I : RT - qPCR of IFNB IFNG in Kms27 MM transiently transfected as in H . Values were normalized to Actin expression . Error bars represent standard error of three different biological experiments performed in duplicate ( Two T * * P < 0 . 01 , * * * P < 0 . 005 . )

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

I: RT-qPCR analysis of IFNB and IFNG levels in Kms27 MM cells transiently transfected as in H. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test **P<0.01, ***P<0.005.)


A : Representative S9 . 6 immunofluorescence in healthy CD138 + plasma purified from bone marrow . Nuclei were visualized by with Hoechst dye . Scale bar 10 μm .

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

A: Representative S9.6 immunofluorescence levels in healthy and tumor CD138+ plasma cells purified from patient bone marrow. Nuclei were visualized by staining with Hoechst dye. Scale bar 10 μm.


B : Dot blot performed to evaluate enrichment of : hybrids formation using S9 . 6 antibody by serial dilutions of genomic starting at 1 . 0 micrograms . Methylene blue normalization of S9 . 6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd .

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

B: Dot blot performed to evaluate the enrichment of RNA:DNA hybrids formation using the S9.6 antibody by serial dilutions of genomic DNA starting at 1.0 micrograms. Methylene blue normalization of S9.6 signal image acquired by using Alliance Mini HD6 system by UVITEC Ltd.


C : Boxplot showing relative S9 . 6 mean in CD138 + plasma from 15 MM ( red ) 5 healthy ( blue ) . Dot plot representing each considered overlay boxplot . Y - axis : S9 . 6 Mean ( AU ) . center denotes median value ( 50th percentile ) while boxes contain 25th to 75th percentiles of data . whiskers mark 5th 95th percentiles .

O O O O O O O O O O B-GENEPROD O B-CELL_TYPE O O B-DISEASE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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: Boxplot showing relative S9.6 nuclear mean intensity in CD138+ plasma cells from 15 MM patients (red) and 5 healthy (light blue). Dot plot representing each cell considered overlay the boxplot. Y-axis: S9.6 Nuclear Mean Intensity (AU). The center line denotes the median value (50th percentile) while the boxes contain the 25th to 75th percentiles of data. The whiskers mark the 5th and 95th percentiles.


D : Representative WB with indicated antibodies of healthy neoplastic CD138 + purified plasma from .

O O O B-EXP_ASSAY O O O O O O B-GENEPROD O O B-CELL_TYPE O O

D: Representative WB analysis with the indicated antibodies of healthy and neoplastic CD138+ purified plasma cells from patients.


F : Unsupervised clustering showing enrichment profiles of 9 IFN signature genes . From top : ISS stages 1 , 2 , 3 of each ( N = 687 ) . IFN signature High ( red , N = 71 ) Low ( , N = 614 ) assigned to each . Color scheme representing up - ( red ) down - ( blue ) regulation of selected genes across dataset . Colors are proportional to normalized ( cpm ) read enrichment - relative read enrichment median of each gene within full cohort .

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

F: Unsupervised clustering heatmap showing enrichment profiles of 9 IFN signature genes. From the top: ISS stages 1,2,3 of each patient (N=687). IFN signature High (red, N=71) and Low (white, N=614) assigned to each patient. Color scheme representing up- (red) and down- (blue) regulation of the selected genes across the patient dataset. Colors are proportional to normalized (cpm) read enrichment - the relative read enrichment median of each gene within the full patient cohort.


A : WB of total extracts from Kms27 U266 human MM with indicated abs .

O O B-EXP_ASSAY O O O O B-CELL_LINE I-CELL_LINE B-ORGANISM B-DISEASE O O O O

A: WB analysis of total extracts from Kms27 and U266 human MM cells with the indicated abs.


B : ( Left ) Kms27 U266 MM were subjected to immunostaining with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 mean in nucleus . 100 were counted in each replicate . Median scores per sample are written at top of median red . P value : 3 . 3 - 13 . T - was performed to evaluate statistical significance .

