei were analyzed separately. A, B (A) Representative dot plots show the degree of aneuploidy and heterogeneity and are quantified in (B, empty symbols). Data are represented as median, statistical significance determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01. N-numbers refer to biological replicates.


( A ) Matched human non - ( NT ) tissue ( T ) samples were analyzed by immunoblotting for CASP2 , PIDD1 RAIDD on membranes a b . PonceauS serves as loading control . asterisk refers to an unspecific band , " C " " CC " indicate functionally active fragments of PIDD1 generated by autoprocessing , PIDD - C PIDD - CC . Densitometric of these blots is shown in Fig . EV4A .

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(A) Matched human patient non-tumor (NT) and tumor tissue (T) samples were analyzed by immunoblotting for CASP2, PIDD1 and RAIDD on membranes a and b. PonceauS protein staining serves as loading control. The asterisk refers to an unspecific band, "C" and "CC" indicate the functionally active fragments of PIDD1 generated by autoprocessing, PIDD-C and PIDD-CC. Densitometric quantification of these blots is shown in Fig. EV4A.


( B ) mRNA expression of PIDDosome components in TCGA Provisional LIHC data set across disease stages . are divided on p53 mutation / deletion ( mut / del ) status . Statistical significance was determined using two - way ANOVA with Tukey ' s post - hoc . central band represents median , boxes indicate 25th 75th percentile , whiskers are 5th 95th percentiles , statistical significance was defined as * p ≤ 0 . 05 , * * p ≤ 0 . 01 .

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(B) mRNA expression analysis of the PIDDosome components in the TCGA Provisional LIHC data set across disease stages. Patients are divided based on the p53 mutation/deletion (mut/del) status. Statistical significance was determined using two-way ANOVA with Tukey's post-hoc analysis. The central band represents the median, the boxes indicate the 25th and 75th percentile, the whiskers are the 5th and 95th percentiles, statistical significance was defined as * p≤0.05, ** p≤0.01.


( C ) same data set was analyzed for recurrence - free survival on high or low expression of CASP2 , RAIDD or PIDD1 divided at median . Statistical significance was determined using a Log - rank .

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(C) The same data set was analyzed for recurrence-free survival based on high or low expression of CASP2, RAIDD or PIDD1 divided at the median. Statistical significance was determined using a Log-rank test.


( B ) E2F target genes expression was analyzed for correlation with expression of CASP2 , RAIDD PIDD1 using Spearman ' s correlation coefficient . central band represents median , boxes indicate 2nd 3rd quartile , whiskers are 1 . 5 times interquartile range ( n = 200 genes ) .

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(B) E2F target genes expression was analyzed for correlation with expression of CASP2, RAIDD and PIDD1 using Spearman's correlation coefficient. The central band represents the median, the boxes indicate the 2nd and 3rd quartile, and the whiskers are 1.5 times the interquartile range (n=200 genes).


Correlation ( Pearson ) of CASP2 mRNA with transcript of Ki67 in TCGA data set .

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Correlation (Pearson) of CASP2 mRNA with transcript levels of Ki67 in the TCGA data set.


Correlation ( Pearson ) of RAIDD PIDD1 mRNA with transcript of Ki67 in TCGA data set .

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Correlation (Pearson) of RAIDD and PIDD1 mRNA with transcript levels of Ki67 in the TCGA data set.


( A ) ploidy index is inferred as 1 / density of tumorous ( T ) non - tumorous ( NT ) tissue of HCC with different liver diseases . ALD - alcoholic liver disease ( n = 65 ) ; HBV - hepatitis B infection ( n = 24 ) ; HCV - hepatitis C infection ( n = 85 ) ; NASH - non - alcoholic steatohepatitis ( n = 33 ) . Statistical significance was determined by one - way ANOVA with multiplicity correction ( Sidak - Holm ) ; * p ≤ 0 . 05 , * * p ≤ 0 . 01 , * * * p ≤ 0 . 001 . n - numbers refer to number of samples analyzed .

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(A) The ploidy index is inferred as 1/cell density of tumorous (T) and non-tumorous (NT) tissue of HCC patients with different liver diseases. ALD - alcoholic liver disease (n=65); HBV - hepatitis B virus infection (n=24); HCV - hepatitis C virus infection (n=85); NASH - non-alcoholic steatohepatitis (n=33). Statistical significance was determined by one-way ANOVA with multiplicity correction (Sidak-Holm); * p≤0.05, ** p≤0.01, *** p≤0.001. n-numbers refer to the number of patient samples analyzed.


( B ) were grouped according to HCC density as surrogate for ploidy . Data were divided at median exact numbers are shown in graphs . Median recurrence - free survival ( RFS ) for a period of 10 years for with low ploidy was 4 . 8 years . For with high ploidy median RFS could not be defined as more than 50 % of survived without relapse . characteristics are shown in Appendix table S1 . A Log - rank was used to determine statistical significance .

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(B) All patients were grouped according to HCC tumor cell density as surrogate for tumor ploidy. Data were divided at the median and exact patient numbers are shown in the graphs. Median recurrence-free survival (RFS) for a period of 10 years for patients with low tumor ploidy was 4.8 years. For patients with high tumor ploidy the median RFS could not be defined as more than 50% of the patients survived without relapse. Patient characteristics are shown in Appendix table S1. A Log-rank test was used to determine statistical significance.


A Baseline blood of granulocytes . One - way ANOVA ; n = 8 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

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A Baseline blood counts of granulocytes. One-way ANOVA; n = 8 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


B Flow cytometry of neutrophils in digested hearts . representative dot plots show gating strategy for cardiacneutrophils ( CD45 + Ly6G + CD11b + ) at ZT5 . One - way ANOVA ; n = 5 mice per ZT ; * P = 0 . 0001 ZT5 vs . ZT13 .

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B Flow cytometry quantification of neutrophils in digested hearts. The representative dot plots show the gating strategy for cardiacneutrophils (CD45+Ly6G+CD11b+) at ZT5. One-way ANOVA; n = 5 mice per ZT; *P = 0.0001 ZT5 vs. ZT13.


C Representative immunostainings for neutrophils in myocardium ( left ventricle ) , identified as Ly6G - positive ( 20x magnification ) .

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C Representative immunostainings for neutrophils in the myocardium (left ventricle), identified as Ly6G-positive (20x magnification).


