( C ) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK - 461 , showing the 25th percentile ( bottom line of each box ) , median ( middle line of each box ) , 75th percentile ( top line of each box ) , maximum and minimum ( each whisker ) . Adenovirus expressing EGFP - CLIP - 170 ( adCLIP ) mutant was used ( WT , S311A , S311D ) . Numbers in the graph indicate n number of the cells from 2 independent experiments . Control ( - / - ) : n = 784 , MYK - 461 : n = 939 , CLIP WT : n = 656 , CLIP WT + MYK - 461 : n = 619 , CLIP S311A : n = 405 , CLIP S311A + MYK - 461 : n = 635 , CLIP S311D : n = 613 , CLIP S311D + MYK - 461 : n = 735 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using the Tukey method . * * , P < 0 . 01 versus Control , † † , P < 0 . 01 versus CLIP WT , n . s . , not significant .

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

(C) Box and whisker plots of the cell size of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK-461, showing the 25th percentile (bottom line of each box), median (middle line of each box), 75th percentile (top line of each box), maximum and minimum (each whisker). Adenovirus expressing EGFP-CLIP-170 (adCLIP) mutant was used (WT, S311A, S311D). Numbers in the graph indicate n number of the cells from 2 independent experiments. Control (-/-): n=784, MYK-461: n=939, CLIP WT: n=656, CLIP WT + MYK-461: n=619, CLIP S311A: n=405, CLIP S311A + MYK-461: n=635, CLIP S311D: n=613, CLIP S311D + MYK-461: n=735. Data means ±S.D. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. **, P<0.01 versus Control, ††, P<0.01 versus CLIP WT, n.s., not significant.


( A ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) of individual Cre control ( CLIP - 170 S311A + / + ; α - MHC - MerCreMer + / − ) , Control mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer − / − ) , and CLIP - 170 S311A overexpressing mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer + / − ) , before ( Pre ) , 2 weeks ( Tx2w ) , 8 weeks ( Tx8w ) , 26 weeks ( Tx26w ) and over 1 year ( Tx1y ) after tamoxifen induction . Number of mice , ( Cre control , Control , S311A ) , Pre ( 6 , 2 , 3 ) , Tx 2w ( 9 , 5 , 5 ) , Tx 8w ( 9 , 10 , 14 ) , Tx1 2w ( 7 , 10 , 14 ) , Tx 26w ( 3 , 7 , 8 ) , Tx 52w ( 3 , 7 , 9 ) .

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

(A) Beeswarm plots of the echocardiographic parameter (ejection fraction) of individual Cre control (CLIP-170 S311A +/+; α-MHC-MerCreMer+/−), Control mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer−/−), and CLIP-170 S311A overexpressing mice (CLIP-170 S311A flox/+; α-MHC-MerCreMer+/−), before (Pre), 2 weeks (Tx2w), 8 weeks (Tx8w), 26 weeks (Tx26w) and over 1 year (Tx1y) after tamoxifen induction. Number of mice, (Cre control, Control, S311A), Pre (6, 2, 3), Tx 2w (9, 5, 5), Tx 8w (9, 10, 14), Tx1 2w (7, 10, 14), Tx 26w (3, 7, 8), Tx 52w (3, 7, 9).


( B ) Beeswarm plots of another line of CLIP - 170 S311A overexpressing mice ( line 9 ) . Number of mice , ( Control , S311A ) , Pre ( 7 , 3 ) , Tx 2w ( 4 , 7 ) , Tx 8w ( 6 , 4 ) , Tx 12w ( 5 , 4 ) , Tx 52w ( 3 , 3 ) . Data means ± S . D . Statistical significance was determined by two - way ANOVA followed by post hoc Tukey ' s multiple comparison test . # # : p < 0 . 01 vs Duration - matched Cre Control , * : p < 0 . 05 , * * : p < 0 . 01 vs Duration - matched Control ; † : p < 0 . 05 , † † : p < 0 . 01 , S311A TG mice 12 weeks vs 52 weeks .

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(B) Beeswarm plots of another line of CLIP-170 S311A overexpressing mice (line 9). Number of mice, (Control, S311A), Pre (7, 3), Tx 2w (4, 7), Tx 8w (6, 4), Tx 12w (5, 4), Tx 52w (3, 3). Data means ±S.D. Statistical significance was determined by two-way ANOVA followed by post hoc Tukey's multiple comparison test. ##: p < 0.01 vs Duration-matched Cre Control, *: p < 0.05, **: p < 0.01 vs Duration-matched Control; †: p < 0.05, ††: p < 0.01, S311A TG mice 12 weeks vs 52 weeks.


( C ) Representative long axis four - chamber cardiac magnetic resonance images of CLIP - 170 S311A TG mice and control mice over 1 year after tamoxifen induction . Left column showed systole images and right column represented diastole images . Three mice from each group were analyzed for cardiac MRI . The representative images were shown .

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(C) Representative long axis four-chamber cardiac magnetic resonance images of CLIP-170 S311A TG mice and control mice over 1 year after tamoxifen induction. Left column showed systole images and right column represented diastole images. Three mice from each group were analyzed for cardiac MRI. The representative images were shown.


( D ) Beeswarm plots showed the ejection fraction from cardiac MRI . LV , left ventricular , RV , right ventricular . Number of mice , ( Control , S311A ) , 1y ( 3 , 3 ) . Data means ± S . D . Two - tailed Student ' s t - test was used to analyze differences between two groups . * * , P < 0 . 01 versus Control .

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(D) Beeswarm plots showed the ejection fraction from cardiac MRI. LV, left ventricular, RV, right ventricular. Number of mice, (Control, S311A), 1y (3, 3). Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus Control.


( E ) Beeswarm plots of the echocardiographic parameter ( ejection fraction ) from CLIP - 170 S311D overexpressing mice and the control . Number of mice , ( Control , S311D ) , Tx 2w ( 3 , 4 ) , Tx 8w ( 5 , 11 ) , Tx 26w ( 4 , 11 ) . Data means ± S . D . There was no significant difference .

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(E) Beeswarm plots of the echocardiographic parameter (ejection fraction) from CLIP-170 S311D overexpressing mice and the control. Number of mice, (Control, S311D), Tx 2w (3, 4), Tx 8w (5, 11), Tx 26w (4, 11). Data means ±S.D. There was no significant difference.


( A ) Masson ' s trichrome staining of the heart of CLIP - 170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment .

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(A) Masson's trichrome staining of the heart of CLIP-170 S311A overexpressing mice and the control mice from 3 months and 1 year after tamoxifen treatment.


( B ) Quantitative analysis of cardiac fibrosis . Quantification was made as percentage of fibrosis area ( in blue ) in the left ventricle . Number of mice , ( Control , S311A ) , 3m ( 7 , 6 ) , 1y ( 5 , 6 ) . * , P < 0 . 05 versus Control .

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(B) Quantitative analysis of cardiac fibrosis. Quantification was made as percentage of fibrosis area (in blue) in the left ventricle. Number of mice, (Control, S311A), 3m (7, 6), 1y (5, 6). *, P<0.05 versus Control.


( C ) Representative immunostained images with an α - tubulin antibody of CLIP - 170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment . ( D ) Quantitative analysis of intensity of α - tubulin in ( C ) . Number of mice , ( Control : n = 7 , S311A : n = 8 ) . Average of 5 sections per animal were analyzed . * * , P < 0 . 01 versus Control .

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(C) Representative immunostained images with an α-tubulin antibody of CLIP-170 S311A overexpressing mice heart and control mice heart over 1 year after tamoxifen treatment. (D) Quantitative analysis of intensity of α-tubulin in (C). Number of mice, (Control: n=7, S311A: n=8). Average of 5 sections per animal were analyzed. **, P<0.01 versus Control.


( E ) Representative images of western blot for an α - tubulin antibody . GAPDH was used as a loading control . ( F ) Quantitative analysis of intensity of α - tubulin in ( E ) . Number of mice , ( Control : n = 4 , S311A : n = 5 ) . * , P < 0 . 05 versus Control .

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(E) Representative images of western blot for an α-tubulin antibody. GAPDH was used as a loading control. (F) Quantitative analysis of intensity of α-tubulin in (E). Number of mice, (Control: n=4, S311A: n=5). *, P<0.05 versus Control.


( G ) Representative immunostained images of CLIP - 170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment . These were stained with WGA ( red ) and a plakoglobin antibody ( green ) .

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(G) Representative immunostained images of CLIP-170 S311A overexpressing mice heart and the control mice heart over 1 year after tamoxifen treatment. These were stained with WGA (red) and a plakoglobin antibody (green).


( H ) Quantitative analysis of ( G ) Cell width , length and aspect ratio ( length / width ) were measured . Number of cells analyzed , Control , n = 251 , S311A , n = 310 , * * , P < 0 . 01 versus Control .

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(H) Quantitative analysis of (G) Cell width, length and aspect ratio (length/width) were measured. Number of cells analyzed, Control, n=251, S311A, n=310, **, P<0.01 versus Control.