O O O O O B-CELL_LINE I-CELL_LINE B-DISEASE O O O B-EXP_ASSAY O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O B-SUBCELLULAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

B: (Left) Kms27 and U266 MM cells were subjected to immunostaining with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity cell in the nucleus. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 3.3-13. T-test was performed to evaluate statistical significance.


C : Different expression of IFNG IFNB in Kms27 U266 MM was assessed by RT - qPCR . Values were normalized to Actin expression . Error bars represent standard error of three different biological experiments performed in duplicate ( Two T * * * P < 0 . 005 ) .

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

C: Different expression of IFNG and IFNB in Kms27 and U266 MM cells was assessed by RT-qPCR. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005).


D : WB with indicated abs of total extracts from U266 MM treated or not with Thapsigargin ( THAP ) 0 . 5 μg / ml for 16 hours . G : WB with indicated abs of total extracts from Kms27 MM treated or not with ISRIB 1μM for 6 hours .

O O B-EXP_ASSAY O O O O O O O B-CELL_LINE B-DISEASE O O O O B-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-CELL_LINE B-DISEASE O O O O B-SMALL_MOLECULE O O O O O

D: WB analysis with the indicated abs of total extracts from U266 MM cells treated or not with Thapsigargin (THAP) 0.5 μg/ml for 16 hours. G: WB with indicated abs of total extracts from Kms27 MM cells treated or not with ISRIB 1μM for 6 hours.


E : ( Left ) U266 MM treated or not with THAP as in D immunostained with S9 . 6 antibody Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 mean per . 100 were counted in each replicate . Median scores per sample are written at top of median red . P value : 1 . 1 - 20 . T - was performed to evaluate statistical significance . H : ( Left ) Immunostaining with S9 . 6 antibody of Kms27 MM treated or not with ISRIB as in G Scale bar 10 μm . ( Right ) Dot plot showing relative S9 . 6 mean per . 100 were counted in each replicate Median scores per sample are written at top of median red . P value : 4 . 7 - 23 . T - was performed to evaluate statistical significance .

O O O O O B-CELL_LINE B-DISEASE O O O O B-SMALL_MOLECULE O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O B-CELL_LINE B-DISEASE O O O O B-SMALL_MOLECULE O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E: (Left) U266 MM cells treated or not with THAP as in D and immunostained with S9.6 antibody Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate. Median scores per sample are written at the top of the median red line. P value: 1.1-20. T-test was performed to evaluate statistical significance. H: (Left) Immunostaining with S9.6 antibody of Kms27 MM cells treated or not with ISRIB as in G Scale bar 10 μm. (Right) Dot plot showing relative S9.6 nuclear mean intensity per cell. 100 cells were counted in each replicate Median scores per sample are written at the top of the median red line. P value: 4.7-23. T-test was performed to evaluate statistical significance.


F : RT - qPCR of IFNG IFNB in U266 MM treated or not with THAP . Values were normalized to Actin expression . Error bars represent standard error of three different biological experiments performed in duplicate ( Two T * * * P < 0 . 005 , * * * * P < 0 . 001 ) I : RT - qPCR of IFNG IFNB in Kms27 MM treated as in G . Values were normalized to Actin expression . Error bars represent standard error of three different biological experiments performed in duplicate ( Two T * * * * P < 0 . 001 ) .

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

F: RT-qPCR analysis of IFNG and IFNB in U266 MM cells treated or not with THAP. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ***P<0.005, ****P<0.001) I: RT-qPCR analysis of IFNG and IFNB levels in Kms27 MM cells treated as in G. Values were normalized to Actin expression. Error bars represent the standard error of three different biological experiments performed in duplicate (Two Tailed T test ****P<0.001).


( A , B ) Immunoblot of phosphorylation level of CLIP - 170 , AMPK ACC in heart ( A ) liver ( B ) along with developmental stages . α - tubulin was used as a loading control . ( C - E ) Quantitative of western blotting shown in Fig 1A . ( F - H ) Quantitative of western blotting shown in Fig 1B .