D Cardiac mRNA expression normalized to HPRT . One - way ANOVA ; n = 5 mice per ZT ; ZT5 vs . ZT17 : * P = 0 . 0064 ( CXCL1 ) , * P = 0 . 0007 ( CXCL2 ) , * P = 0 . 0007 ( CXCL5 ) , * P = 0 . 0001 ( ICAM - 1 ) , * P = 0 . 0009 ( VCAM - 1 ) , * P = 0 . 0181 ( CCL3 ) , * P = 0 . 0360 ( CCL5 ) .

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D Cardiac mRNA expression levels normalized to HPRT. One-way ANOVA; n = 5 mice per ZT; ZT5 vs. ZT17: *P = 0.0064 (CXCL1), *P = 0.0007 (CXCL2), *P = 0.0007 (CXCL5), *P = 0.0001 (ICAM-1), *P = 0.0009 (VCAM-1), *P = 0.0181 (CCL3), *P = 0.0360 (CCL5).


E Mean ( MFI ) of CXCR2 expression by neutrophils in blood heart . One - way ANOVA ; n = 3 mice for ZT1 , ZT17 , ZT21 n = 5 for ZT5 , ZT9 , ZT13 ; ZT5 vs . ZT13 : * P = 0 . 0425 ( blood ) , * P = 0 . 0078 ( heart ) .

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E Mean fluorescence intensity (MFI) of CXCR2 expression by neutrophils in blood and heart. One-way ANOVA; n = 3 mice for ZT1, ZT17, ZT21 and n = 5 for ZT5, ZT9, ZT13; ZT5 vs. ZT13: *P = 0.0425 (blood), *P = 0.0078 (heart).


A Flow cytometry of neutrophils in hearts representative immunostainings for neutrophils in infarct , identified as Ly6G - positive ( 5x 20x magnifications ) . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 3 for 24h post MI n = 3 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0161 ( 12h MI ) , * P = 0 . 003 ( 24h MI ) .

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A Flow cytometry analysis of neutrophils in hearts and representative immunostainings for neutrophils in the infarct area, identified as Ly6G-positive (5x and 20x magnifications). Two-way ANOVA followed by Bonferroni post-hoc test; n=5 mice for no MI, n = 5 for 12h post MI, n = 3 for 24h post MI and n = 3 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0161 (12h MI), *P = 0.003 (24h MI).


B Flow cytometry of neutrophils in bone marrow . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice for no MI , n = 5 for 12h post MI , n = 4 for 24h post MI n = 4 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0471 ( 12h MI ) , * P = 0 . 0035 ( 24h MI ) .

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B Flow cytometry analysis of neutrophils in bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice for no MI, n = 5 for 12h post MI, n = 4 for 24h post MI and n = 4 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0471 (12h MI), *P = 0.0035 (24h MI).


C Blood of granulocytes . Two - way ANOVA followed by Bonferroni post - hoc ; n = 9 mice for no MI , n = 10 for 12h post MI , n = 13 for 24h post MI n = 10 for 72h post MI in both ZT groups ; ZT5 vs . ZT13 : * P = 0 . 0364 ( 24h MI ) .

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C Blood counts of granulocytes. Two-way ANOVA followed by Bonferroni post-hoc test; n= 9 mice for no MI, n = 10 for 12h post MI, n = 13 for 24h post MI and n = 10 for 72h post MI in both ZT groups; ZT5 vs. ZT13: *P = 0.0364 (24h MI).


D Flow cytometry of neutrophils in hearts bone marrow under inverted cycle conditions . Two - way ANOVA followed by Bonferroni post - hoc ; n = 3 mice for no MI in both ZT groups , n = 5 for ZT5 n = 6 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0147 ( 24h MI ) .

D Flow cytometry analysis of neutrophils in hearts and bone marrow under inverted light cycle conditions. Two-way ANOVA followed by Bonferroni post-hoc test; n = 3 mice for no MI in both ZT groups, n = 5 for ZT5 and n = 6 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0147 (24h MI).


E Representative gating strategy for GMPs in bone marrow , identified as identified as lineage negative ( CD11b - , Gr1 - , B220 - , CD3 - Ter119 - ) Sca - 1 - , c - kit + , CD16 / 32 + , CD34 + . Flow cytometry of GMP in bone marrow . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice for no MI 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

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E Representative gating strategy for GMPs in the bone marrow, identified as identified as lineage negative (CD11b-, Gr1-, B220-, CD3- and Ter119-) and Sca-1-, c-kit+, CD16/32+, CD34+. Flow cytometry quantification of GMP in the bone marrow. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for no MI and 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


F CXCL12 in bone marrow lavage . Two - way ANOVA followed by Bonferroni post - hoc ; n = 7 mice for no MI n = 6 for 24h post MI in both groups ; ZT5 vs . ZT13 : * P = 0 . 0077 ( no MI ) , * P = 0 . 0013 ( 24h MI ) .

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F CXCL12 levels in bone marrow lavage. Two-way ANOVA followed by Bonferroni post-hoc test; n = 7 mice for no MI and n = 6 for 24h post MI in both groups; ZT5 vs. ZT13: *P = 0.0077 (no MI), *P = 0.0013 (24h MI).


G Plasma of pro - inflammatory cytokines chemokines . Two - way ANOVA followed by Bonferroni post - hoc ; n = 11 mice for no MI in both ZT groups , n = 7 for ZT5 n = 8 for ZT13 24h post MI ; ZT5 vs . ZT13 : * P = 0 . 0271 ( CXCL12 , no MI ) , * P = 0 . 0108 ( TNFα , 24h MI ) , * P = 0 . 001 ( G - CSF , 24h MI ) , * P = 0 . 005 ( CXCL1 , 24h MI ) , * P = 0 . 0005 ( CXCL2 , 24h MI ) , * P = 0 . 0016 ( CCL3 24h MI ) , * P = 0 . 0144 ( CCL5 , 24h MI ) .

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

G Plasma levels of pro-inflammatory cytokines and chemokines. Two-way ANOVA followed by Bonferroni post-hoc test; n = 11 mice for no MI in both ZT groups, n = 7 for ZT5 and n = 8 for ZT13 24h post MI; ZT5 vs. ZT13: *P = 0.0271 (CXCL12, no MI), *P = 0.0108 (TNFα, 24h MI), *P = 0.001 (G-CSF, 24h MI), *P = 0.005 (CXCL1, 24h MI), *P = 0.0005 (CXCL2, 24h MI), *P = 0.0016 (CCL3 24h MI), *P = 0.0144 (CCL5, 24h MI).