( A ) Swelling of neurons in the vestibular nucleus due to fine vesiculation ( arrows ) and clear vacuolization ( arrowhead ) of the cytoplasm . HE , scale bar 100 μm .

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(A) Swelling of neurons in the vestibular nucleus due to fine vesiculation (arrows) and clear vacuolization (arrowhead) of the cytoplasm. HE, scale bar 100 μm.


( B ) Clear cytoplasmic vacuolization ( arrows ) in cerebellar corticalPurkinje cells . HE , scale bar 100 μm .

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(B) Clear cytoplasmic vacuolization (arrows) in cerebellar corticalPurkinje cells. HE, scale bar 100 μm.


( C ) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells ( arrows ) and a dense granular cell layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present the cerebellar cortex of an affected dog . The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain ( arrow ) . HE , scale bar 100 μm .

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(C) Normal cerebellar cortex of an unaffected dog shows viable Purkinje cells (arrows) and a dense granular cell layer. HE, scale bar 100 μm. (D) Marked neuronal loss is present the cerebellar cortex of an affected dog. The number of neurons in the granular cell layer is reduced and only scattered Purkinje cells remain (arrow). HE, scale bar 100 μm.


( E ) Axonal spheroids of varying quality were seen in the white matter ( arrows ) of cerebellum and brainstem . HE , scale bar 100 μm .

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(E) Axonal spheroids of varying quality were seen in the white matter (arrows) of cerebellum and brainstem. HE, scale bar 100 μm.


( F ) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells . HE , scale bar 100 μm .

(F) Diffuse cytoplasmic vacuolization of the exocrine pancreatic acinar cells. HE, scale bar 100 μm.


( G ) Purkinje cell with numerous single - membrane bound , cytoplasmic vacuoles tethering to each other ( arrows , inset ) . Electronmicrograph , scale bar 2 μm . Inset : scale bar 1 μm .

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(G) Purkinje cell with numerous single-membrane bound, cytoplasmic vacuoles tethering to each other (arrows, inset). Electronmicrograph, scale bar 2 μm. Inset: scale bar 1 μm.


( H ) Axonal spheroid containing aggregated degenerated mitochondria , occasional double - membrane - bound autophagosomes ( arrows ) and free electron dense material , compressed by a peripheral clear vacuolar space . Electronmicrograph , scale bar 0 . 5 μm . Abbreviations : n , nucleus ; ml , molecular layer ; pl , Purkinje cell layer ; gl , granular cell layer .

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(H) Axonal spheroid containing aggregated degenerated mitochondria, occasional double-membrane-bound autophagosomes (arrows) and free electron dense material, compressed by a peripheral clear vacuolar space. Electronmicrograph, scale bar 0.5 μm. Abbreviations: n, nucleus; ml, molecular layer; pl, Purkinje cell layer; gl, granular cell layer.


( A ) Axonal spheroids stain diffusely positive for LC3B . IHCLC3B , scale bar 100 μm . ( B , C ) The granular cores of the spheroids are positive for ( B ) ubiquitin ( arrow ) and ( C ) p62 . IHCubiquitin and p62 , scale bars 20 and 100 μm , respectively . ( D ) Smooth axonal swellings in the cerebellar cortex contain ATG4D . Inset : control . IHCATG4D , scale bar 100 μm . ( E ) Affected neurons show increased perinuclear granular LC3B positivity . Inset : control . IHCLC3B , scale bar 100 μm . ( F ) Neuronalvacuoles are partially LAMP2 positive ( arrow ) and partially negative ( arrow head ) . Inset : control . IHCLAMP2 , scale bar 20 μm .

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(A) Axonal spheroids stain diffusely positive for LC3B. IHCLC3B, scale bar 100 μm. (B,C) The granular cores of the spheroids are positive for (B) ubiquitin (arrow) and (C) p62. IHCubiquitin and p62, scale bars 20 and 100 μm, respectively. (D) Smooth axonal swellings in the cerebellar cortex contain ATG4D. Inset: control. IHCATG4D, scale bar 100 μm. (E) Affected neurons show increased perinuclear granular LC3B positivity. Inset: control. IHCLC3B, scale bar 100 μm. (F) Neuronalvacuoles are partially LAMP2 positive (arrow) and partially negative (arrow head). Inset: control. IHCLAMP2, scale bar 20 μm.


( A - F ) Lateral views of control and morphant zebrafishembryos . ( A , B ) Control embryos appear normal at 1 dpf . ( C , D ) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf . Black arrowheads denote the developing cerebellum . Purple arrows indicate the hindbrain irregularities . ( E ) Control embryo at 2 dpf . ( F ) A morphant embryo at 2 dpf displaying hydrocephalus ( yellow arrow ) and small head and eye .

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(A-F) Lateral views of control and morphant zebrafishembryos. (A,B) Control embryos appear normal at 1 dpf. (C,D) Morphant embryos show severe abnormalities in different regions of the brain at 1 dpf. Black arrowheads denote the developing cerebellum. Purple arrows indicate the hindbrain irregularities. (E) Control embryo at 2 dpf. (F) A morphant embryo at 2 dpf displaying hydrocephalus (yellow arrow) and small head and eye.


( G ) RT - PCR assay showing the efficiency of the atg4daSMO .

(G) RT-PCR assay showing the efficiency of the atg4daSMO.


Immunostaining with ( A - C ) anti - Pvalb7 and ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants . ( A , D ) Control embryos at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons , which are laterally located in the cerebellum . ( G - I ) Labeling of cerebellar granule cells with anti - Vglut1 antibody in control and morphant embryos . ( H ) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum . ( I ) Vglut1 expression is strongly reduced in embryos showing severe phenotype . White arrowheads indicate the region of cerebellum . Abbreviations : hb , hindbrain .

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Immunostaining with (A-C) anti-Pvalb7 and (D-F) anti-zebrinII antibody show loss of cerebellarPurkinje cells in atg4da morphants. (A,D) Control embryos at 4.5 dpf. (B,E) Morphants with mild phenotype show partial loss of cerebellarPurkinje cells. (C,F) Morphants with strong phenotype show either total loss of Purkinje cells or presence of few differentiated neurons, which are laterally located in the cerebellum. (G-I) Labeling of cerebellar granule cells with anti-Vglut1 antibody in control and morphant embryos. (H) Mildly affected morphants show reduced expression of Vglut1 in the cerebellum. (I) Vglut1 expression is strongly reduced in embryos showing severe phenotype. White arrowheads indicate the region of cerebellum. Abbreviations: hb, hindbrain.


C : FACS plots from a representative experiment showing gates for purification of the cell populations . Upper left : GFP + cells in the population of ESCs infected with pGIPZ - shRNA library lentiviruses . Upper right : Isolation of mesodermal ( SSEA1 - Flk1 + Cxcr4 - ) and endodermal ( SSEA1 - Flk1 - Cxcr4 + ) cells . Lower panels : Isolation of D20LS ( Lin - Sca1 + c - Kit - ) , D20LK ( Lin - Sca1 - c - Kit + ) , and D20LSK ( Lin - Sca1 + c - Kit + ) cells .

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C: FACS plots from a representative experiment showing gates for purification of the cell populations. Upper left: GFP+ cells in the population of ESCs infected with pGIPZ-shRNA library lentiviruses. Upper right: Isolation of mesodermal (SSEA1-Flk1+Cxcr4-) and endodermal (SSEA1-Flk1-Cxcr4+) cells. Lower panels: Isolation of D20LS (Lin-Sca1+c-Kit-), D20LK (Lin-Sca1-c-Kit+), and D20LSK (Lin-Sca1+c-Kit+) cells.


A : mRNA analysis of expression of endothelial ( Tie2 , CD31 , Cdh5 ) , epithelial ( Cdh1 ) , and early hematopoietic ( Scl , CD41 ) marker genes in the indicated sorted cell populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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A: mRNA analysis of expression of endothelial (Tie2, CD31, Cdh5), epithelial (Cdh1), and early hematopoietic (Scl, CD41) marker genes in the indicated sorted cell populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


B : Confirmation of the purity of sorted populations by qRT - PCR : c - Kit is highly expressed only in the c - Kit + ( D20LSK and D20LK ) populations , and Sca1 is highly expressed only in the in Sca1 + ( D20LSK and D20LS ) populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

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B: Confirmation of the purity of sorted populations by qRT-PCR: c-Kit is highly expressed only in the c-Kit+ (D20LSK and D20LK) populations, and Sca1 is highly expressed only in the in Sca1+ (D20LSK and D20LS) populations. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


C : qRT - PCR analysis of Gata1 , Gata2 , Lyz2 , and Scl expression in the sorted cell populations on day 20 . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test . * p < 0 . 05 .

C: qRT-PCR analysis of Gata1, Gata2, Lyz2, and Scl expression in the sorted cell populations on day 20. Error bars represent standard deviation (SD), n=3. Student's t-test. *p<0.05.


D : FACS analysis of ESC - derived cells confirming that D20LS , D20LK , and D20LSK cell populations express the early hematopoietic marker CD41 and the pan - hematopoietic marker CD45 .