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

(A, B) Immunoblot analysis of the phosphorylation level of CLIP-170, AMPK and ACC in heart (A) and liver (B) along with the developmental stages. α-tubulin was used as a loading control. (C-E) Quantitative analysis of western blotting shown in Fig 1A. (F-H) Quantitative analysis of western blotting shown in Fig 1B.


( I ) Immunostained of adult mouse heart tissue . These were stained with a phosphorylated AMPK antibody ( I , upper ) , a phosphorylated CLIP - 170 antibody ( I , lower ) a plakoglobin antibody ( middle ) . representative from more than 5 analyzed tissues were shown .

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

(I) Immunostained images of adult mouse heart tissue. These were stained with a phosphorylated AMPK antibody (I, upper), a phosphorylated CLIP-170 antibody (I, lower) and a plakoglobin antibody (middle). The representative images from more than 5 analyzed tissues were shown.


( A , B ) Immunostained of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , middle row ) 4 hours after washing out of MYK - 461 ( Wash out , bottom row ) . These were stained with a phosphorylated AMPK antibody , an AMPKβ2 antibody a N - cadherin antibody .

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

(A, B) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, middle row) and 4 hours after washing out of MYK-461 (Wash out, bottom row). These cells were stained with a phosphorylated AMPK antibody, an AMPKβ2 antibody and a N-cadherin antibody.


( C , D ) Immunostained of neonatal rat cardiomyocytes 2 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) stained with a connexin43 antibody , a plakoglobin antibody , an LKB - 1 antibody an N - cadherin antibody . representative from 4 independent experiments were shown .

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

(C, D) Immunostained images of neonatal rat cardiomyocytes 2 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row) and stained with a connexin43 antibody, a plakoglobin antibody, an LKB-1 antibody and an N-cadherin antibody. The representative images from 4 independent experiments were shown.


( E ) of pAMPK , AMPKβ2 , Connexin43 or LKB1 that was localized with a - junction marker ( N - cadherin or Plakoglobin ) . Box whisker plots showed 25th percentile ( bottom of each box ) , median ( middle of each box ) , 75th percentile ( top of each box ) , maximum minimum ( each whisker ) . Numbers in brackets indicate that were analyzed for . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using Tukey method . Two - Student ' s t - was used to analyze differences between two groups . * , P < 0 . 05 * * , P < 0 . 01 . n . s . , not significant .

O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of pAMPK, AMPKβ2, Connexin43 or LKB1 that was localized with a cell-cell junction marker (N-cadherin or Plakoglobin). Box and whisker plots showed 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). Numbers in the brackets indicate the cells that were analyzed for quantification. Differences among multiple groups were compared by one-way ANOVA, followed by a post hoc comparison using the Tukey method. Two-tailed Student's t-test was used to analyze differences between two groups. *, P<0.05 **, P<0.01. n.s., not significant.


( A ) GFP time - lapse of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 min 15 min after treatment with 20 μM Compound C ( left side panel ) expressing EGFP - CLIP - 170 S311A ( right side panel ) . Higher magniﬁcation of square ( upper row ) showing CLIP - 170 migrated longitudinally toward - junctions .

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

(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0 min and 15 min after treatment with 20 μM Compound C (left side panel) and expressing EGFP-CLIP-170 S311A (right side panel). Higher magniﬁcation of white square (upper row) showing CLIP-170 migrated longitudinally toward the cell-cell junctions.


( B ) Beeswarm plots of comet of EGFP - CLIP - 170 . comet from multiple in different fields was analyzed . Number of comets analyzed , Pre : n = 32 , Cpd . C : n = 36 , CLIP S311A : n = 68 . Data means ± S . D . Differences among multiple groups were compared by one - way ANOVA , followed by a post hoc comparison using Tukey method . * * , P < 0 . 01 versus Pre .

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

(B) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells in different fields was analyzed. Number of comets analyzed, Pre: n=32, Cpd. C: n=36, CLIP S311A: n=68. 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 Pre.