A Permanent LAD occlusion was perfomed at ZT5 or ZT13 . TTC ( , infarct ; red , vital myocardium ) of infarct normalized to left ventricle ( LV ) . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0041 .

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A Permanent LAD occlusion was perfomed at ZT5 or ZT13. TTC staining (white, infarct; red, vital myocardium) and quantification of infarct size normalized to the left ventricle (LV). Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0041.


B Plasma troponin I 24 hours after MI . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0007 .

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B Plasma troponin I levels 24 hours after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0007.


C Flow cytometry of dead cardiomyocytes ( CD45 - , zombie + ) 24 hours after MI . Student ` s t - ; n = 3 mice in both groups ; * P = 0 . 0072 .

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C Flow cytometry analysis of dead cardiomyocytes (CD45-, zombie+) 24 hours after MI. Student`s t-test; n = 3 mice in both groups; *P = 0.0072.


D Survival rates after MI cause of . Log - rank ; n = 87 mice in both groups ; * P = 0 . 0006 .

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D Survival rates after MI and cause of death. Log-rank test; n = 87 mice in both groups; *P = 0.0006.


E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin ( αSMA ) as between stained total of random fields . Student ` s t - ; n = 4 mice in both groups ; * P = 0 . 0134 .

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E Myofibroblasts within infarcts were quantified by alpha smooth muscle actin (αSMA) staining as ratio between stained and total area of random fields. Student`s t-test; n = 4 mice in both groups; *P = 0.0134.


F Masson ´ s trichome of fibrosis ( blue , collagen ; red , vital myocardium ) relative to total LV ( * P = 0 . 0095 ) as well as LV anterior wall thickness ( * P = 0 . 0068 ) 7 days after MI . Student ` s t - ; n = 6 mice for ZT5 n = 7 for ZT13 MI .

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F Masson´s trichome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total LV (*P = 0.0095) as well as LV anterior wall thickness (*P = 0.0068) 7 days after MI. Student`s t-test; n = 6 mice for ZT5 and n = 7 for ZT13 MI.


G of relative collagen type I content identified by Sirius Redstaining 7 days after MI . Student ` s t - ; n = 4 mice for ZT5 n = 5 for ZT13 MI ; * P = 0 . 0175 .

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G Analysis of relative collagen type I content identified by Sirius Redstaining 7 days after MI. Student`s t-test; n = 4 mice for ZT5 and n = 5 for ZT13 MI; *P = 0.0175.


H Echocardiography assessment of ejection fraction ( EF ) , end - systolic - volume ( ESV ) end - diastolic - volume ( EDV ) . Two - way ANOVA ; n = 6 mice for no MI for both groups , n = 6 for ZT5 n = 9 for ZT13 at 72h post MI , n = 8 for both groups at 7d post MI n = 7 for ZT5 n = 5 for ZT13 14d post MI ; ZT5 vs . ZT13 : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0042 ( EF , 7d ) , * P = 0 . 0121 ( EF , 14d ) ; * P = 0 . 0253 ( ESV , 3d ) , * P = 0 . 0421 ( ESV , 7d ) , * P = 0 . 0005 ( ESV , 14d ) ; * P = 0 . 0053 ( EDV , 14d ) .

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H Echocardiography assessment of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV). Two-way ANOVA; n = 6 mice for no MI for both groups, n = 6 for ZT5 and n = 9 for ZT13 at 72h post MI, n = 8 for both groups at 7d post MI and n = 7 for ZT5 and n = 5 for ZT13 14d post MI; ZT5 vs. ZT13: *P = 0.0001 (EF, 3d), *P = 0.0042 (EF, 7d), *P = 0.0121 (EF, 14d); *P = 0.0253 (ESV, 3d), *P = 0.0421 (ESV, 7d), *P = 0.0005 (ESV, 14d); *P = 0.0053 (EDV, 14d).


B Flow cytometry of cardiacneutrophils 24 hours after ZT13 MI . dotted indicates cardiacneutrophil 24h after ZT5 MI , shown in Fig . 2A . Student ` s t - ; n = 3 mice for isotype n = 5 for Ly6G injected mice ; * P = 0 . 0004 .

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B Flow cytometry analysis of cardiacneutrophils 24 hours after ZT13 MI. The dotted line indicates cardiacneutrophil counts 24h after ZT5 MI, shown in Fig. 2A. Student`s t-test; n = 3 mice for isotype and n = 5 for Ly6G injected mice; *P = 0.0004.


C Plasma TNFα 24 hours after ZT13 MI . Student ` s t - . n = 8 independent samples for isotype n = 6 for Ly6G injected mice ; * P = 0 . 0019 .

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C Plasma TNFα levels 24 hours after ZT13 MI. Student`s t-test. n=8 independent samples for isotype and n=6 for Ly6G injected mice; *P = 0.0019.


D Infarct relative to left ventricular ( LV ) plasmatroponin I 24 hours after ZT5 or ZT13 MI . Student ` s t - ; For infarct n = 3 mice in both groups for ZT5 n = 4 mice for isotype n = 3 mice for Ly6G at ZT13 ; Isotype vs . Ly6G : * P = 0 . 0158 ( ZT13 ) . For troponin n = 8 mice for isotype n = 6 mice for Ly6G with ZT13 MI ; * P = 0 . 0012 .

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D Infarct size relative to left ventricular area (LV) and plasmatroponin I levels 24 hours after ZT5 or ZT13 MI. Student`s t-test; For infarct size n = 3 mice in both groups for ZT5 and n = 4 mice for isotype and n = 3 mice for Ly6G at ZT13; Isotype vs. Ly6G: *P = 0.0158 (ZT13). For troponin levels n = 8 mice for isotype and n = 6 mice for Ly6G with ZT13 MI; *P = 0.0012.


E Masson ' s - trichrome of fibrosis ( blue , collagen ; red , vital myocardium ) relative to total of LV ( * P = 0 . 0084 ) ; morphometric of LV anterior wall thickness ( * P = 0 . 0027 ) 7 days after ZT13 MI . Student ` s t - ; n = 5 mice for isotype n = 4 mice for Ly6G .