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D: FACS analysis of ESC-derived cells confirming that D20LS, D20LK, and D20LSK cell populations express the early hematopoietic marker CD41 and the pan-hematopoietic marker CD45.


A : Clustering and enrichment analyses of shRNA reads in D6C ( endoderm ) , D6F ( HM / E ) , D20LS , D20LK , and D20LSK ( HSPC ) populations compared with ESCs . Specifically enriched / depleted shRNAs are grouped according to their target gene function ( I - X ) . Color coding bar is shown below .

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A: Clustering and enrichment analyses of shRNA reads in D6C (endoderm), D6F (HM/E), D20LS, D20LK, and D20LSK (HSPC) populations compared with ESCs. Specifically enriched/depleted shRNAs are grouped according to their target gene function (I-X). Color coding bar is shown below.


B : Heat map of shRNA reads in D6C and D6F populations compared with ESCs . shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII , respectively .

B: Heat map of shRNA reads in D6C and D6F populations compared with ESCs. shRNAs specifically depleted in D6C and D6F are labeled as Groups XI and XII, respectively.


E : Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples .

E: Heat map showing depletion of shRNAs targeting Cdc and Cdk genes in all day 20 samples.


A : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Esrra , S100a8 , or Hcfc2 and induced to form EBs . The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) and Endoderm cells ( Cxcr4 + / Flk1 - ) . The percentages of Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) were decreased on day 6 with shRNA knockdown of Group XII genes Esrra , S100a8 , and Hcfc2 compared to control as quantified by flow cytometric analysis . B : Knockdown of Esrra , S100a8 , and Hcfc2 showed that the ratios of percentage of Mesoderm - Endothelium population vs Endoderm population were decreased as measured by Flk1 + / Cxcr4 - vs . Flk1 - / Cxcr4 + on day 6 . The data were generated based on the quantification of flow cytometry analyses . Error bars represent standard deviation ( SD ) , n = 2 . Student ' s t - test , * p < 0 . 05 .

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A: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Esrra, S100a8, or Hcfc2 and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). The percentages of Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were decreased on day 6 with shRNA knockdown of Group XII genes Esrra, S100a8, and Hcfc2 compared to control as quantified by flow cytometric analysis.B: Knockdown of Esrra, S100a8, and Hcfc2 showed that the ratios of percentage of Mesoderm-Endothelium population vs Endoderm population were decreased as measured by Flk1+/Cxcr4- vs. Flk1-/Cxcr4+ on day 6. The data were generated based on the quantification of flow cytometry analyses. Error bars represent standard deviation (SD), n=2. Student's t-test, *p<0.05.


C : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Atp5g3 , Bub1b , Eci1 , or Dis3l and induced to form EBs . The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) and Endoderm cells ( Cxcr4 + / Flk1 - ) . Flow cytometric analysis showed that Endoderm cells ( Cxcr4 + / Flk1 - ) were deceased while Mesoderm - Endothelium cells ( Flk1 + / Cxcr4 - ) were slightly increased in the cells with shRNA knockdown of Group XI genes Atp5g3 , Bub1b , Eci1 , and Dis3l compared to control . D : Knockdown of Atp5g3 , Bub1b , Eci1 , or Dis3l led to increase of Mesoderm - Endothelium / Endoderm population ratio as quantified based on the flow cytometry data . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test , * p < 0 . 05 .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Atp5g3, Bub1b, Eci1, or Dis3l and induced to form EBs. The day 6 EB cells were analyzed by flow cytometry to measure the Mesoderm-Endothelium cells (Flk1+/Cxcr4-) and Endoderm cells (Cxcr4+/Flk1-). Flow cytometric analysis showed that Endoderm cells (Cxcr4+/Flk1-) were deceased while Mesoderm-Endothelium cells (Flk1+/Cxcr4-) were slightly increased in the cells with shRNA knockdown of Group XI genes Atp5g3, Bub1b, Eci1, and Dis3l compared to control.D: Knockdown of Atp5g3, Bub1b, Eci1, or Dis3l led to increase of Mesoderm-Endothelium/Endoderm population ratio as quantified based on the flow cytometry data. Error bars represent standard deviation (SD), n=3. Student's t-test, *p<0.05.


E : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 and differentiated towards HSPCs . The differentiated cells on day 20 were analyzed by flow cytometry . Silencing of group IV genes including Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 decreased overall LK , LS , and LSK population on day 20 . F : Quantification of LK + LSK + LS cell fractions with Wbp5 , Rbm26 , Gdpd4 , and Nrxn1 silencing based on flow cytometric analyses . Error bars represent standard deviation ( SD ) , n = 2 . Student ' s t - test , * p < 0 . 05 .

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E: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Wbp5, Rbm26, Gdpd4, and Nrxn1 and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Silencing of group IV genes including Wbp5, Rbm26, Gdpd4, and Nrxn1 decreased overall LK, LS, and LSK population on day 20.F: Quantification of LK+LSK+LS cell fractions with Wbp5, Rbm26, Gdpd4, and Nrxn1 silencing based on flow cytometric analyses. Error bars represent standard deviation (SD), n=2. Student's t-test, *p<0.05.


G : The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1 , Mettl22 , Lrsam1 , or Hal and differentiated towards HSPCs . The differentiated cells on day 20 were analyzed by flow cytometry . Flow cytometric analysis showed notable reduction of LSK population on day 20 with Group X genes Ap2a1 , Mettl22 , Lrsam1 , or Hal knockdown . H : Quantification of flow cytometric analyses showed knockdown of Ap2a1 , Mettl22 , Lrsam1 , and Hal significantly impaired generation of LSK population on day 20 in vitro compared to control . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - test , * p < 0 . 05 .

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G: The E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1, Mettl22, Lrsam1, or Hal and differentiated towards HSPCs. The differentiated cells on day 20 were analyzed by flow cytometry. Flow cytometric analysis showed notable reduction of LSK population on day 20 with Group X genes Ap2a1, Mettl22, Lrsam1, or Hal knockdown.H: Quantification of flow cytometric analyses showed knockdown of Ap2a1, Mettl22, Lrsam1, and Hal significantly impaired generation of LSK population on day 20 in vitro compared to control. Error bars represent standard deviation (SD), n=3. Student's t-test, *p<0.05.


B : Heat map showing bona fide HSC regulators in Group IV target genes .

B: Heat map showing bona fide HSC regulators in Group IV target genes.


C : Heat map showing the most depleted shRNAs in pair - wise comparisons between the D20LSK and D20LK populations .

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C: Heat map showing the most depleted shRNAs in pair-wise comparisons between the D20LSK and D20LK populations.


D : Validation of the involvement of Group X target genes Lrsam1 , Ap2a1 , Mettl22 , and Hal in zebrafish hematopoiesis by MO - mediated knockdown . Whole - mount in situ hybridization of Runx1 and c - Myb expression is shown at 26 hpf and 30hpf , respectively . Prdm16 knockdown serves as a positive control . Note that nonspecific staining for Runx1 and c - Myb was largely unaffected by targeted MO knockdown , whereas HSC markers Runx1 and c - Myb staining were notably reduced in discrete cells in the ventral region of the dorsal aorta ( arrows ) . Scale bar = 100 µm .

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D: Validation of the involvement of Group X target genes Lrsam1, Ap2a1, Mettl22, and Hal in zebrafish hematopoiesis by MO-mediated knockdown. Whole-mount in situ hybridization of Runx1 and c-Myb expression is shown at 26 hpf and 30hpf, respectively. Prdm16 knockdown serves as a positive control. Note that nonspecific staining for Runx1 and c-Myb was largely unaffected by targeted MO knockdown, whereas HSC markers Runx1 and c-Myb staining were notably reduced in discrete cells in the ventral region of the dorsal aorta (arrows). Scale bar = 100 µm.


A : Representative flow cytometric analysis showed that CD34 + cells were significantly decreased at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , or HAL . The human cord blood CD34 + cells were transduced with pLKO - shRNAs targeting human AP2A1 , METTL22 , LRSAM1 , or HAL and cultured for 5 days . Then , the flow cytometric analysis was performed to examine the percentage of CD34 + cells in culture . B : Quantification of CD34 + cells at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HALshowed significant decrease of CD34 + cells compared to control . Error bars represent standard deviation ( SD ) from duplicates , * p < 0 . 05 .

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A: Representative flow cytometric analysis showed that CD34+cells were significantly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, or HAL. The human cord blood CD34+cells were transduced with pLKO-shRNAs targeting human AP2A1, METTL22, LRSAM1, or HAL and cultured for 5 days. Then, the flow cytometric analysis was performed to examine the percentage of CD34+cells in culture.B: Quantification of CD34+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+ cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.


C : Flow cytometric analysis showed CD34 + / CD90 + cells were strikingly decreased at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HAL . D : Quantification of CD34 + CD90 + cells at 5dpi with knockdown of AP2A1 , METTL22 , LRSAM1 , and HALshowed significant decrease of CD34 + CD90 + cells compared to control . Error bars represent standard deviation ( SD ) from duplicates , * p < 0 . 05 .