( C ) GFP time - lapse of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT treated with control siRNA ( siCL , left ) or siRNA targeting both AMPKα1 α2 ( siAMPKα1α2 , right ) . dotted lines in showed connected cardiomyocyte not expressing EGFP - CLIP - 170 WT .

O O O B-GENEPROD O O O O O B-ORGANISM B-CELL_TYPE O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O B-CELL_TYPE O O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O

(C) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT treated with control siRNA (siCL, left) or siRNA targeting both AMPKα1 and α2 (siAMPKα1α2, right). White dotted lines in the images showed the connected cardiomyocyte not expressing EGFP-CLIP-170 WT.


( D ) Beeswarm plots of comet of EGFP - CLIP - 170 . comet from multiple from different fields was analyzed . Number of comets analyzed , siCL : n = 178 , siAMPKα1α2 : n = 184 . Data means ± S . D . Two - Student ' s t - was used to analyze differences between two groups . * * , P < 0 . 01 versus siCL .

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

(D) Beeswarm plots of comet length of EGFP-CLIP-170. The comet length from multiple cells from different fields was analyzed. Number of comets analyzed, siCL: n=178, siAMPKα1α2: n=184. Data means ±S.D. Two-tailed Student's t-test was used to analyze differences between two groups. **, P<0.01 versus siCL.


( A ) GFP time - lapse of neonatal rat cardiomyocytes expressing EGFP - CLIP - 170 WT 0 , 2 3 hours after treatment with 2 μM MYK - 461 .

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

(A) GFP time-lapse images of neonatal rat cardiomyocytes expressing EGFP-CLIP-170 WT 0, 2 and 3 hours after treatment with 2 μM MYK-461.


( B ) Immunostained of neonatal rat cardiomyocytes 4 hours after treatment with 0 . 01 % DMSO ( Control , upper row ) or 2 μM MYK - 461 ( MYK - 461 , bottom row ) . These were stained with an α - tubulin ( green ) a plakoglobin ( red ) antibody .

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

(B) Immunostained images of neonatal rat cardiomyocytes 4 hours after treatment with 0.01% DMSO (Control, upper row) or 2 μM MYK-461(MYK-461, bottom row). These cells were stained with an α-tubulin (green) and a plakoglobin (red) antibody.


( C ) Box whisker plots of of a cardiomyocyte 2 hours after treatment with or without 2 μM MYK - 461 , showing 25th percentile ( bottom of each box ) , median ( middle of each box ) , 75th percentile ( top of each box ) , maximum minimum ( each whisker ) . Adenovirus expressing EGFP - CLIP - 170 ( adCLIP ) mutant was used ( WT , S311A , S311D ) . Numbers in graph indicate n number of 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 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 B-CELL_TYPE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O B-GENEPROD O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-GENEPROD O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O 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 echocardiographic parameter ( ejection fraction ) of Cre control ( CLIP - 170 S311A + / + ; α - MHC - MerCreMer + / − ) , Control mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer − / − ) , CLIP - 170 S311A overexpressing mice ( CLIP - 170 S311A flox / + ; α - MHC - MerCreMer + / − ) , before ( Pre ) , 2 ( Tx2w ) , 8 ( Tx8w ) , 26 ( Tx26w ) 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 B-EXP_ASSAY O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O O B-ORGANISM O B-GENEPROD I-GENEPROD I-GENEPROD O O O O O B-GENEPROD I-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O B-ORGANISM O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 of CLIP - 170 S311A overexpressing mice ( 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 . # # : 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 vs 52 .