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

E Masson's-trichrome staining of fibrosis (blue, collagen; red, vital myocardium) and quantification relative to total area of the LV (*P = 0.0084); morphometric quantification of the LV anterior wall thickness (*P = 0.0027) 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G.


F of collagen type I within infarcts identified by Sirius Red 7 days after ZT13 MI . Student ` s t - ; n = 5 mice for isotype n = 4 mice for Ly6G ; * P = 0 . 0375 .

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F Analysis of collagen type I fibers within infarcts identified by Sirius Red staining 7 days after ZT13 MI. Student`s t-test; n = 5 mice for isotype and n = 4 mice for Ly6G; *P = 0.0375.


G Echocardiography measurement of ejection fraction ( EF ) , end - systolic - volume ( ESV ) end - diastolic - volume ( EDV ) before after ZT13 MI . Two - way ANOVA ; n = 6 mice for no MI in both groups , n = 9 for isotype n = 5 for Ly6G at 72h post MI , n = 8 for isotype n = 4 for Ly6G at 7d post MI . Isotype vs . Ly6G : * P = 0 . 0001 ( EF , 3d ) , * P = 0 . 0227 ( EF , 7d ) ; MI * P = 0 . 0133 ( ESV , 3d ) , * P = 0 . 0004 ( ESV , 7d ) ; * P = 0 . 0017 ( EDV , 7d ) .

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

G Echocardiography measurement of ejection fraction (EF), end-systolic-volume (ESV) and end-diastolic-volume (EDV) before and after ZT13 MI. Two-way ANOVA; n = 6 mice for no MI in both groups, n = 9 for isotype and n = 5 for Ly6G at 72h post MI, n = 8 for isotype and n = 4 for Ly6G at 7d post MI. Isotype vs. Ly6G: *P = 0.0001 (EF, 3d), *P = 0.0227 (EF, 7d); MI *P = 0.0133 (ESV, 3d), *P = 0.0004 (ESV, 7d); *P = 0.0017 (EDV, 7d).


A Representative flow cytometry of CXCR2highneutrophils in blood after ZT5 ZT13 MI . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice for time points in both groups ; ZT5 vs . ZT13 : * P = 0 . 0001 ( 4h ) , * P = 0 . 0001 ( 8h ) , * P = 0 . 0447 ( 12h ) .

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

A Representative flow cytometry analysis and quantification of CXCR2highneutrophils in the blood after ZT5 and ZT13 MI. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice for all time points in both groups; ZT5 vs. ZT13: *P = 0.0001 (4h), *P = 0.0001 (8h), *P = 0.0447 (12h).


B mean ( MFI ) of CXCX2 expression by cardiacneutrophils 12h post - MI after ZT5 ZT13 MI . Student ` s t - ; n = 4 mice in both groups ; * P = 0 . 0001 .

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

B Percentage and mean fluorescence intensity (MFI) of CXCX2 expression by cardiacneutrophils 12h post-MI after ZT5 and ZT13 MI. Student`s t-test; n = 4 mice in both groups; *P = 0.0001.


C of CXCR2highneutrophils in blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0005 ( ZT13 ) .

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C Percentage of CXCR2highneutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0005 (ZT13).


D Flow cytometry of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0079 ( ZT13 ) .

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D Flow cytometry quantification of neutrophils in hearts 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0079 (ZT13).


E Flow cytometry of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 4 mice in both groups ; DMSO vs . SB225002 : * P = 0 . 0002 ( ZT13 ) .

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E Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 4 mice in both groups; DMSO vs. SB225002: *P = 0.0002 (ZT13).


F Mean ( MFI ) of CXCX2 expression at ZT5 in blood 24h after MI in wildtype ( WT ) CXCR2 KO mice . Student ` s t - . N = 6 mice in both groups ; * P = 0 . 0001 .

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F Mean fluorescence intensity (MFI) of CXCX2 expression at ZT5 in the blood 24h after MI in wildtype (WT) and CXCR2 KO mice. Student`s t-test. N = 6 mice in both groups; *P = 0.0001.


G of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT CXCR2 KO mice . Two - way ANOVA followed by Bonferroni post - hoc ; for ZT5 n = 6 mice in both groups , for ZT13 n = 7 WT mice n = 5 CXCR2 KO mice ; WT vs . CXCR2 KO : * P = 0 . 0001 ( ZT13 ) .

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G Percentage of CXCR2hi bloodneutrophils 24h after ZT5 or ZT13 MI in WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0001 (ZT13).


H Flow cytometry of neutrophils in hearts 24h after MI in ZT5 ZT13 operated WT CXCR2 KO mice . Two - way ANOVA followed by Bonferroni post - hoc ; for ZT5 n = n = 6 mice in both groups , for ZT13 n = 7 WT mice n = 5 CXCR2 KO mice ; WT vs . CXCR2 KO : * P = 0 . 0461 ( ZT13 ) , ns = not significant .

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H Flow cytometry quantification of neutrophils in hearts 24h after MI in ZT5 and ZT13 operated WT and CXCR2 KO mice. Two-way ANOVA followed by Bonferroni post-hoc test; for ZT5 n= n = 6 mice in both groups, for ZT13 n = 7 WT mice and n = 5 CXCR2 KO mice; WT vs. CXCR2 KO: *P = 0.0461 (ZT13), ns = not significant.


B of CXCR2hineutrophils in blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 . 0046 ( ZT13 ) ; ZT5 vs . ZT13 : * P = 0 . 0153 ( DMSO ) .

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B Percentage of CXCR2hineutrophils in the blood 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0046 (ZT13); ZT5 vs. ZT13: *P = 0.0153 (DMSO).


C Flow cytometry of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 . 0006 ( ZT13 ) ; ZT5 vs . ZT13 : * P = 0 . 0001 ( DMSO ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry quantification of cardiacneutrophils 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0.0006 (ZT13); ZT5 vs. ZT13: *P = 0.0001 (DMSO).


D Flow cytometry of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle . Two - way ANOVA followed by Bonferroni post - hoc ; n = 5 mice in both groups at ZT5 , n = 5 mice for vehicle n = 6 mice for SB225002 at ZT13 ; DMSO vs . SB225002 : * P = 0 , 0486 ( ZT13 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Flow cytometry quantification of neutrophils in bone marrow 24h after ZT5 or ZT13 MI in mice receiving CXCR2 antagonist SB225002 or vehicle. Two-way ANOVA followed by Bonferroni post-hoc test; n = 5 mice in both groups at ZT5, n = 5 mice for vehicle and n = 6 mice for SB225002 at ZT13; DMSO vs. SB225002: *P = 0,0486 (ZT13).