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C: Flow cytometric analysis showed CD34+/CD90+cells were strikingly decreased at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HAL.D: Quantification of CD34+CD90+cells at 5dpi with knockdown of AP2A1, METTL22, LRSAM1, and HALshowed significant decrease of CD34+CD90+cells compared to control. Error bars represent standard deviation (SD) from duplicates, *p<0.05.


( A ) Akt is deubiquitinated during fasting . Left : Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS - PAGE and immunoblot using Akt antibody . Right : densitometric measurement of presented blots ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

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(A) Akt is deubiquitinated during fasting. Left: Soluble fractions of TA muscles from fed and fasted mice were analyzed by SDS-PAGE and immunoblot using Akt antibody. Right: densitometric measurement of presented blots (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( B ) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted ( 2d ) mice . Percipitated were analyzed by immunoblotting using an anti - ubiquitin conjugates . Mouse IgG was used as a control for non - specific binding .

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(B) Akt was immunoprecipitated from the soluble fraction of muscles from fed or fasted (2d) mice. Percipitated were analyzed by immunoblotting using an anti-ubiquitin conjugates. Mouse IgG was used as a control for non-specific binding.


) D ( Ubiquitinated Akt is a substrate for USP1 in vitro . Top : Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice , and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi - well plate . The dividing line indicates the removal of an intervening lane for presentation purposes . Bottom : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each well .

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)D( Ubiquitinated Akt is a substrate for USP1 in vitro. Top: Akt was immunoprecipitated from the soluble fraction of TA muscle from fed mice, and was subjected to an in vitro deubiquitination by a panel of purified DUBs arrayed in a multi-well plate. The dividing line indicates the removal of an intervening lane for presentation purposes. Bottom: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each well.


( E ) shRNA - mediated knockdown of USP1 in Hela cells . Soluble extracts were analyzed by immunoblotting .

(E) shRNA-mediated knockdown of USP1 in Hela cells. Soluble extracts were analyzed by immunoblotting.


( F ) USP1 deubiquitinates Akt during fasting in vivo . Left : Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS - PAGE and immunoblotting using anti - Akt . The actin blot serves as a loading control . Right : densitometric measurements of presented blots ( n = 3 ) . Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

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(F) USP1 deubiquitinates Akt during fasting in vivo. Left: Soluble fractions of TA muscles transfected with shLacz control or shUSP1 from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using anti-Akt. The actin blot serves as a loading control. Right: densitometric measurements of presented blots (n=3). Data is presented as ratio of ubiquitinated Akt to total Akt normalized to actin. * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( G ) USP1 removes K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1 ( C90S ) or control plasmids . Mouse IgG was used as a control for non - specific binding . Protein precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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(G) USP1 removes K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing USP1(C90S) or control plasmids. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( H ) USP1 removes polyubiquitin chains linked to K8 on Akt . Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1 ( C90S ) , HA - Akt ( K8R ) or control from fasted mice , and protein precipitates were analyzed by immunoblotting with anti - Akt antibody . Mouse IgG was used as a control for non - specific binding . Right : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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(H) USP1 removes polyubiquitin chains linked to K8 on Akt. Akt was immunoprecipitated from the soluble fraction of TA muscles transfected with USP1(C90S) , HA-Akt(K8R) or control from fasted mice, and protein precipitates were analyzed by immunoblotting with anti-Akt antibody. Mouse IgG was used as a control for non-specific binding. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( A ) Inhibition of USP1 increases PI3K - Akt - FoxO signaling during fasting . Soluble fractions of normal and atrophying muscles expressing shLacz or USP1 ( C90S ) were analyzed by SDS - PAGE and immunoblot . The black line indicate the removal of intervening lanes for presentation purposes . Right : densitometric measurements of presented pAkt ( T308 ) , pAkt ( S473 ) , and Akt blots . Data is presented as ratio of phosphorylated Akt to total Akt ( n = 3 ) . * p < 0 . 05 vs . fed by one - tailed t - test . Data are represented as mean ± SEM .

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(A) Inhibition of USP1 increases PI3K-Akt-FoxO signaling during fasting. Soluble fractions of normal and atrophying muscles expressing shLacz or USP1(C90S) were analyzed by SDS-PAGE and immunoblot. The black line indicate the removal of intervening lanes for presentation purposes. Right: densitometric measurements of presented pAkt (T308), pAkt (S473), and Akt blots. Data is presented as ratio of phosphorylated Akt to total Akt (n=3). * p<0.05 vs. fed by one-tailed t-test. Data are represented as mean ±SEM.


( B ) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting . Quantitative RT - PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 ( 80 % transfection efficiency ) using primers for MuRF1 and Atrogin1 . Data are plotted as the mean fold change relative to fed control . n = 4 . * , P < 0 . 05 vs . shLacz in fed . # , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(B) Downregulation of USP1 reduces MuRF1 and Atrogin1 expression during fasting. Quantitative RT-PCR of mRNA preparations from atrophying and control muscles expressing shLacz or shUSP1 (80% transfection efficiency) using primers for MuRF1 and Atrogin1. Data are plotted as the mean fold change relative to fed control. n = 4. *, P < 0.05 vs. shLacz in fed. #, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( C ( Downregulation of USP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 fibers transfected with shUSP1 ( that express GFP , green bars ) vs . 500 nontransfected fibers ( black bars ) in the same muscle . n = 5 mice . A representative image is shown ; Laminin staining is in red ; Bar , 50μm .

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(C( Downregulation of USP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shUSP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 5 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) Downregulation of USP1attenuates the loss of muscle mass during fasting . shUSP1 was delivered to more than 60 % of muscle fibers . Mean weights of electroporated muscles are plotted as the percent weight loss . n = 6 . # , P < 0 . 05 vs . shLacz in fed ; * , P < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(D) Downregulation of USP1attenuates the loss of muscle mass during fasting. shUSP1 was delivered to more than 60% of muscle fibers. Mean weights of electroporated muscles are plotted as the percent weight loss. n = 6. #, P < 0.05 vs. shLacz in fed; *, P < 0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( E ) USP1 inhibition significantly increases glucose tolerance in mice during fasting . Left : mice injected i . p . with specific USP1 Inhibitor ( 12ug / gr body weight ) or saline during fasting ( 2d ) were subjected to glucose tolerance test . Blood glucose levels were measured at the indicated time points following glucose injection ( 1 mg / gr body weight ) . Data is depicted in a graph as mg / dL glucose ( n = 3 ) . * p < 0 . 05 , * * p < 0 . 005 , * * * p < 0 . 0005 vs . mice injected with saline by one - tailed t - test . Data are represented as mean ± SEM . Right : soluble fractions of muscles from injected mice were analyzed by SDS - PAGE and immunoblot .

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

(E) USP1 inhibition significantly increases glucose tolerance in mice during fasting. Left: mice injected i.p. with specific USP1 Inhibitor (12ug/gr body weight) or saline during fasting (2d) were subjected to glucose tolerance test. Blood glucose levels were measured at the indicated time points following glucose injection (1 mg/gr body weight). Data is depicted in a graph as mg/dL glucose (n=3). * p<0.05, ** p<0.005, *** p<0.0005 vs. mice injected with saline by one-tailed t-test. Data are represented as mean ±SEM. Right: soluble fractions of muscles from injected mice were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , downregulation of PHLPP1 enhances Akt phosphorylation at S473 . Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice ( 2d ) were analyzed by immunoblotting . Right : densitometric measurements of presented blots . Data is presented as ratio of shPHLPP1 to shLacz ( n = 3 for shLacz and n = 4 for shPHLPP1 ) . * p < 0 . 05 vs . shLacz in fasting by one - tailed t - test . Data are represented as mean ± SEM .

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(A) During fasting, downregulation of PHLPP1 enhances Akt phosphorylation at S473. Soluble fractions of muscles transfected with shLacz or shPHLPP1 from fasted mice (2d) were analyzed by immunoblotting. Right: densitometric measurements of presented blots. Data is presented as ratio of shPHLPP1 to shLacz (n=3 for shLacz and n=4 for shPHLPP1). * p<0.05 vs. shLacz in fasting by one-tailed t-test. Data are represented as mean ±SEM.


( B ) PHLPP1 downregulation increases rates of protein synthesis during fasting . Mice were injected with puromycin , and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody . Analysis of right ( R ) and left ( L ) limbs for each mouse is shown .

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(B) PHLPP1 downregulation increases rates of protein synthesis during fasting. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Analysis of right (R) and left (L) limbs for each mouse is shown.


( C ) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy . Cross - sectional areas of 500 fibers transfected with shPHLPP1 ( that express GFP , green bars ) vs . 500 nontransfected fibers ( black bars ) in the same muscle . n = 4 mice . A representative image is shown ; Laminin staining is in red ; Bar , 50μm .

(C) Downregulation of PHLPP1 markedly reduces muscle fiber atrophy. Cross-sectional areas of 500 fibers transfected with shPHLPP1 (that express GFP, green bars) vs. 500 nontransfected fibers (black bars) in the same muscle. n = 4 mice. A representative image is shown; Laminin staining is in red; Bar, 50μm.