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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 of CLIP - 170 S311A TG mice control mice over 1 year after tamoxifen induction . Left column showed systole right column represented diastole . Three mice from each group were analyzed for cardiac MRI . representative 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 ejection fraction from cardiac MRI . LV , left ventricular , RV , right ventricular . Number of mice , ( Control , S311A ) , 1y ( 3 , 3 ) . Data means ± S . D . Two - Student ' s t - 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 echocardiographic parameter ( ejection fraction ) from CLIP - 170 S311D overexpressing mice 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 of heart of CLIP - 170 S311A overexpressing mice control mice from 3 months 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 of cardiac fibrosis . was made as of fibrosis ( in blue ) in 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 with an α - tubulin antibody of CLIP - 170 S311A overexpressing mice heart control mice heart over 1 year after tamoxifen treatment . ( D ) Quantitative of of α - tubulin in ( C ) . Number of mice , ( Control : n = 7 , S311A : n = 8 ) . Average of 5 sections per 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 of western blot for an α - tubulin antibody . GAPDH was used as a loading control . ( F ) Quantitative of 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 of CLIP - 170 S311A overexpressing mice heart control mice heart over 1 year after tamoxifen treatment . These were stained with WGA ( red ) 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 of ( G ) width , aspect ( / width ) were measured . Number of 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 vestibular nucleus due to fine vesiculation ( arrows ) clear vacuolization ( arrowhead ) of 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 vacuolization ( arrows ) in cerebellar corticalPurkinje . 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 ( arrows ) a dense granular layer . HE , scale bar 100 μm . ( D ) Marked neuronal loss is present cerebellar cortex of an affected dog . number of neurons in granular layer is reduced only scattered Purkinje 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 matter ( arrows ) of cerebellum 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 vacuolization of exocrine pancreatic acinar . HE , scale bar 100 μm .

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


( G ) Purkinje with numerous single - bound , 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 - - bound autophagosomes ( arrows ) 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 layer ; gl , granular 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 ) granular cores of spheroids are positive for ( B ) ubiquitin ( arrow ) ( C ) p62 . IHCubiquitin p62 , scale bars 20 100 μm , respectively . ( D ) Smooth axonal swellings in 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 ) 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 morphant zebrafishembryos . ( A , B ) Control appear normal at 1 dpf . ( C , D ) Morphant show severe abnormalities in different regions of brain at 1 dpf . Black arrowheads denote developing cerebellum . Purple arrows indicate hindbrain irregularities . ( E ) Control at 2 dpf . ( F ) A morphant at 2 dpf displaying hydrocephalus ( yellow arrow ) small head 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 showing efficiency of atg4daSMO .

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


Immunostaining with ( A - C ) anti - Pvalb7 ( D - F ) anti - zebrinII antibody show loss of cerebellarPurkinje in atg4da morphants . ( A , D ) Control at 4 . 5 dpf . ( B , E ) Morphants with mild phenotype show partial loss of cerebellarPurkinje . ( C , F ) Morphants with strong phenotype show either total loss of Purkinje or presence of few differentiated neurons , which are laterally located in cerebellum . ( G - I ) Labeling of cerebellar granule with anti - Vglut1 antibody in control morphant . ( H ) Mildly affected morphants show reduced expression of Vglut1 in cerebellum . ( I ) Vglut1 expression is strongly reduced in showing severe phenotype . arrowheads indicate 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 populations . Upper left : GFP + in population of ESCs infected with pGIPZ - shRNA library lentiviruses . Upper right : Isolation of mesodermal ( SSEA1 - Flk1 + Cxcr4 - ) endodermal ( SSEA1 - Flk1 - Cxcr4 + ) . Lower panels : Isolation of D20LS ( Lin - Sca1 + c - Kit - ) , D20LK ( Lin - Sca1 - c - Kit + ) , D20LSK ( Lin - Sca1 + c - Kit + ) .

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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 of expression of endothelial ( Tie2 , CD31 , Cdh5 ) , epithelial ( Cdh1 ) , early hematopoietic ( Scl , CD41 ) marker genes in indicated sorted populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - . * 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 purity of sorted populations by qRT - PCR : c - Kit is highly expressed only in c - Kit + ( D20LSK D20LK ) populations , Sca1 is highly expressed only in in Sca1 + ( D20LSK D20LS ) populations . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - . * 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 of Gata1 , Gata2 , Lyz2 , Scl expression in sorted populations on day 20 . Error bars represent standard deviation ( SD ) , n = 3 . Student ' s t - . * p < 0 . 05 .