GPS reporter expressing peptide libraries ending with a random ( X ) or Gly ( G ) residue were treated with dominant - negative ( DN ) cullins analyzed . Data are presented as mean ± standard deviation from nine replicates . GPS reporter carrying indicated libraries were treated with shRNAs against various BC - box analyzed . VHL FEM1C serve as unrelated BC - box controls . Data are presented as mean ± standard deviation from nine replicates .

GPS reporter cells expressing peptide libraries ending with a random (X) or Gly (G) residue were treated with dominant-negative (DN) cullins and analyzed. Data are presented as mean ± standard deviation from nine replicates. GPS reporter cells carrying indicated libraries were treated with shRNAs against various BC-box proteins and analyzed. VHL and FEM1C serve as unrelated BC-box protein controls. Data are presented as mean ± standard deviation from nine replicates.


Comparisons of stability among wild - type mutants indicated on right .

Comparisons of protein stability among wild-type protein and the mutants indicated on the right.


( A ) relative frequency of Gly in final 50 residues of functional human with different subcellular localizations . number on each graph denotes left - p - value of - 1 residue . CT - inside CT - outside represent with their C - termini facing cytosol or extracellular space , respectively .

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(A) The relative frequency of Gly in the final 50 residues of functional human proteins with different subcellular localizations. The number on each graph denotes the left-tailed p-value of the -1 residue. CT-inside and CT-outside represent membrane proteins with their C-termini facing the cytosol or extracellular space, respectively.


( G ) Confocal of U2OS expressing MTSGLUL - GFP with or without a C - terminal diGly degron . MitoTracker shows mitochondria . Scale bar = 20 µm . ( H ) Confocal of U2OS expressing MTSGLUL - GFP - diGly degron fusion co - stained with MitoTracker , with or without DNCul2 treatment . Scale bar = 20 µm .

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(G) Confocal images of U2OS cells expressing MTSGLUL-GFP proteins with or without a C-terminal diGly degron. MitoTracker staining shows mitochondria. Scale bar = 20 µm. (H) Confocal images of U2OS cells expressing MTSGLUL-GFP-diGly degron fusion proteins co-stained with MitoTracker, and with or without DNCul2 treatment. Scale bar = 20 µm.


Stability of indicated in treated with shRNAs against various BC - box . Since GFP was tagged at N - terminus signal peptide MTSs need to be exposed to be functional , tested were mislocalized . stability analyses of wild - type ( WT ) mutant lacking their extreme terminal Gly ( ∆ G ) or having it masked by adding Leu ( + L ) in with or without BC - box knockdown ( C ) or overexpression ( D ) .

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Stability analysis of indicated proteins in cells treated with shRNAs against various BC-box proteins. Since GFP was tagged at the protein N-terminus and signal peptide and MTSs need to be exposed to be functional, all tested proteins were mislocalized. Protein stability analyses of wild-type (WT) and mutant proteins lacking their extreme terminal Gly (∆G) or having it masked by adding Leu (+L) in cells with or without BC-box protein knockdown (C) or overexpression (D).


Fractionation Western blot of wild - type Flag - tagged PDP2 ( top ) , wild - type MTS - deleted ( ∆ MTS ) MRPL28 ( bottom ) . Flag - PDP2 ∆ MTS MRPL28 were mislocalized . W , C , M denote whole lysates , cytosol fraction mitochondrial fraction , respectively . Tubulin VDAC serve as fractionation quality controls .

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Fractionation and Western blot analysis of wild-type and Flag-tagged PDP2 (top), and wild-type and MTS-deleted (∆MTS) MRPL28 (bottom). Flag-PDP2 and ∆MTS MRPL28 were mislocalized. W, C, and M denote whole cell lysates, cytosol fraction and mitochondrial fraction, respectively. Tubulin and VDAC serve as fractionation quality controls.


abundance of HA - tagged wild - type or mutant secretory with their signal peptide deleted ( ∆ SP ) in with or without KLHDC3 or APPBP2 expression . HA epitope was inserted immediately after signal peptide ( SP ) , which had no impact on secretion .

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Protein abundance analysis of HA-tagged wild-type or mutant secretory proteins with their signal peptide deleted (∆SP) in cells with or without KLHDC3 or APPBP2 expression. The HA epitope was inserted immediately after the signal peptide (SP), which had no impact on protein secretion.


Fractionation Western blot of endogenous MIC19 in U2OS with or without DNKLHDC2 , DNCul2 or IMP - 1088 treatments . IMP - 1088 inhibits NMTs . C M denote cytosol mitochondrial fractions , respectively . Tubulin VDAC serve as fractionation quality controls . relative abundance of MIC19 / tubulin was normalized to that of lane 1 is indicated under each lane in red . Another replicate of this experiment is shown in Fig . EV3B .

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Fractionation and Western blot analysis of endogenous MIC19 in U2OS cells with or without DNKLHDC2, DNCul2 or IMP-1088 treatments. IMP-1088 inhibits NMTs. C and M denote cytosol and mitochondrial fractions, respectively. Tubulin and VDAC serve as fractionation quality controls. The relative abundance of cytosolic MIC19/tubulin was normalized to that of lane 1 and is indicated under each lane in red. Another replicate of this experiment is shown in Fig. EV3B.


Abundance comparison of GFP N - or C - terminal tagged with wild - type or mutant N - terminal ( aa . 1 - 24 ) or C - terminal ( a . a . 218 - 227 ) motif of MIC19 in with indicated treatments . Data from sh # 1 is shown here .

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Abundance comparison of GFP N- or C-terminal tagged with the wild-type or mutant N-terminal (aa. 1-24) or C-terminal (a.a. 218-227) motif of MIC19 in cells with indicated treatments. Data from sh#1 is shown here.


Fractionation of wild - type HA - MIC19 in U2OS treated with IMP - 1088 with or without inhibition of ZYG11B or KLHDC2 . normalized relative abundance of HA - MIC19 / tubulin is indicated under each lane in red .