( D ) USP1 / UAF1 interact with PHLPP1 in normal muscle . USP1 was immunoprecipitated from the soluble fraction of normal muscles . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting .

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(D) USP1/UAF1 interact with PHLPP1 in normal muscle. USP1 was immunoprecipitated from the soluble fraction of normal muscles. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting.


( C ( In fasting , Akt sediments to glycerol gradient fractions containing USP1 / UAF1 , PHLPP1 , and TSC1 / TSC2 . Muscle homogenates from fed or fasted mice were separated on 10 % - 40 % glycerol gradient , and alternate fractions were analyzed by SDS - PAGE and immunoblot . Red rectangle marks fractions containing USP1 / UAF1 , PHLPP1 , TSC1 and TSC2 in fed conditions .

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(C( In fasting, Akt sediments to glycerol gradient fractions containing USP1/UAF1, PHLPP1, and TSC1/TSC2. Muscle homogenates from fed or fasted mice were separated on 10%-40% glycerol gradient, and alternate fractions were analyzed by SDS-PAGE and immunoblot. Red rectangle marks fractions containing USP1/UAF1, PHLPP1, TSC1 and TSC2 in fed conditions.


( D ) During fasting , Akt is recruited to USP1 - PHLPP1 - TSC1 complex . TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1 ( C90S ) from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Black lines indicate the removal of intervening lanes for presentation purposes .

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(D) During fasting, Akt is recruited to USP1-PHLPP1-TSC1 complex. TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or USP1(C90S) from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Black lines indicate the removal of intervening lanes for presentation purposes.


( E ) During fasting , inhibition of USP1 reduces rates of protein synthesis . Mice were injected with puromycin , and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody . Right : Densitometric measurement of the presented blot . n = 3 . * , P < 0 . 05 vs . shLacz by one - tailed t - test . Data are represented as mean ± SEM .

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(E) During fasting, inhibition of USP1 reduces rates of protein synthesis. Mice were injected with puromycin, and soluble fractions of electroporated muscles were analyzed by immunoblotting using puromycin antibody. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( F ) Inhibition of USP1 results in TSC1 accumulation . Soluble fraction of muscles expressing shLacz or USP1 ( C90S ) from fasted mice were analyzed by SDS - PAGE and immunoblotting . Right : Densitometric measurement of the presented blot . n = 3 . * , P < 0 . 05 vs . shLacz . Data is presented as protein content ( AU ) in USP1 ( C90S ) vs . shLacz by one - tailed t - test . Data are represented as mean ± SEM .

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(F) Inhibition of USP1 results in TSC1 accumulation. Soluble fraction of muscles expressing shLacz or USP1(C90S) from fasted mice were analyzed by SDS-PAGE and immunoblotting. Right: Densitometric measurement of the presented blot. n = 3. *, P < 0.05 vs. shLacz. Data is presented as protein content (AU) in USP1(C90S) vs. shLacz by one-tailed t-test. Data are represented as mean ±SEM.


( G ) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308 . However , a simultaneous downregulation of USP1 is required to promote TSC1 accumulation . Soluble fraction of muscles expressing shLacz or gUSP1 ( in vivo CRISPR ) from fed and fasted mice injected i . p . with rapamycin ( 6mg / kg body weight ) or saline were analyzed by SDS - PAGE and immunoblot .

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(G) mTOR inhibition with rapamycin results in reduced USP1 protein content and enhanced Akt phosphorylation at T308. However, a simultaneous downregulation of USP1 is required to promote TSC1 accumulation. Soluble fraction of muscles expressing shLacz or gUSP1 (in vivo CRISPR) from fed and fasted mice injected i.p. with rapamycin (6mg/kg body weight) or saline were analyzed by SDS-PAGE and immunoblot.


( A ) During fasting , inhibition of Dab2 - Akt association reduces the interaction of Akt with USP1 - PHLPP1 - TSC1 complex . Left : TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed or fasted mice . Mouse IgG was used as a control for non - specific binding . Precipitates were analyzed by immunoblotting . Right : densitometric measurement of PHLPP1 and TSC1 blots , and data is presented as PHLPP1 / TSC1 ratio .

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(A) During fasting, inhibition of Dab2-Akt association reduces the interaction of Akt with USP1-PHLPP1-TSC1 complex. Left: TSC1 was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed or fasted mice. Mouse IgG was used as a control for non-specific binding. Precipitates were analyzed by immunoblotting. Right: densitometric measurement of PHLPP1 and TSC1 blots, and data is presented as PHLPP1/TSC1 ratio.


( B ) Dab2 promotes Akt deubiquitination in vivo . Left : Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed and fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . Protein precipitates were analyzed by Western blot analysis using Akt antibody . Right : Densitometric measurements of presented blots . Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

(B) Dab2 promotes Akt deubiquitination in vivo. Left: Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were analyzed by Western blot analysis using Akt antibody. Right: Densitometric measurements of presented blots. Data is presented as the ratio between ubiquitinated Akt to total Akt in each lane (n=2).


( C ( Dab2 promotes cleavage of K63 - linked polyubiquitin chains on Akt in vivo . Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2 - DN from fed and fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . Protein precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

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(C( Dab2 promotes cleavage of K63-linked polyubiquitin chains on Akt in vivo. Akt was immunoprecipitated from the soluble fraction of muscles expressing shLacz or Dab2-DN from fed and fasted mice using a specific antibody. Mouse IgG was used as a control for non-specific binding. Protein precipitates were subjected to immunoblotting with an antibody against K63-linked polyubiquitin conjugates.


( D ) Inhibition of Dab2 - Akt association enhances Akt phosphorylation in fasting . Soluble fraction of TA muscles transfected with shLacz or Dab2 - DN from fed and fasted mice were analyzed by SDS - PAGE and immunoblotting using the indicated antibodies . Right : densitometric measurement of presented blots . Data are mean ± SEM , n = 3 . * p < 0 . 05 . vs . shLacz in fed conditions , by one - tailed t - test .

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(D) Inhibition of Dab2-Akt association enhances Akt phosphorylation in fasting. Soluble fraction of TA muscles transfected with shLacz or Dab2-DN from fed and fasted mice were analyzed by SDS-PAGE and immunoblotting using the indicated antibodies. Right: densitometric measurement of presented blots. Data are mean ±SEM, n=3. * p<0.05. vs. shLacz in fed conditions, by one-tailed t-test.


( b ) Experimental validation of predicted synergistic SR - based combinational therapies in head and neck cancer : A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer - targeting treatments in 5 HNSC cell lines , based on drug treatment experiments . Synergism was estimated using standard Fa - CI analysis . The table displays the average combination index ( CI ; synergism CI < 1 , additivity effect CI = 1 , antagonism CI > 1 , NAN indeterminate CI ) at 50 % growth inhibition ( Fraction affected ) . Combinations that are synergistic are colored blue ( black otherwise ) for each cell lines tested . The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix ( number indicates % cell viability at 48h , n = 3 ) , and Fa - CI curve .

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(b) Experimental validation of predicted synergistic SR-based combinational therapies in head and neck cancer: A table summarizing the experimentally observed synergism between primary drugs and their predicted rescuer-targeting treatments in 5 HNSC cell lines, based on drug treatment experiments. Synergism was estimated using standard Fa-CI analysis. The table displays the average combination index (CI; synergism CI < 1, additivity effect CI = 1, antagonism CI > 1, NAN indeterminate CI) at 50% growth inhibition (Fraction affected). Combinations that are synergistic are colored blue (black otherwise) for each cell lines tested. The inset shows an example of CI calculation for BYL719 and Dasatinib combination in HN12 cell lines based on the corresponding dose matrix (number indicates % cell viability at 48h, n = 3), and Fa-CI curve.


( a ) Experimental testing of the predicted SR ( DU ) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine . The matrix displays drug interactions between Decitabine , a DNMT1 inhibitor , and inhibitors of its predicted rescuer genes ( X - axis ) across 18 NSCLC cell lines ( Y - axis ) that are insensitive to Decitabine . Row labels present rescuer genes and their inhibitors . Colors in the matrix show whether the interactions found are significantly synergistic ( red ) , antagonistic ( green ) or non - significant ( in gray ) . Values in the matrix show average synergism ( < 1 synergism and > 1 antagonism Thirteen predicted DU - SR rescuers ( red lines ) , two predicted DD SR rescuers ( green lines ) of DNMT1 and one random control ( JAK3i ) were tested .

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(a) Experimental testing of the predicted SR (DU) rescuers of DNMT1 via drug combination experiments in 18 NSCLC cell lines insensitive to Decitabine. The matrix displays drug interactions between Decitabine, a DNMT1 inhibitor, and inhibitors of its predicted rescuer genes (X-axis) across 18 NSCLC cell lines (Y-axis) that are insensitive to Decitabine. Row labels present rescuer genes and their inhibitors. Colors in the matrix show whether the interactions found are significantly synergistic (red), antagonistic (green) or non-significant (in gray). Values in the matrix show average synergism (<1 synergism and >1 antagonism Thirteen predicted DU-SR rescuers (red lines), two predicted DD SR rescuers (green lines) of DNMT1 and one random control (JAK3i) were tested.