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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 of ESC - derived confirming that D20LS , D20LK , D20LSK populations express early hematopoietic marker CD41 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 enrichment analyses of shRNA reads in D6C ( endoderm ) , D6F ( HM / E ) , D20LS , D20LK , 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 D6F populations compared with ESCs . shRNAs specifically depleted in D6C D6F are labeled as Groups XI XII , respectively .

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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 Cdk genes in day 20 samples .

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


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

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

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

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

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

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 : E14tg2a ESCs were transduced with lentiviral shRNAs targeting genes Ap2a1 , Mettl22 , Lrsam1 , or Hal differentiated towards HSPCs . differentiated on day 20 were analyzed by flow cytometry . Flow cytometric showed notable reduction of LSK population on day 20 with Group X genes Ap2a1 , Mettl22 , Lrsam1 , or Hal knockdown . H : of flow cytometric analyses showed knockdown of Ap2a1 , Mettl22 , Lrsam1 , 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 - , * p < 0 . 05 .

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

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 most depleted shRNAs in pair - wise comparisons between D20LSK D20LK populations .

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


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

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

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

O O O O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-GENEPROD O O O O O O O O O O O O O O B-GENEPROD O O O O O O O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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

O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O B-GENEPROD O B-GENEPROD O O O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O

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 from fed fasted mice were analyzed by SDS - PAGE immunoblot using Akt antibody . Right : densitometric measurement of presented blots ( n = 3 ) . * p < 0 . 05 vs . fed by one - t - . Data are represented as mean ± .

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

(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 soluble fraction of 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 .

O O O B-GENEPROD O B-EXP_ASSAY O O O O O O O O O O O B-ORGANISM O B-EXP_ASSAY O O O B-EXP_ASSAY O O O O B-SMALL_MOLECULE O O B-ORGANISM O O O O O O O O O O O O

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

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

)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 . Soluble extracts were analyzed by immunoblotting .

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

(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 transfected with shLacz control or shUSP1 from fed fasted mice were analyzed by SDS - PAGE immunoblotting using anti - Akt . actin blot serves as a loading control . Right : densitometric measurements of presented blots ( n = 3 ) . Data is presented as of ubiquitinated Akt to total Akt normalized to actin . * p < 0 . 05 vs . fed by one - t - . Data are represented as mean ± .

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

(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 soluble fraction of expressing USP1 ( C90S ) or control plasmids . Mouse IgG was used as a control for non - specific binding . precipitates were subjected to immunoblotting with an antibody against K63 - linked polyubiquitin conjugates .

O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O B-GENEPROD O O O B-GENEPROD O B-EXP_ASSAY O O O O O B-GENEPROD O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE O O

(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 soluble fraction of TA transfected with USP1 ( C90S ) , HA - Akt ( K8R ) or control from fasted mice , 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 between ubiquitinated Akt to total Akt in each lane ( n = 2 ) .

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

(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 atrophying expressing shLacz or USP1 ( C90S ) were analyzed by SDS - PAGE immunoblot . black indicate removal of intervening lanes for presentation purposes . Right : densitometric measurements of presented pAkt ( T308 ) , pAkt ( S473 ) , Akt blots . Data is presented as of phosphorylated Akt to total Akt ( n = 3 ) . * p < 0 . 05 vs . fed by one - t - . Data are represented as mean ± .

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

(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 Atrogin1 expression during fasting . Quantitative RT - PCR of mRNA preparations from atrophying control expressing shLacz or shUSP1 ( 80 % transfection efficiency ) using primers for MuRF1 Atrogin1 . Data are plotted as 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 - t - . Data are represented as mean ± .

O O O O O B-GENEPROD O B-GENEPROD I-GENEPROD B-EXP_ASSAY O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O O O O O O B-GENEPROD I-GENEPROD O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) 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 transfected with shUSP1 ( that express GFP , green bars ) vs . 500 nontransfected ( black bars ) in same muscle . n = 5 mice . A representative image is shown ; Laminin is in red ; Bar , 50μm .