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Fractionation analysis of wild-type HA-MIC19 in U2OS cells treated with IMP-1088 and with or without inhibition of ZYG11B or KLHDC2. The normalized relative abundance of cytosolic HA-MIC19/tubulin is indicated under each lane in red.


GST pull - down using expressing GST or GST - tagged wild - type or mutant KLHDC2 HA - tagged SDE2 , FAU or TUG .

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GST pull-down assay using cells expressing GST or GST-tagged wild-type or mutant KLHDC2 and HA-tagged SDE2, FAU or TUG.


Relative stability of GAPDH C - terminally tagged with last 12 residues of indicated with or without DNKLHDC2 treatment by GPS assays . - 3 , - 4 or - 5 residue of SARS - CoV NSP was mutated to Ala ( A ) as indicated . experiment was done in triplicate using three independently prepared to express DNKLHDC2 . Data are presented as mean value of GFP / RFP + / - standard deviation from triplicate experiments .

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Relative stability analysis of GAPDH C-terminally tagged with the last 12 residues of indicated proteins with or without DNKLHDC2 treatment by GPS assays. The -3, -4 or -5 residue of SARS-CoV NSP proteins was mutated to Ala (A) as indicated. The experiment was done in triplicate using three independently prepared viruses to express DNKLHDC2. Data are presented as the mean value of GFP/RFP +/- standard deviation from triplicate experiments.


a , Wild - type ( WT ) Park2 - / - BMDMs were infected with mCherry - expressing M . tuberculosis ( Mtb ) for 4 h immunostained using anti - parkin antibodies .

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a, Wild-type (WT) and Park2-/- BMDMs were infected with mCherry-expressing M. tuberculosis (Mtb) for 4 h and immunostained using anti-parkin antibodies.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h immunostained for polyubiquitin .

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b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin.


b , Wild - type BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h immunostained for polyubiquitin . c , of ubiquitin - positive M . tuberculosis from b . Results are means ± s . e . m . of three independent experiments ( * * P < 0 . 001 , paired Student ' s t - ) .

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b, Wild-type BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for polyubiquitin. c, Quantification of ubiquitin-positive M. tuberculosis from b. Results are means ± s.e.m. of three independent experiments (**P < 0.001, paired Student's t-test).


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h immunostained for polyubiquitin .

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d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin.


d , U937 human macrophages expressing a scrambled shRNA ( Control ) or one of two different shRNAs targeting PARK2 ( shRNA no . 1 , shRNA no . 2 ) were infected with mCherry - expressing M . tuberculosis for 12 h immunostained for polyubiquitin . e , of ubiquitin positive M . tuberculosis from d , results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , Student ' s t - ) . f , parkin actin expression in from e was determined by western blotting .

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d, U937 human macrophages expressing a scrambled shRNA (Control) or one of two different shRNAs targeting PARK2 (shRNA no. 1, shRNA no. 2) were infected with mCherry-expressing M. tuberculosis for 12 h and immunostained for polyubiquitin. e, Quantification of ubiquitin positive M. tuberculosis from d, results are means ± s.e.m. of three independent experiments (**P  0.005, Student's t-test). f, The parkin and actin expression in cells from e was determined by western blotting.


g , Park2 − / − BMDMs were transduced with lentivirus expressing BFP ( − ) , wild - type parkin , or two separate mutant parkin alleles ( T240R , P437L ) . were infected with M . tuberculosis for 4 h ubiquitin M . tuberculosis co - localization was quantified expressed relative to control BMDMs . Results are means ± s . e . m . of three independent experiments ( * * P 0 . 005 , paired Student ' s t - ) . h , parkin actin expression in from g was determined by western blotting .

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g, Park2−/− BMDMs were transduced with lentivirus expressing BFP (−), wild-type parkin, or two separate mutant parkin alleles (T240R, P437L). Cells were infected with M. tuberculosis for 4 h and ubiquitin and M. tuberculosis co-localization was quantified and expressed relative to control BMDMs. Results are means ± s.e.m. of three independent experiments (**P  0.005, paired Student's t-test). h, The parkin and actin expression in cells from g was determined by western blotting.


a , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h immunostained using linkage - specific ubiquitin antibodies . b , of K63 K48 ubiquitin - positive M . tuberculosis from a .

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a, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained using linkage-specific ubiquitin antibodies. b, Quantification of K63 and K48 ubiquitin-positive M. tuberculosis from a.


c , BMDMs were infected with mCherry - expressing M . tuberculosis for 4 h immunostained for NDP52 , p62 , phospho - TBK1 or NBR1 . d , of co - localization from c ( n = 3 per group , * * P 0 . 001 ) . errors , s . e . m .

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c, BMDMs were infected with mCherry-expressing M. tuberculosis for 4 h and immunostained for NDP52, p62, phospho-TBK1 or NBR1. d, Quantification of co-localization from c (n = 3 per group, **P  0.001). All errors, s.e.m.


a , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 4 h immunostained for LC3 ( LC3B antibody ) or ATG12 . b , of results from a ( n = 3 per group , * * P 0 . 001 ) . c , Western blot of LC3 ( LC3B antibody ) from lysates from a .

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a, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 4 h and immunostained for LC3 (LC3B antibody) or ATG12. b, Quantification of results from a (n = 3 per group, **P  0.001). c, Western blot analysis of LC3 (LC3B antibody) from cell lysates from a.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h immunostained for LAMP1 .

d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1.


d , BMDMs were infected with mCherry - expressing M . tuberculosis ( arrows ) for 6 h immunostained for LAMP1 . e , of results from d expressed relative to control BMDMs ( n = 3 per group , * * P 0 . 001 ) .

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d, BMDMs were infected with mCherry-expressing M. tuberculosis (arrows) for 6 h and immunostained for LAMP1. e, Quantification of results from d expressed relative to control BMDMs (n = 3 per group, **P  0.001).


f , BMDMs were infected with M . tuberculosis colony - forming units ( c . f . u . ) at t = 0 t = 16 h were determined by plating . Results were normalized to t = 0 ( n = 3 per group , * P < 0 . 02 ) .

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f, BMDMs were infected with M. tuberculosis and colony-forming units (c.f.u.) at t = 0 and t = 16 h were determined by plating. Results were normalized to t = 0 (n = 3 per group, *P < 0.02).


g , RAW264 . 7 macrophages transduced with lentivirus expressing green ( GFP ) or parkin were infected with M . tuberculosis c . f . u . were determined after 16 h . Results were normalized to GFP - expressing ( n = 3 per group , * * P < 0 . 0076 ) .