The transcriptomic alterations of rescuer genes post PD1 / PDL1 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - PD1 ( b ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-PD1 (b) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - CTLA4 ( c ) Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after anti-CTLA4 (c) Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


The transcriptomic alterations of rescuer genes post PD1 / PDL1 and CTLA4 blockade in patient tumor biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after PD1 + CTLA4 combination therapies ( d ) . Each panel displays the expression fold change of each predicted rescuer gene ( rows ) for different tumor samples ( columns ) and the P - value of over - all paired Wilcoxon test of the expression changes observed in paired samples . Significantly altered up / down - regulated genes are marked by ( * ) . Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti - PD1 blockade in mice models .

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The transcriptomic alterations of rescuer genes post PD1/PDL1 and CTLA4 blockade in patient tumor biopsies: Their post (vs. pre) treatment expression changes of DU/DD rescuers after PD1 + CTLA4 combination therapies (d). Each panel displays the expression fold change of each predicted rescuer gene (rows) for different tumor samples (columns) and the P-value of over-all paired Wilcoxon test of the expression changes observed in paired samples. Significantly altered up/down-regulated genes are marked by (*). Genes marked in red are those whose CRISPR knockdown enhances melanoma sensitivity to anti-PD1 blockade in mice models.


a , Phase partitioning of Apg8 . Wild - type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning ( see Methods ) .

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a, Phase partitioning of Apg8. Wild-type and Δapg4Δapg8 cells producing Apg8FG and Δapg7 cells were subjected to phase partitioning (see Methods).


b , MALDI mass spectrum of His6 - Apg8 . Purified His6 - Apg8 ( predominantly Apg8FG - X and a small amount of Apg8FG ) was extracted with 0 . 4 % β - octylglycoside from a polyacrylamide gel and desalted with ZipTipC4 ( Millipore ) before mass measurement . The principal MH + ion signal at m / z 15 , 003 is shifted in mass by about + 635 from the MH + value corresponding to His6 - Apg8FG .

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b, MALDI mass spectrum of His6-Apg8. Purified His6-Apg8 (predominantly Apg8FG-X and a small amount of Apg8FG) was extracted with 0.4% β-octylglycoside from a polyacrylamide gel and desalted with ZipTipC4(Millipore) before mass measurement. The principal MH+ ion signal at m/z 15,003 is shifted in mass by about +635 from the MH+ value corresponding to His6-Apg8FG.


c , MS / MS spectra of the C - terminal peptides of Apg8FG - X ( top ) and Apg8FG ( bottom ) . C - terminal fragments were generated from Apg8FG - X and Apg8FG ( see Methods ) . Each fragment that was observed only as non - 18O - labelled signals was subjected to MS / MS . The MS / MS spectra , deconvoluted with MaxEnt3 , were interpreted by SeqMS software26 . Arrows show the sequences from the N and C terminus based on y ″ m and bl ions , respectively ( where m and l denote positions counted from the C and N terminus ) that were produced by cleavage of peptide bonds during MS / MS . Amino acids shown in three - letter code denote immonium ions . The nomenclature of these ions is as in previous work28 .

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c, MS/MS spectra of the C-terminal peptides of Apg8FG-X (top) and Apg8FG (bottom). C-terminal fragments were generated from Apg8FG-X and Apg8FG (see Methods). Each fragment that was observed only as non-18O-labelled signals was subjected to MS/MS. The MS/MS spectra, deconvoluted with MaxEnt3, were interpreted by SeqMS software26. Arrows show the sequences from the N and C terminus based on y″m and bl ions, respectively (where m and l denote positions counted from the C and N terminus) that were produced by cleavage of peptide bonds during MS/MS. Amino acids shown in three-letter code denote immonium ions. The nomenclature of these ions is as in previous work28.


d , Thioester formation between Apg8 and Apg7 . Cell extracts of Δapg7Δapg8 cells expressing Apg7 - Myc and / or HA - Apg8 from 2µ plasmids were immunoprecipitated with anti - HA antibody , and immunoblotted ( IB ) with anti - Myc and anti - HA antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg7 conjugate .

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d, Thioester formation between Apg8 and Apg7. Cell extracts of Δapg7Δapg8 cells expressing Apg7-Myc and/or HA-Apg8 from 2µ plasmids were immunoprecipitated with anti-HA antibody, and immunoblotted (IB) with anti-Myc and anti-HA antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg7 conjugate.


b , Interaction between Apg8 and Apg7 . Δapg7Δapg8 cells were transformed with APG7 - Myc and / or APG8 on 2µ plasmids . Cell lysates were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc or anti - Apg8 antibody . Apg8 co - immunoprecipitated with Apg7 - Myc .

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b, Interaction between Apg8 and Apg7. Δapg7Δapg8 cells were transformed with APG7-Myc and/or APG8 on 2µ plasmids. Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc or anti-Apg8 antibody. Apg8 co-immunoprecipitated with Apg7-Myc.


c , Effect of Apg8 C - terminal processing on the Apg8 - Apg7 interaction . Δapg4 cells co - expressing Apg7 - Myc and HA - Apg8 ( FGR , FG and F ) from 2µ plasmids . FGR , wild - type Apg8 ; FG , Apg8 lacking C - terminal arginine ; F , Apg8 lacking C - terminal glycine and arginine . Cell lysates were immunoprecipitated with anti - HA antibody , and immunoblotted with anti - HA and anti - Myc antibody .

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c, Effect of Apg8 C-terminal processing on the Apg8-Apg7 interaction. Δapg4 cells co-expressing Apg7-Myc and HA-Apg8 (FGR, FG and F) from 2µ plasmids. FGR, wild-type Apg8; FG, Apg8 lacking C-terminal arginine; F, Apg8 lacking C-terminal glycine and arginine. Cell lysates were immunoprecipitated with anti-HA antibody, and immunoblotted with anti-HA and anti-Myc antibody.


b , Interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing APG3 and / or Myc - APG8 from 2µ plasmids were immunoprecipitated with anti - Apg3 antibody , and immunoblotted with anti - Apg3 and anti - Myc antibody .

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b, Interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing APG3 and/or Myc-APG8 from 2µ plasmids were immunoprecipitated with anti-Apg3 antibody, and immunoblotted with anti-Apg3 and anti-Myc antibody.


c , Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3 . Lysates of Δapg3 cells expressing Myc - APG8 and either wild - type ( wt ) APG3 or mutant APG3 ( C234S and C234A ) from 2µ plasmids were immunoprecipitated with anti - Myc antibody , and immunoblotted with anti - Myc and anti - Apg3 antibodies .

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c, Effect of mutated Cys 234 in Apg3 on the interaction between Apg8 and Apg3. Lysates of Δapg3 cells expressing Myc-APG8 and either wild-type (wt) APG3 or mutant APG3 (C234S and C234A) from 2µ plasmids were immunoprecipitated with anti-Myc antibody, and immunoblotted with anti-Myc and anti-Apg3 antibodies.


d , Thioester formation between Apg8 and Apg3 . Δapg3 , Δapg7 and Δapg10 cells were transformed with both Myc - APG8 and Apg3 , or only Apg3 , on 2µ plasmids . Cell extracts were immunoprecipitated with anti - Myc antibody , and subjected to immunoblot ( IB ) using anti - Myc and anti - Apg3 antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg3 conjugate ; diamond , possible Myc - Apg8 dimer .

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d, Thioester formation between Apg8 and Apg3. Δapg3, Δapg7 and Δapg10 cells were transformed with both Myc-APG8 and Apg3, or only Apg3, on 2µ plasmids. Cell extracts were immunoprecipitated with anti-Myc antibody, and subjected to immunoblot (IB) using anti-Myc and anti-Apg3 antibody. DTT-, no DTT; DTT+, 100 mM DTT; asterisk, Apg8-Apg3 conjugate; diamond, possible Myc-Apg8 dimer.


a , Apg7 and Apg3 are necessary for attachment of Apg8 to PE . Δapg cell lysates were subjected to SDS - PAGE containing 6 M urea , and immunoblotted with anti - Apg8 antibody .

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a, Apg7 and Apg3 are necessary for attachment of Apg8 to PE. Δapg cell lysates were subjected to SDS-PAGE containing 6 M urea, and immunoblotted with anti-Apg8 antibody.


b , Autophagic activity in apg3 mutants . Wild - type , Δapg3 and apg3C243A cells were grown in rich medium ( open bar ) and subjected to nitrogen starvation for 4 h ( filled bar ) . The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity . The apg3C243A mutant showed defective autophagy .

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b, Autophagic activity in apg3 mutants. Wild-type, Δapg3 and apg3C243A cells were grown in rich medium (open bar) and subjected to nitrogen starvation for 4 h (filled bar). The autophagic activity of each cell was assessed by measuring the alkaline phosphatase activity. The apg3C243A mutant showed defective autophagy.