O O O O O B-GENEPROD O O B-TISSUE I-TISSUE B-DISEASE O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O B-TISSUE O O O O B-ORGANISM O O O B-EXP_ASSAY O O O B-GENEPROD O O O O O O O O

(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 loss of muscle mass during fasting . shUSP1 was delivered to more than 60 % of muscle . Mean weights of electroporated are plotted as loss . n = 6 . # , P < 0 . 05 vs . shLacz in fed ; * , P < 0 . 05 vs . shLacz in fasting by one - t - . Data are represented as mean ± .

O O O O O B-GENEPROD O O B-TISSUE B-EXP_ASSAY O O O O O O O O O O O O B-TISSUE O O B-EXP_ASSAY O O O O O B-EXP_ASSAY O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) or saline during fasting ( 2d ) were subjected to glucose tolerance . Blood glucose were measured at indicated time points following glucose injection ( 1 mg / gr ) . 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 - t - . Data are represented as mean ± . Right : soluble fractions of from injected mice were analyzed by SDS - PAGE immunoblot .

O O O B-GENEPROD O O O B-EXP_ASSAY I-EXP_ASSAY O B-ORGANISM O O O O O B-ORGANISM O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O B-TISSUE B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O O O O O O O O O O O O O O O O O O O O O B-ORGANISM O O O B-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY I-EXP_ASSAY 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 transfected with shLacz or shPHLPP1 from fasted mice ( 2d ) were analyzed by immunoblotting . Right : densitometric measurements of presented blots . Data is presented as of shPHLPP1 to shLacz ( n = 3 for shLacz n = 4 for shPHLPP1 ) . * p < 0 . 05 vs . shLacz in fasting by one - t - . Data are represented as mean ± .

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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 synthesis during fasting . Mice were injected with puromycin , soluble fractions of electroporated were analyzed by immunoblotting using puromycin antibody . of right ( R ) 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 transfected with shPHLPP1 ( that express GFP , green bars ) vs . 500 nontransfected ( black bars ) in same muscle . n = 4 mice . A representative image is shown ; Laminin 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 soluble fraction of normal . 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 , TSC1 / TSC2 . Muscle homogenates from fed or fasted mice were separated on 10 % - 40 % glycerol gradient , alternate fractions were analyzed by SDS - PAGE immunoblot . Red rectangle marks fractions containing USP1 / UAF1 , PHLPP1 , TSC1 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 . TSC1 was immunoprecipitated from soluble fraction of 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 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 synthesis . Mice were injected with puromycin , soluble fractions of electroporated were analyzed by immunoblotting using puromycin antibody . Right : Densitometric measurement of presented blot . n = 3 . * , P < 0 . 05 vs . shLacz by one - t - . Data are represented as mean ± .

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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 expressing shLacz or USP1 ( C90S ) from fasted mice were analyzed by SDS - PAGE immunoblotting . Right : Densitometric measurement of presented blot . n = 3 . * , P < 0 . 05 vs . shLacz . Data is presented as content ( AU ) in USP1 ( C90S ) vs . shLacz by one - t - . Data are represented as mean ± .

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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 content enhanced Akt phosphorylation at T308 . However , a simultaneous downregulation of USP1 is required to promote TSC1 accumulation . Soluble fraction of expressing shLacz or gUSP1 ( in vivo CRISPR ) from fed fasted mice injected i . p . with rapamycin ( 6mg / kg ) or saline were analyzed by SDS - PAGE 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 interaction of Akt with USP1 - PHLPP1 - TSC1 . Left : TSC1 was immunoprecipitated from soluble fraction of 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 TSC1 blots , data is presented as PHLPP1 / TSC1 .

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

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(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 soluble fraction of expressing shLacz or Dab2 - DN from fed fasted mice using a specific antibody . Mouse IgG was used as a control for non - specific binding . 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 transfected with shLacz or Dab2 - DN from fed fasted mice were analyzed by SDS - PAGE immunoblotting using indicated antibodies . Right : densitometric measurement of presented blots . Data are mean ± , n = 3 . * p < 0 . 05 . vs . shLacz in fed conditions , by one - t - .