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g, RAW264.7 macrophages transduced with lentivirus expressing green fluorescent protein (GFP) or parkin were infected with M. tuberculosis and c.f.u. were determined after 16 h. Results were normalized to GFP-expressing cells (n = 3 per group, **P < 0.0076).


h , U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M . tuberculosis for 36 h c . f . u were determined . Results were normalized to t = 0 ( N = 3 per group , * P < 0 . 02 ) . errors , s . e . m .

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h, U937 human macrophages expressing either scrambled or PARK2 shRNAs were infected with M. tuberculosis for 36 h and c.f.u were determined. Results were normalized to t = 0 (N = 3 per group, *P< 0.02). All errors, s.e.m.


a , WT Park2 - / - mice were infected with wild - type M . tuberculosis by aerosol lung burdens were determined by plating ( means ± s . d . , n = 5 per group , * P < 0 . 02 by Student ' s t - ) .

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a, WT and Park2-/- mice were infected with wild-type M. tuberculosis by aerosol and lung bacterial burdens were determined by plating (means ± s.d., n = 5 per group, *P < 0.02 by Student's t-test).


b , Enumeration of liver spleenc . f . u . from mice infected in a , 21 days post - infection ( means ± s . d , n = 5 per group , * P < 0 . 03 by Student ' s t - ) .

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b, Enumeration of liver and spleenc.f.u. from mice infected in a, 21 days post-infection (means ± s.d, n = 5 per group, *P < 0.03 by Student's t-test).


c , Survival of M . tuberculosis - infected wild - type Park2 - / - mice ( n = 10 , * * P < 0 . 001 by log - rank ) .

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c, Survival of M. tuberculosis-infected wild-type and Park2-/- mice (n = 10, **P < 0.001 by log-rank test).


d , Immunohistochemistry of lung sections from infected mice21 days post - infection , scale bar , 50 µm .

d, Immunohistochemistry staining of lung sections from infected mice21 days post-infection, scale bar, 50 µm.


e , Immunohistochemistry of lung sections from a human with active tuberculosis . Scale bars , 500 µm ( × 4 ) , 50 µm ( × 40 ) .

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e, Immunohistochemistry staining of lung sections from a human patient with active tuberculosis. Scale bars, 500 µm (×4), 50 µm (×40).


f , Confocal microscopy of humanlung sections from e immunostained for M . tuberculosis parkin ; was visualized using DAPI ( 4 ′ , 6 - diamidino - 2 - phenylindole ) . Scale bars , 25 µm .

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f, Confocal microscopy images of humanlung sections from e immunostained for M. tuberculosis and parkin; DNA was visualized using DAPI (4′,6-diamidino-2-phenylindole). Scale bars, 25 µm.


g , Mice were infected with wild - type L . monocytogenes by i . p . injection burdens in livers spleens were determined by plating ( means ± s . d . , n = 7 per group , * P < 0 . 04 by Student ' s t - ) .

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g, Mice were infected with wild-type L. monocytogenes by i.p. injection and bacterial burdens in livers and spleens were determined by plating (means ± s.d., n = 7 per group, *P < 0.04 by Student's t-test).


a , Wild - type two independent parkin - deficient D . melanogaster lines ( parkc00062 , parkf01950 ) were infected with L . monocytogenes by anterior abdomen injection . Atg8 processing was monitored in whole - fly lysates by western blotting .

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a, Wild-type and two independent parkin-deficient D. melanogaster lines (parkc00062, parkf01950) were infected with L. monocytogenes by anterior abdomen injection. Atg8 processing was monitored in whole-fly protein lysates by western blotting.


b , L . monocytogenes c . f . u . from a were determined by plating ( means ± s . d . , n = 3 - 5 , * * P < 0 . 001 by Student ' s t - ) .

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b, L. monocytogenes c.f.u. from a were determined by plating (means ± s.d., n = 3-5, **P < 0.001 by Student's t-test).


c , Survival of D . melanogaster was determined ( n = 33 , * * P < 0 . 001 by log - rank ) .

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c, Survival of D. melanogaster was determined (n = 33, **P < 0.001 by log-rank test).


d , e , Flies were infected with S . enterica serovar Typhimurium c . f . u . ( d ) ( means ± s . d . , n = 3 - 5 per group , * * P 0 . 009 by Student ' s t - ) fly survival ( e ) were determined ( n = 59 , * * P 0 . 001 by log - rank ) .

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d, e, Flies were infected with S. enterica serovar Typhimurium and bacterial c.f.u. (d) (means ± s.d., n = 3-5 per group, **P  0.009 by Student's t-test) and fly survival (e) were determined (n = 59, **P  0.001 by log-rank test).


f , Survival of flies infected with M . marinum ( n = 37 , * * P 0 . 0045 by log - rank ) .

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f, Survival of flies infected with M. marinum (n = 37, **P  0.0045 by log-rank test).


Immunofluorescence of U2OS expressing FLAG - HA - FAM134 after 24hrs doxycycline induction stained for HA ( green ) endogenous calnexin ( CANX ; red ) . Scale bar : 20μm .

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Immunofluorescence images of U2OS cells expressing FLAG-HA-FAM134 after 24hrs doxycycline induction stained for HA (green) and endogenous calnexin (CANX; red). Scale bar: 20μm.


Lysates of HEK293T transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) purified GST , wild type GST - LC3s , GST - GABARAPs , GST - Ubiquitin ( Ub ) , GST - Tetra - Ubiquitin , A representative Ponceau is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) purified GST, wild type GST-LC3s, GST-GABARAPs, GST-Ubiquitin (Ub), GST-Tetra-Ubiquitin, A representative Ponceau staining is shown.


Lysates of HEK293T transiently transfected with HA - tagged wild - type wild - type or ∆ LIR mutant FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) A representative Ponceau is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type and wild-type or ∆LIR mutant FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) A representative Ponceau staining is shown.


Lysates of HEK293T transiently transfected with HA - tagged wild - type FAM134 genes were subjected to pulldown experiments ( representative data ; WB detection against HA ; n = 3 ) using GST - LC3B GST - LC3B F52A - V53A as baits . A representative Ponceau is shown .