( A , B ) BMDCs were infected with Pru parasites ( at a MOI of 4 parasites / BMDC ) or treated with tunicamycin ( TN ) for 16h and the expression of UPR target genes were analyzed by RT - qPCR . The results are expressed as ( Log2 ) Fold change ( A ) or relative mRNA level ( B ) compared to non - infected ( NI ) conditions . IRE1α / XBP1s pathway : Ern1 ( IRE1α ) , Xbp1s , Dnajb9 ( ERdj4 ) , Sec24D , Bloc1s1 and Sec61a1 ; PERK / ATF4 pathway : Atf4 , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) ; ATF6 pathway : Atf6 and Herpud1 ; classical ER chaperons : Hspa5 ( BIP ) and Hsp90b1 ( GRP94 ) . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 2 - 5 independent experiments depending of the studied gene , 3 mice / independent experiment ) .

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(A, B) BMDCs were infected with Pru parasites (at a MOI of 4 parasites/BMDC) or treated with tunicamycin (TN) for 16h and the expression of UPR target genes were analyzed by RT-qPCR. The results are expressed as (Log2) Fold change (A) or relative mRNA level (B) compared to non-infected (NI) conditions. IRE1α/XBP1s pathway: Ern1 (IRE1α), Xbp1s, Dnajb9 (ERdj4), Sec24D, Bloc1s1 and Sec61a1; PERK/ATF4 pathway: Atf4, Ddit3 (CHOP) and Ppp1r15a (GADD34); ATF6 pathway: Atf6 and Herpud1; classical ER chaperons: Hspa5 (BIP) and Hsp90b1 (GRP94). Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=2-5 independent experiments depending of the studied gene, 3 mice / independent experiment).


( C , D ) Immunoblot analysis of IRE1α , PERK , Phospho - PERK ( detected by the presence of a second upper band after migration on a 6 % acrylamide gel ) , Phospho - eIF2α , CHOP and XBP1s protein expression in non - infected BMDCs , BMDCs treated with TN and Pru infected BMDCs ( C ) and in non - infected and Pru infected Flt3 / Notch - DCs ( D ) for the indicated times . GAPDH and tubulin were used as loading controls . The same blots are shown in Figure EV3B - C .

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

(C, D) Immunoblot analysis of IRE1α, PERK, Phospho-PERK (detected by the presence of a second upper band after migration on a 6% acrylamide gel), Phospho-eIF2α, CHOP and XBP1s protein expression in non-infected BMDCs, BMDCs treated with TN and Pru infected BMDCs (C) and in non-infected and Pru infected Flt3/Notch-DCs (D) for the indicated times. GAPDH and tubulin were used as loading controls. The same blots are shown in Figure EV3B-C.


( D ) mRNA relative expression level of Xbp1s , Dnajb9 ( ERdj4 ) , Ddit3 ( CHOP ) and Ppp1r15a ( GADD34 ) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 , * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 4 - 5 mice pooled from 2 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) mRNA relative expression level of Xbp1s, Dnajb9 (ERdj4), Ddit3 (CHOP) and Ppp1r15a (GADD34) from WT and MyD88KO BMDCs infected with Pru parasites for 16h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001, ****: p<0.0001; mean ± S.E.M (n=4-5 mice pooled from 2 independent experiments).


( A ) BMDCs ( CD11c + ) were incubated with Pru - Tomato parasites for 16h . Infected cells ( red gating ) and non - infected bystander cells ( green gating ) were sorted - purified and further analyzed . ( B ) The expression of UPR target genes were analyzed by RT - qPCR in infected and non - infected bystander cells compared to non - infected BMDCs ( NI ) . The results are expressed as relative mRNA level compared to non - infected conditions . Results are normalized to the housekeeping gene Gapdh . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; mean ± S . E . M ( n = 6 mice pooled from 2 independent experiments ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) BMDCs (CD11c+) were incubated with Pru-Tomato parasites for 16h. Infected cells (red gating) and non-infected bystander cells (green gating) were sorted-purified and further analyzed. (B) The expression of UPR target genes were analyzed by RT-qPCR in infected and non-infected bystander cells compared to non-infected BMDCs (NI). The results are expressed as relative mRNA level compared to non-infected conditions. Results are normalized to the housekeeping gene Gapdh. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; mean ± S.E.M (n=6 mice pooled from 2 independent experiments).


( E ) mRNA relative expression level of Il - 6 , Il - 12 and Il - 23 from XBP1fl / flIRE1fl / fl , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non - infected ( NI ) conditions . Results are normalized to the housekeeping gene Gapdh . Two - way ANOVA Tukey ' s multiple comparisons , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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

(E) mRNA relative expression level of Il-6, Il-12 and Il-23 from XBP1fl/flIRE1fl/fl, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs infected with Pru parasites for 16 h compared to non-infected (NI) conditions. Results are normalized to the housekeeping gene Gapdh. Two-way ANOVA Tukey's multiple comparisons, ns: p>0.05; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( I ) Graph depicts the expression ( indicated as the Mean Fluorescence Intensity ) of H - 2Kb by Pru Tomato SAG1 ‐ OVA infected XBP1fl / fl IRE1fl / fl treated or not with 4μ8C , XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs . Bar graphs depict mean ± S . E . M ( n = 3 mice , one representative experiment from 2 independent experiments ) . Tukey ' s ANOVA one - way test , ns : p > 0 . 05 .

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

(I) Graph depicts the expression (indicated as the Mean Fluorescence Intensity) of H-2Kb by Pru Tomato SAG1‐OVA infected XBP1fl/fl IRE1fl/fl treated or not with 4μ8C, XBP1ΔDC IRE1truncDC and XBP1ΔDC BMDCs. Bar graphs depict mean ± S.E.M (n=3 mice, one representative experiment from 2 independent experiments). Tukey's ANOVA one-way test, ns: p>0.05.


( A , B ) Expression of VenusFP by splenic cDC1 ( CD26 + CD11c + MHCII + XCR1 + CD172a - , upper panels ) and cDC2 ( CD26 + CD11c + MHCII + XCR1 - CD172a + , lower panel ) from WT ( A ) and ERAI ( B ) mice non - infected ( NI ) or infected with Pru ( 6 days post - infection ( dpi ) ) reporting on IRE1α pathway activation .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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, B) Expression of VenusFP by splenic cDC1 (CD26+ CD11c+ MHCII+ XCR1+ CD172a-, upper panels) and cDC2 (CD26+ CD11c+ MHCII+ XCR1- CD172a+, lower panel) from WT (A) and ERAI (B) mice non-infected (NI) or infected with Pru (6 days post-infection (dpi)) reporting on IRE1α pathway activation.


( E , F ) RT - qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 ( E ) and cDC2 ( F ) sorted by flow cytometry 6 dpi from wild - type mice , non - infected ( NI ) or infected with Pru parasites . Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition . Bar graphs depict mean ± S . E . M . Unpaired Student T - test , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; mean ± S . E . M ( n = 3 - 5 mice pooled from 2 independent experiments ) .

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

(E, F) RT-qPCR analysis of the indicated UPR genes among total RNA prepared from splenic cDC1 (E) and cDC2 (F) sorted by flow cytometry 6 dpi from wild-type mice, non-infected (NI) or infected with Pru parasites. Results were normalized to the housekeeping gene Gapdh and are expressed as relative mRNA level compared to the NI condition. Bar graphs depict mean ± S.E.M. Unpaired Student T-test, ns: p>0.05; *: p<0.05; **: p<0.01; mean ± S.E.M (n=3-5 mice pooled from 2 independent experiments).


( A ) Survival curves of XBP1fl / flIRE1fl / fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites . Log - rank ( Mantel - Cox ) test , p = 0 , 0004 ; n = 10 mice per genotype .

O O O O O O O O O O O O O O O O O O O O O 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) Survival curves of XBP1fl/flIRE1fl/fl and XBP1ΔDC IRE1truncDC mice infected intraperitoneally with 500 Pru parasites. Log-rank (Mantel-Cox) test, p=0,0004; n=10 mice per genotype.


( D , E ) Quantification of the number of total ( left histogram ) and activated ( right histogram ) ( IFNγ - positive ) CD4 + T cells ( D ) and CD8 + T cells ( E ) in the spleen of control mice ( XBP1fl / flIRE1fl / fl ) and XBP1ΔDC IRE1truncDC infected for 6 days . Two - way ANOVA Tukey ' s multiple compare , ns : p > 0 . 05 ; * : p < 0 . 05 ; * * : p < 0 . 01 ; * * * : p < 0 . 001 ; * * * * : p < 0 . 0001 ; mean ± S . E . M ( n = 2 independent experiments combined ) .