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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 - combinational therapies in head neck cancer : A table summarizing experimentally observed synergism between drugs their predicted rescuer - targeting treatments in 5 HNSC lines , on drug treatment experiments . Synergism was estimated using standard Fa - CI . table displays 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 lines tested . inset shows an example of CI calculation for BYL719 Dasatinib combination in HN12 lines on corresponding dose matrix ( number indicates % viability at 48h , n = 3 ) , 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 predicted SR ( DU ) rescuers of DNMT1 via drug combination experiments in 18 NSCLC lines insensitive to Decitabine . matrix displays drug interactions between Decitabine , a DNMT1 inhibitor , inhibitors of its predicted rescuer genes ( X - axis ) across 18 NSCLC lines ( Y - axis ) that are insensitive to Decitabine . Row labels present rescuer genes their inhibitors . Colors in matrix show whether interactions found are significantly synergistic ( red ) , antagonistic ( green ) or non - significant ( in gray ) . Values in matrix show average synergism ( < 1 synergism > 1 antagonism Thirteen predicted DU - SR rescuers ( red lines ) , two predicted DD SR rescuers ( green lines ) of DNMT1 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.


transcriptomic alterations of rescuer genes post PD1 / PDL1 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - PD1 ( b ) Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of 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.


transcriptomic alterations of rescuer genes post CTLA4 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after anti - CTLA4 ( c ) Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of 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.


transcriptomic alterations of rescuer genes post PD1 / PDL1 CTLA4 blockade in biopsies : Their post ( vs . pre ) treatment expression changes of DU / DD rescuers after PD1 + CTLA4 combination therapies ( d ) . Each panel displays expression fold change of each predicted rescuer gene ( rows ) for different samples ( columns ) P - value of over - paired Wilcoxon of 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 Δapg4Δapg8 producing Apg8FG Δapg7 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 a small amount of Apg8FG ) was extracted with 0 . 4 % β - octylglycoside from a polyacrylamide gel desalted with ZipTipC4 ( Millipore ) before mass measurement . principal MH + ion signal at m / z 15 , 003 is shifted in mass by about + 635 from 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 C - terminal peptides of Apg8FG - X ( top ) Apg8FG ( bottom ) . C - terminal fragments were generated from Apg8FG - X Apg8FG ( see Methods ) . Each fragment that was observed only as non - 18O - labelled signals was subjected to MS / MS . MS / MS spectra , deconvoluted with MaxEnt3 , were interpreted by SeqMS software26 . Arrows show sequences from N C terminus on y ″ m bl ions , respectively ( where m l denote positions counted from C N terminus ) that were produced by cleavage of peptide bonds during MS / MS . shown in three - letter code denote immonium ions . 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 Apg7 . extracts of Δapg7Δapg8 expressing Apg7 - Myc / or HA - Apg8 from 2µ plasmids were immunoprecipitated with anti - HA antibody , immunoblotted ( IB ) with anti - Myc anti - HA antibody . DTT - , no DTT ; DTT + , 100 mM DTT ; asterisk , Apg8 - Apg7 conjugate .

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

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

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

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 Apg8 - Apg7 interaction . Δapg4 co - expressing Apg7 - Myc HA - Apg8 ( FGR , FG F ) from 2µ plasmids . FGR , wild - type Apg8 ; FG , Apg8 lacking C - terminal arginine ; F , Apg8 lacking C - terminal glycine arginine . lysates were immunoprecipitated with anti - HA antibody , immunoblotted with anti - HA anti - Myc antibody .

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

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 Apg3 . Lysates of Δapg3 expressing APG3 / or Myc - APG8 from 2µ plasmids were immunoprecipitated with anti - Apg3 antibody , immunoblotted with anti - Apg3 anti - Myc antibody .

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

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 interaction between Apg8 Apg3 . Lysates of Δapg3 expressing Myc - APG8 either wild - type ( wt ) APG3 or mutant APG3 ( C234S C234A ) from 2µ plasmids were immunoprecipitated with anti - Myc antibody , immunoblotted with anti - Myc anti - Apg3 antibodies .

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

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

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

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.