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Lysates of HEK293T cells transiently transfected with HA-tagged wild-type FAM134 genes were subjected to pulldown experiments (representative data; WB detection against HA; n=3) using GST-LC3B and GST- LC3B F52A-V53A as baits. A representative Ponceau staining is shown.


Immunofluorescence of U2OS expressing wild - type or ∆ LIR mutant FLAG - HA - FAM134 after 24hrs doxycycline induction under different conditions ( basal = DMSO , Baf . A1 = 2h 200nM Bafilomycin A1 , EBSS = 2h starvation in EBSS ) Scale bar : 10μm ; against FAM134 ( HA ; green ) endogenous LC3B ( red ) .

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Immunofluorescence images of U2OS expressing wild-type or ∆LIR mutant FLAG-HA-FAM134 proteins after 24hrs doxycycline induction under different conditions (basal=DMSO, Baf.A1=2h 200nM Bafilomycin A1, EBSS=2h starvation in EBSS) Scale bar: 10μm; staining against FAM134 (HA; green) and endogenous LC3B (red).


Small sections of sample pictures of U2OS stably expressing ER - phagy reporter ssRFP - GFP - KDEL . Pictures were obtained by IncuCyte ® S3 ( 10x ) show overlaid GFP RFP signal from DMSO Torin1 treated over time . Scale bar 25µm . Time lapse of representative total image views can be found in Movie EV7 Movie EV8 .

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Small sections of sample pictures of U2OS cells stably expressing the ER-phagy reporter ssRFP-GFP-KDEL. Pictures were obtained by the IncuCyte® S3 (10x) and show the overlaid GFP and RFP signal from DMSO and Torin1 treated cells over time. Scale bar 25µm. Time lapse of representative total image views can be found in Movie EV7 and Movie EV8.


Representative Western blot of total lysates of U2OS overexpressing indicated FAM134 upon 24hrs Dox induction . Autophagy flux was induced blocked by treatment with Torin1 ( 8hrs ) Bafilomycin A1 ( Baf . A1 , 8hrs ) , respectively . n =

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Representative Western blot of total cell lysates of U2OS cells overexpressing indicated FAM134 proteins upon 24hrs Dox induction. Autophagy flux was induced and blocked by treatment with Torin1 (8hrs) and Bafilomycin A1 (Baf.A1, 8hrs), respectively. n=3


Representative Western blot for Fam134 in single knockout mouse embryonic fibroblasts ( MEFs ) .

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Representative Western blot for Fam134 proteins in single knockout mouse embryonic fibroblasts (MEFs).


Western blot for Fam134 in indicated MEFs , expressing ER - phagy reporter ssRFP - GFP - KDEL being reconstituted with respective wild - type or ∆ LIR mutant Fam134 .

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Western blot for Fam134 proteins in indicated MEFs, expressing the ER-phagy reporter ssRFP-GFP-KDEL and being reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein.


Profile plots showing Log2 LFQ ( G ) representative immunofluorescence ( H ) of Climp63 ( green ) in wild - type Fam134 knockout MEFs .

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Profile plots showing the Log2 LFQ intensity (G) and representative immunofluorescence (H) of Climp63 (green) in wild-type and Fam134 knockout MEFs.


Profile plots showing Log2 LFQ ( I ) representative immunofluorescence ( J ) for collagen I ( red ) expression in wild - type Fam134 knockout MEFs . Scale bar : 10μm . Inset scale bar : 5μm . Nuclei were stained with Hoechst 33342 .

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Profile plots showing the Log2 LFQ intensity (I) and representative immunofluorescence (J) for collagen I (red) expression in wild-type and Fam134 knockout MEFs. Scale bar: 10μm. Inset scale bar: 5μm. Nuclei were stained with Hoechst 33342.


Electron microscopy of wild - type Fam134 single knockout MEFs ( ER = endoplasmic reticulum ) . Scale bar : 500nm .

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Electron microscopy of wild-type and Fam134 single knockout MEFs (ER=endoplasmic reticulum). Scale bar: 500nm.


Immunofluorescence stained for endogenous Calnexin ( Canx ) or Climp63 in wild - type ( WT ) , Fam134 knockout MEFs , Fam134 knockout MEFs reconstituted with respective wild - type or ∆ LIR mutant Fam134 . Scale bar : 10μm . Inset scale bar : 5μm .

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Immunofluorescence images stained for endogenous Calnexin (Canx) or Climp63 in wild-type (WT), Fam134 knockout MEFs, and Fam134 knockout MEFs reconstituted with the respective wild-type or ∆LIR mutant Fam134 protein. Scale bar: 10μm. Inset scale bar: 5μm.


Representative Western blots of total lysate derived from MEFs used in ( B ) detected for endogenous Fam134 Actin , respectively .

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Representative Western blots of total cell lysate derived from MEFs used in (B) and detected for endogenous Fam134 and Actin, respectively.


Representative Western Blot of Collagen I in wild - type MEFs Fam134 knockout MEFs reconstituted for respective wild - type ( WT ) or ∆ LIR mutant Fam134 . Vinculin has been used as reference for calculation . statistical significance was estimated by unpaired Student ' s t . data are represented as mean ± s . e . m . of n = 3 biological experiments statistical significance is defined as * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Representative Western Blot of Collagen I in wild-type MEFs and Fam134 knockout MEFs reconstituted for the respective wild-type (WT) or ∆LIR mutant Fam134 protein. Vinculin has been used as reference for ratio calculation. The statistical significance was estimated by unpaired Student's t test. All data are represented as mean ± s.e.m. of n=3 biological experiments and the statistical significance is defined as * p<0.05, ** p<0.01, *** p<0.001.


Representative Western Blot ( D ) relative bar plot ( E ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type ∆ LIR Fam134a .

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

Representative Western Blot (D) and the relative bar plot (E) showing collagen expression in Fam134b knockout MEFs overexpressing wild type and the ∆LIR Fam134a protein.


Representative Western Blot ( F ) relative bar plot ( G ) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a Fam134c . Representative Western Blot ( H ) relative bar plot ( I ) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a Fam134b .

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

Representative Western Blot (F) and the relative bar plot (G) showing collagen expression in Fam134b knockout MEFs overexpressing wild type Fam134a and Fam134c. Representative Western Blot (H) and the relative bar plot (I) showing collagen expression in Fam134c knockout MEFs overexpressing wild type Fam134a and Fam134b.