(D, E) Quantification of the number of total (left histogram) and activated (right histogram) (IFNγ-positive) CD4+ T cells (D) and CD8+ T cells (E) in the spleen of control mice (XBP1fl/flIRE1fl/fl) and XBP1ΔDC IRE1truncDC infected for 6 days. Two-way ANOVA Tukey's multiple compare, ns: p> 0.05; *: p <0.05; **: p <0.01; ***: p <0.001; ****: p <0.0001; mean ± S.E.M (n=2 independent experiments combined).


b , c Phospho - sites identified after TiO2 enrichment b and phospho - tyrosine immunoprecipitation ( pY - IP ) c . Left panel : pie chart presenting the proportion of identified phosphorylated serines ( pS ) , threonines ( pT ) and tyrosines ( pY ) in the two data sets . Right panel : cumulative number of unique identified phospho - sites across the biological replicates ( 3 and 4 independent experiments for the TiO2 and pY - IP , respectively ) . Phosphorylated serines , threonines and tyrosines are presented individually next to the total number of identified sites ( pSTY ) . The phospho - sites that were not previously reported in any species in the PhosphoSitePlus database ( www . phosphosite . org ) are reported in isolation in the graph on the right .

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

b, c Phospho-sites identified after TiO2 enrichment b and phospho-tyrosine immunoprecipitation (pY-IP) c. Left panel: pie chart presenting the proportion of identified phosphorylated serines (pS), threonines (pT) and tyrosines (pY) in the two data sets. Right panel: cumulative number of unique identified phospho-sites across the biological replicates (3 and 4 independent experiments for the TiO2 and pY-IP, respectively). Phosphorylated serines, threonines and tyrosines are presented individually next to the total number of identified sites (pSTY). The phospho-sites that were not previously reported in any species in the PhosphoSitePlus database (www.phosphosite.org) are reported in isolation in the graph on the right.


a , b Output of the statistical analyses of the TiO2 - fractionated a and the pY - IP - fractionated b data sets . Left panel : number of phospho - sites significantly regulated upon TCR stimulation presented in bar plots for each time point , next to the total number of regulated sites across the entire time course ( total ) . The number of phosphorylated threonines ( pT ) and serines ( pS ) regulated in the TiO2 data set are indicated in yellow and green , respectively . Right panel : representative volcano plot presenting the statistical significance distribution against the log2 - transformed fold change between 300 seconds and the unstimulated control . For each condition , phospho - sites were considered significantly up - ( red ) or down - ( blue ) regulated when displaying a corrected P - value ≤ 0 . 05 ( Anova test ) and an absolute fold change ≥ 1 . 75 ( see Materials and Methods for more detailed information ) .

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

a, b Output of the statistical analyses of the TiO2-fractionated a and the pY-IP-fractionated b data sets. Left panel: number of phospho-sites significantly regulated upon TCR stimulation presented in bar plots for each time point, next to the total number of regulated sites across the entire time course (total). The number of phosphorylated threonines (pT) and serines (pS) regulated in the TiO2 data set are indicated in yellow and green, respectively. Right panel: representative volcano plot presenting the statistical significance distribution against the log2-transformed fold change between 300 seconds and the unstimulated control. For each condition, phospho-sites were considered significantly up- (red) or down- (blue) regulated when displaying a corrected P-value ≤ 0.05 (Anova test) and an absolute fold change ≥ 1.75 (see Materials and Methods for more detailed information).


a Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster . Black dots indicate clusters with significantly enriched kinase activity ( hypergeometric test P - value ≤ 0 . 05 , fold - change ≥ 2 , number of annotated sites ≥ 2 ; Grey squares : fold - change < 1 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Heatmap showing kinase activities based on enrichment of their specific substrates for each cluster. Black dots indicate clusters with significantly enriched kinase activity (hypergeometric test P-value ≤ 0.05, fold-change ≥ 2, number of annotated sites ≥ 2; Grey squares: fold-change < 1).


d Immunoblot analysis of equal amounts of proteins from total lysates of CD4 + T cells treated with DMSO , U0126 ( 10 μM ) or AKTi VIII ( 10 μM ) and left unstimulated ( - ) or stimulated for 15 , 120 , or 300 sec with anti - CD3 and anti - CD4 antibodies , probed with the indicated phospho - specific antibodies . Anti - ERK1 / 2 immunoblot served as a loading control .

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

d Immunoblot analysis of equal amounts of proteins from total lysates of CD4+ T cells treated with DMSO, U0126 (10 μM) or AKTi VIII (10 μM) and left unstimulated (-) or stimulated for 15, 120, or 300 sec with anti-CD3 and anti-CD4 antibodies, probed with the indicated phospho-specific antibodies. Anti-ERK1/2 immunoblot served as a loading control.


e Equal amounts of proteins from total lysates of CD4 + T cells left unstimulated ( - ) or stimulated for 120 , 300 or 600 sec with anti - CD3 and anti - CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho - sites / proteins .

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

e Equal amounts of proteins from total lysates of CD4+ T cells left unstimulated (-) or stimulated for 120, 300 or 600 sec with anti-CD3 and anti-CD4 antibodies were analyzed by immunoblot with antibodies probing the indicated phospho-sites/proteins.


b Cas9 - EGFP OT - I CD8 + T cells were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers ( 0 . 1 nM ) in presence or absence of soluble anti - CD28 antibody or with IL - 7 as control . Cell surface expression of CD69 and CD5 were analyzed by flow cytometry ( Z - score normalization of the Geometric Means of fluorescence MFI per experiment ) in four independent experiments . Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t - test . Boxplot elements : center line corresponds to median , box limits correspond to the first and third quartiles , whiskers indicate variability from Q1 - 1 . 5 . IQR to Q3 + 1 . 5 IQR . Outliers are shown as black dots .

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

b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC tetramers (0.1 nM) in presence or absence of soluble anti-CD28 antibody or with IL-7 as control. Cell surface expression of CD69 and CD5 were analyzed by flow cytometry (Z-score normalization of the Geometric Means of fluorescence MFI per experiment) in four independent experiments. Comparison between sgEGFP and sgITSN2 conditions were performed using a paired t-test. Boxplot elements: center line corresponds to median, box limits correspond to the first and third quartiles, whiskers indicate variability from Q1-1.5. IQR to Q3+1.5 IQR. Outliers are shown as black dots.


b Cas9 - EGFP OT - I CD8 + T cells were transfected with control sgRNA ( sgEGFP ) or with two different sgRNA targeting Itsn2 ( sgITSN2 - 1 or sgITSN2 - 2 ) . Transfected cells were stimulated for 48 h with N4 peptide

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

b Cas9-EGFP OT-I CD8+ T cells were transfected with control sgRNA (sgEGFP) or with two different sgRNA targeting Itsn2 (sgITSN2-1 or sgITSN2-2). Transfected cells were stimulated for 48 h with N4 peptide MHC


c Proliferation of OT - I Cas9 - EGFP CD8 + T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers ( 0 . 01 - 0 . 1 nM ) in the presence or absence of soluble anti - CD28 antibody or with PMA and ionomycin ( PMA / Iono ) or with IL - 7 . Data are presented as mean ± standard deviation ( SD ) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections . Data are representative of three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Proliferation of OT-I Cas9-EGFP CD8+ T cells transfected with EGFP or with ITSN2 sgRNA activated for 48 h with N4 peptide MHC tetramers (0.01-0.1 nM) in the presence or absence of soluble anti-CD28 antibody or with PMA and ionomycin (PMA/Iono) or with IL-7. Data are presented as mean ± standard deviation (SD) from four proliferative measures of two independent sgEGFP and sgITSN2 transfections. Data are representative of three independent experiments.


d IFN - γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation .

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

d IFN-γ production of cells treated as in c was assessed by intracellular staining after 24 h of stimulation.


e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti - CD28 antibody .

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

e Proliferation of similar cells as in c activated for 48 h in vitro with a range of increasing doses of N4 peptide MHC tetramers in the presence of soluble anti-CD28 antibody.


a Immunoblot analysis of equal amounts of proteins from total lysates of Cas9 - EGFP OT - I CD8 + T cells transfected with sgEGFP , sgITSN2 - 1 or sgITSN2 - 2 and left unstimulated ( - ) or stimulated for 2 or 5 min with N4 tetramers ( 20 nM ) . Upper panel : membrane probed with antibody to phosphorylated tyrosine ( Anti - p - Tyr ) or anti - LAT ( loading control ) . Lower panel : membrane probed with antibodies to anti - CBL - pY774 , anti - ZAP70 - pY319 / 352 , anti - AKT - pT308 , anti - ERK1 / 2 - pY204 / T202 or anti - ERK1 / 2 ( loading control ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-MEASURED_VAR O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Immunoblot analysis of equal amounts of proteins from total lysates of Cas9-EGFP OT-I CD8+ T cells transfected with sgEGFP, sgITSN2-1 or sgITSN2-2 and left unstimulated (-) or stimulated for 2 or 5 min with N4 tetramers (20 nM). Upper panel: membrane probed with antibody to phosphorylated tyrosine (Anti-p-Tyr) or anti-LAT (loading control). Lower panel: membrane probed with antibodies to anti-CBL-pY774, anti-ZAP70-pY319/352, anti-AKT-pT308, anti-ERK1/2-pY204/T202 or anti-ERK1/2 (loading control).